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Клінічні випробування препаратів "Екомеду" при лікуванні ВІЛ інфекції та туберкульозу (оригінал на англ.мові)

Effect of Solid Formulation of Immunoxel (Dzherelo) as an Adjuvant Immunotherapy in Treatment of TB and TB/HIV Co-infected Patients.

Olga V. Arjanoval, Nathalia D. Phhoda2, Larisa V. Yurchenko3, Nina I.Sokolenko4, LyudmilaA. Vihrova5, VolodymyrS. Pylypchuk6, ValeryM. Frolov7, GalynaA. Kutsyna8 Lisichansk Tuberculosis Dispensary, Lisichansk, Ukrainel; Lisichansk Regional Hospital, Lisichansk, Ukraine!; Ekomed LLC, Kiev, Ukraine3; Luhansk Regional AIDS Center and Luhansk State Medical University, Luhansk, Ukraine kutsynagalyna@yahoo. com

Background: In previous studies Immunoxel - an oral immunomodulator used as an adjunct to TB therapy - was formulated as alcohol-water extract of medicinal herbs. In this study a solid, sublingual, sugar-based formulation of Immunoxel was tested to determine whether it will be as effective as liquid formula.
Methods: TB and TB/HIV co-infected patients received either first-line anti-TB therapy (ATT consisting of HRZES; Arm A; N=20) or ATT+ once daily dose of Immunoxel (Arm B; N=19).
Results: At the end of one-month of follow-up, 2 (10.5%) vs 17 (89.5%) patients had cleared M. tuberculosis in sputum samples and 7 (35%) vs 13 (65%) had culture conversion (P=0.0006) in arms A and B respectively. In ATT arm the body weight at baseline was 62.6±8.3 kg, after one month the average gain was 1.1 kg (P=0.03). The mean starting weight in Immunoxel arm was 54.1±7.5 kg, the immunotherapeutic intervention helped them to gain 2.8 kg (P=0.0000004). The hemoglobin (Hb) had increased by 2.95 g/L from 116.1 to 119 (P=0.03) in arm A, whereas those who received Immunoxel had Hb risen by 10.4 g/L from 117.3 to 127.7 (P=0.00007). Erythrocyte sedimentation rate in arms A and B decreased from 19.1 to 13.1 (P=0.00001) and 20.5 to 10.5 (P=0.0003) respectively. The leukocyte count decreased from 10.8 to 9 x109 (P=0.0007) and 9.5 to 5.8 x109 (P=0.00005). Clinical improvement was seen in 7 out 20 and 18 oat 19 patients in arms A and B. In each arm one patient died at the end of follow-up. The arms A and B had 7 and 2 patients co­ntacted with HIV who seemed to respond to therapy at the same rate as TB patients. Other solid formulations of Immunoxel are now being tested and results will be available in near future.
Conclusion: The preliminary findings indicate that solid formulation of Dzherelo is equally or perhaps even more effective than liquid formulation and improves significantly the clinical efficacy of anti-TB drugs and shortens treatment duration.

Effect of Immunomodulator Dzherelo In HIV/TB co-Infected patients receiving anti-tuberculosis therapy under DOTS

Lyudmila G. Nikolaeva1 *, Tatyana V. Maystat1, Volodymyr S. Pylypchuk2, Yuri L. Volyanskii3, Lllla A Masyuk4. Galyna Kutavna5*. 1 Kharkov Regional AIDS Prophylaxis and Prevention Center, Kharkov Medical Academy of Postgraduate Education, 6 Bor'by street, Kharkov 61044, Ukraine; 2Ekomed LLC. Prospect Pravdy 80-A, Kiev 04208, Ukraine: 3I.I. Mechnlkov inslltute of Microbiology and Immunology, Kharkov 61057, Ukraine; 'Jovtnevsky Correctional Colony No.17, State Department of the Penitentiary of Ukraine In Kharkov Region, Kharkov, Ukraine; 5Luhansk Regional AIDS Center and Luhansk State Medical University, Luhansk 91045, Ukraine; "Corresponding author: E-mail: kutsyna@list.ru
Open-label, phase II clinical trial was conducted in 40 HIV/TB dually infected patients to evaluate the effect of oral Immunomodulator Dzherelo on immune and viral parameters. The anti-retrovlral therapy naive patients were randomized into two equal groups, to be given anti-tuberculosis therapy (ATT) under DOTS. The arm A, which served as a conlrol, received Isonlazid (H); Rimfapicin (R); Pyrazinamide (Z); Streptomycin (S); and Ethambutol (E), and arm B received 50 drops of Dzherelo twice per day in addition to HR2SE. After 2 months the total CD3 lymphocytes increased from 728 to 921 cells/ul (P=0.025) in Dzherelo recipients, whereas in the control they decreased from 651 to 585 cells (P=0.25). The population of CD4 T-cells expanded in Dzherelo arm (174 to 283; P=0.00003) but declined In ATT group (182 to 174; P=0.34). The CD8 cells fluctuated slightly upward In both groups: 159>180 (P=0.17) and 159>183 (P=0.13).The ratio between CD4/CD8 cells deteriorated In arm A (1.213>0.943; P=0.002) but improved in arm B (1.244>1.536; P=0.007). The percent of CD3+HLA-QH+ activated lymphocytes fell in ATT (22.6>20.S; P=0.004), but rose in Dzherelo recipients (21.5>30.5; P=0.0001).The changes in CD20 B lymphocytes were Insignificant In both arms (28.4%>28.6%; P=0.4) and (27.2%>26.7%; P=0.38). No differences were seen in the amount of CD3-CD16+CD56+ natural killer (NK) cells In arm A (21.3%>22.6%; P=0.1), while in Dzherelo recipients they declined (19.9%>14.5%; P=0.003).The average viral load, as measured by plasma RMA-PCR, decreased in Dzherelo group (2,174>1,558 copies/ml; P=0.002), but increased In ATT group (1,907>2,076; P=0.03). Dzherelo has a favorable effect on the Immune status and viral burden in HIV/TB patients when given as an Immunomodulating adjunct to ATT.

Open label trial of adjuvant immunotherapy with Dzherelo, Svitanok and Lizorm, in MDR-TB, XDR-TB and TB/HIV co-infected patients receiving anti-tuberculosis therapy under DOT

Journal of Medicinal Plants Research Vol. 1 (5), pp. 117-122, December 2007 Available online at http://www.academicjournals.org/JMPR ISSN 1996-0875 © 2007 Academic Journals
Full Length Research Paper
Nathalia D. Prihoda1, Olga V. Arjanova1, Lyudmila V. Yurchenko1, Nina I. Sokolenko1, Lyudmila A. Vihrova2, Volodymyr S. Pylypchuk3, Galyna A. Kutsyna4*1Lisichansk Tuberculosis Dispensary, Lisichansk, Ukraine. 2Lisichansk Regional Hospital, Lisichansk, Ukraine. 3Ekomed LLC, Kiev, Ukraine. 4Luhansk Regional AIDS Center and Luhansk State Medical University, Luhansk, Ukraine.
Accepted 04, November 2007
Open label trial of anti-tuberculosis therapy (ATT) combined with oral immunomodulators derived from medicinal plants, Dzherelo, Svitanok , and  Lizorm, was conducted in a representative group of 14 Ukrainian patients, half of whom (7) were dually infected with HIV. Among them 9 individuals had multidrug-resistant form of TB (MDR-TB) including 2 (22%) patients who presented with extensively drug-resistant TB (XDR-TB). Patients hospitalized in our TB dispensary were treated under directly observed therapy (DOT) until they became culture negative and their radiological and clinical symptoms improved. All patients, except one, gained weight, ranging between 3-17 kg with median gain of 9 kg (P=0.0002). The liver function tests revealed that the level of total bilirubin had decreased from 15.5 to 12 umol/L - an improvement that was statistically significant (P=0.03). Alanine transaminase (ALT), another marker of hepatic damage, declined from abnormally high 55.4 IU/L to a normal 38.2 IU/L level (P=0.03). The median time to bacterial clearance was 32 days. The mean duration of therapy was 3.9 months - shorter than average 12 months time needed to treat drug-resistant TB. These findings indicate that the combination of Ekomed's phytopreparations with ATT enhances the efficacy of TB therapy and is safe and beneficial even to patients with poor prognosis due to drug resistance and/or co-infection with HIV.
Keywords: MDR-TB, XDR-TB, Mycobacterium tuberculosis, HIV, herbal, phytotherapy.
INTRODUCTION
The TB epidemic is on the rise in most countries, include-ing Ukraine. This problem is further compounded by HIV co-infection, since one-third of HIV/AIDS-related deaths results from TB. Ukraine has the highest preva-lence of TB/HIV co-infection in Eastern Europe (van der Werf et al., 2006). The effectiveness of TB therapy is significantly lower among patients with HIV/AIDS. The World Health Organization (WHO) estimates that a per-son with both HIV and TB infection is thirty times more likely to become ill with TB than a person with Mycobacterium tuberculosis infection alone (Reid et al., 2006).
The rate of relapse and mortality are consistently higher even when TB/HIV patients are treated with anti-tuberculosis therapy (ATT) under directly observed treatment regimen (DOT) (Khauadamova et al., 2001). Drug resistance accompanied by HIV-associated immunodeficiency is the main cause of treatment failure. The recently published survey of Nikolayevskyy et al., (2007) indicates that in Ukraine the multi-drug resistant form of TB (MDR-TB) was found in 27.3% of TB patients and was twice higher (54.8%) among incarcerated individuals.
The first line of TB drugs includes isoniazid (H), rifam-picin (R), ethambutol (E), pyrazinamide (Z), and streptomycin (S). There are six classes of second-line TB drugs including aminoglycosides: amikacin, kanamycin; polypeptides: capreomycin, viomycin, enviomycin; fluoroquinolones: ciprofloxacin, moxifloxacin; thioamides: ethionamide, prothionamide; cycloserine; and para-ami-nosalicylic acid. Other TB drugs, which are not on the WHO list, include: rifabutin; clarithromycin; linezolid; thio-acetazone; thioridazine; arginine; vitamin D; and R207910. MDR-TB is diagnosed when M. tuberculosis is resistant to at least isoniazid (H) and rifampicin (R), the two most powerful, first line drugs. The extensively resistant form of TB (XDR-TB), in addition to lack of sensitivity to H and R, is also resistant to any of fluoroquinolones, and at least one of second-line injectable drugs, e.g., kanamycin and amikacin (Migliori et al., 2007). These emerging strains of drug-resistant TB became of particular concern after recent publication of outbreak in South Africa where 52 of 53 patients with XDR tuberculosis died within 16 days from the time of diagnosis (Gandhi et al., 2006).
Immunomodulators
Dzherelo ,  Svitanok and Lizorm are made from a combination of medicinal plants and are commonly used in Ukraine for the management of TB and HIV infections, including patients with dual infection (Arjanova et al., 2006; Chkhetiany et al., 2007; Prihoda et al., 2006; Zaitzeva, 2006). They have been approved in 1997 by the Ministry of Health of Ukraine as functional supplements with therapeutic indications. In 1999 Dzherelo and  Svitanok were specifically recommended as immune adjuncts for the therapy of pulmonary tuberculosis (Melnik et al., 1999). So far they have been used by several hundred thousand individuals for various indications including chronic bacterial and viral infections such as TB and HIV, autoimmune diseases, and malign-nancy (Chkhetiany et al., 2007). Published studies have demonstrated that Dzherelo can significantly shorten the duration of treatment and helps to achieve higher response rate even in those who are HIV co-infected or have MDR forms of TB (Arjanova et al., 2006; Chkhetiany et al., 2007; Prihoda et al., 2006).  Dzherelo has also been found to decrease the hepatotoxicity associated with ATT (Zaitzeva, 2006). Svitanok is commonly used for counteracting the toxic effect of drugs and in hepatitis therapy. Lizorm  is commonly used for alleviating symptoms of autoimmune disorders. Our study was aimed at evaluating the combined effect of Dzherelo , Svitanok, and Lizorm in a representative sample of hospitalized patients who received the anti-TB therapy under DOT. Patients who had particularly poor prognosis due to drug resistance and/or HIV co-infection were selected to be given ATT in combination with three herbal immunomodulators.
MATERIALS AND METHODS
Patients
Fourteen patients with active TB and poor prognosis due to resistant TB and/or HIV co-infection were selected to be given in addition to ATT the over-the-counter phytopreparations manufactured by Ekomed company. The age of patients ranged between 24 and 58 years with mean/median age of 39 years. The female/male ratio was 3/11. The diagnosis of HIV infection was established by standard ELISA test further confirmed by Western blot. Seven patients presented with first-diagnosed or primary TB and the other half had previously treated, relapsed, or chronic TB. Nine patients had drug-resistant TB and five patients in TB/HIV subgroup were drug-sensitive. All patients with HIV were in advanced stage III of HIV infection. Neither patient has received the anti-retroviral therapy prior to and during follow-up. Active pulmonary tuberculosis was certified by a medical history and clinical findings compatible with pulmonary tuberculosis, a chest X-ray showing lung involvement, and positive sputum smear for acid-fast bacilli or the culture of M. tuberculosis. All patients received anti-tuberculosis therapy administered under DOT schedule. In addition to ATT, patients received a daily dose of
 Dzherelo which was given as 30 drops diluted in a half-glass of water at least 30 minutes before breakfast. Some patients received Dzherelo-PI - a modified form  Dzherelo. The same 30 drops dose of Lizorm  and Svitanok were given before lunch and supper respectively. The conduct of the trial was approved by the Ethical Board of Lisichansk TB dispensary. The participation in this study was voluntary and patients were eligible to enroll only after signing the written consent. Patients were treated until they were discharged from the dispensary. The decision to discharge was based on negative culture findings and satisfactory clinical and radiological findings.
Anti-tuberculosis drugs and phytopreparations
All anti-TB drugs were supplied through the centralized national supply system of Ukraine. Phytopreparations,
Dzherelo  Svitanok, and  Lizorm, were generously provided by Ekomed LLC. Dzherelo contains concentrated aqueous-alcohol extract from medicinal plants such as Aloe (Aloe arborescens), Common knotgrass (Polygonum aviculare), Yarrow (Achillea millefolium), Purple coneflower (Echinacea purpurea), St. John's Wort (Hypericum perforatum), Centaury (Centaurium erythraea), Snowball tree berries (Viburnum opulus), Nettle (Urtica dioica), Dandelion (Taraxacum officinale), Sweet-sedge (Acorus calamus), Oregano (Oreganum majorana), Marigold (Calendula officinalis), Seabuckthorn berries (Hippophae rhamnoides), Elecampane (Inula helenium), Tormentil (Potentilla erecta), Greater plantain (Plantago major), Wormwood (Artemisia sp.), Siberian golden root (Rhodiola rosea), Cudweed (Gnaphalium uliginosum), Licorice (Glycyrrhiza glabra), Fennel (Foeniculum vulgare), Chaga (Inonotus obliquus), Thyme (Thymus vulgaris), Three-lobe Beggarticks (Bidens tripartite), Sage (Salvia officinalis), Dog rose (Rosa canina), and Juniper berries (Juniperus communis). Svitanok contains flowers of Immortelle (Helichrysi arenarii), Barberry roots (Berberis vulgaris), Chicory roots (Cichorium intybus), Coriander seeds (Coriandrum sativum), Marigold (Calendula officinalis), Wormwood (Artemisia sp.), and Maize cores with stigmas (Zea mays).  Lizorm  contains concentrated aqueous-alcohol extract from Barberry roots (Berberis vulgaris), Aronia berries (Aronia melanocarpa), St. John's Wort (Hypericum perforatum), Centaury (Centaurium erythraea), Nettle (Urtica dioica), Common knotgrass (Polygonum aviculare), Wild strawberry leaves (Fragaria vesca), Greater celandine (Chelidonium majus), and Immortelle (Helichrysi arenarii). All phytoconcentrates were approved in 1997 by the Ministry of Health of Ukraine as dietary botanical supplements. In 2006 they have received so-called status of functional food - a special category of herbal supplements that can carry medical claims which were substantiated by clinical evidence.
Test for drug resistance
The cultures of M. tuberculosis derived from sputum of each patient were inoculated into ready-to-use tubes each containing one of first-line and second-line TB drug that were incorporated at predetermined concentrations into standard Lowenstein-Jensen agar slants. The drug resistance was evaluated with commercially available kit (Tulip Diagnostics, Goa, India). The cultures were incubated at 37o C and checked periodically until appearance of colonies in control, drug-free tubes.
Statistical analysis
The obtained results were analyzed with the aid of statistical software STATMOST (Datamost, South Sandy, UT). The baseline cell numbers relative to the end of study were evaluated by paired Student t-test. All statistical calculations were done on total number of patients without subgrouping them into responders and non-responders. When required stratification analysis was carried out to determine the difference between distinct categories of patients. The resulting probability values were considered as significant at P<0.05.
RESULTS
The treatment lasted until patients were discharged from the dispensary upon negative culture findings and satisfactory clinical and radiological findings. The duration of DOT ranged between 10.6-30.3 weeks with average/median 16.7/16.2 weeks (Table 1). While patients with HIV co-infection were treated on average 3 weeks longer, the difference was not statistically significant (P=0.17). The time to negative culture ranged between 10-62 days with mean/median 32/33 days. The patients with HIV became culture negative at 36 instead of 30 days as in patients with TB alone but the difference was not significant (P=0.2). Similarly, no difference was seen between chronic, previously treated TB and first-diagnosed TB cases in terms of days to discharge, i.e., 113.3 vs 120.4 (P=0.38) or days to bacterial clearance, 34.7 vs 31.4 (P=0.33). The comparison of treatment outcomes between 9 drug-resistant and 5 drug-sensitive cases also failed to reveal statistical difference. Time to negative culture was 34 vs 31.4 days and time to discharge 107 vs 135 days with probability values P=0.33 and P=0.18 respectively.
As a result of combination treatment our patients experienced better quality of life and were tolerating ATT at much higher degree than those who received ATT without phytotherapy. This is reflected and supported by intriguing observation that almost every patient had gained substantial lean body mass - an effect that was evident within one month from initiation of the therapy. Except patient #10 who lost 10 kg, all other patients gained weight, ranging between 3 and 17 kg by the end of 3.9 months of follow-up. The average accrual in lean body mass was 8.4 kg (median 9 kg) which was statistically highly significant (P=0.0002).
The improvement of quality of life is further supported by quantitative liver function tests. The level of total biliru-bin had decreased from mean 15.5 to 12 umol/L - a favorable change that was statistically significant (P = 0.03). Similarly the values of alanine transaminase (ALT), another marker of hepatic damage, have declined from elevated (55.4 IU/L) to normal levels (38.2 IU/L) - a change that was also statistically significant (P=0.03).
Another sign of clinical improvement is a recovery from anemia and inflammatory status. Most patients at study entry were anemic and had abnormally elevated leukocyte counts. At the end of treatment these parameters were improved in a statistically significant manner. The levels of hemoglobin had risen from 105±15.6 to 117±5.3 g/L (P=0.003), whereas leukocyte counts had returned back to normal levels from 8.7±2.9 to 6.9±1.7 x109 cells/L
(P = 0.02).
DISCUSSION
Results of this limited, small-scale study indicate that when ATT is combined with immunomodulating herbal preparations,  Dzherelo, Svitanok, and  Lizorm, the patients are cured after about 4 months and complete disappearance of Mycobacterium tuberculosis from spu-tum culture is observed within one month from treatment initiation. The conversion time of sputum mycobacterial culture from positive to negative is an important interim indicator of the efficacy of anti-TB intervention (Holtz et al., 2006). We observed culture conversion at median 32 days. This is twice shorter than the reported conversion time for drug-resistant TB patients in Latvia or Hong Kong (Holtz et al., 2006; Yew et al., 2003). The mean duration of successful chemotherapy in Yew et al., study (2003) was 14.5 months with a range that appears to be anywhere between 11 to more than 24 months as reported by Japanese investigators (Yoshiyama et al., 2007). If these studies are representative of success rates in MDR-TB therapy then our immunomodulatory intervene-tion appears to produce the twofold reduction in culture conversion time and shortens treatment duration by at least three times. These results indicate that the combination of phytoconcentrates with anti-tuberculosis drugs results in significant enhancement of the efficacy of ATT. Our findings agree with earlier clinical studies of ATT that were conducted mostly with   Dzherelo  and occasionally with   Dzherelo and  Svitanok combination. They also indicated that the efficacy of ATT was enhanced and duration of treatment was considerably shortened (Arja-nova et al., 2006; Chkhetiany et al., 2007; Prihoda et al., 2006; Zaitzeva, 2006). The beneficial effects were observed in patients with MDR as well as patients with TB/HIV co-infection. Furthermore, previous studies have shown the amelioration of liver function as evidenced by normalization of ALT, AST, bilirubin and other markers of liver damage. This effect alone is of major significance since many of TB drugs are hepatotoxic and so far there are no effective means to counteract this negative aspect of ATT (Durand et al., 1996). By definition, tuberculosis is a wasting disease (Edwards et al., 1971). This condition is poorly manageable and is one of the leading factors contributing to higher morbidity and mortality (Villamor et al., 2006).
Table 1. Baseline and end-of-study characteristics of TB patients treated with ATT in combination with Dzherelo, Svitanok, and  Lizorm


Khan et al., (2006) reported that patients with under weight problem had higher risk of TB relapse and that changes in weight were an independent predictor of treatment outcome. The remarkable aspect of our therapy is a dramatic body weight gain in 93% of our patients (P = 0.0002). TB drugs seldom enhance body weight. The only known to us report of significant weight gain has been described by Donald et al., (1997). In their placebo-controlled study the increase in body mass, that is, mean gain 8.9 kg, has been described when TB patients were administered beta-sitosterol and sitosterolin - phytoste-rols from a pine tree. However, this intervention had no effect on the rate of mycobacterial clearance. There are other adjunct therapies such as nutritional supple-ments and corticosteroids that can enhance weight but they also had no effect on TB (Paton et al., 2004; Smego and Ahmed, 2003). The weight loss-reversing property of Dzherelo Svitanok and Lizorm along with substantiated therapeutic effect on TB and HIV can be particularly advantageous to those who live in resource-poor countries, where malnutrition is very common and deaths are more prevalent due to this single cause (Farmer et al., 1991). This study includes for the first time Ukrainian patients with XDR form of TB. Despite poor prognosis we were able to achieve mycobacterial clearance and significant clinical and radiological improvement to such an extent that these patients were discharged from the dispensary within average 16 weeks. Our results contrast two available clinical studies of XDR-TB. In a study from South Africa 52 out of 53 (98%) patients had died within 2 weeks from diagnosis (Gandhi et al., 2006). However this mortality rate may be not representative of the situation when more advanced clinical care is available. The clinical survey reported by Kim et al., (2007) indicated that in South Korea the treatment failure due to XDR-TB was 44.2%, whereas 27.4% patients with MDR did not respond to the therapy.
However we had only two individuals with XDR-TB and our results need to be confirmed in larger group of patients. Judging from surveys on global prevalence of XDR-TB among MDR-TB cases we sur-mise that 2 out of 9 (22%) cases of multidrug-resistant TB in our dispensary are in the 10-20% range reported recently (CDC, 2006; Migliori et al., 2007).
The currently available chemo-therapy for the treatment of TB is not perfect (Durand et al., 1996). Multiple tuberculous drugs need to be taken in combination for long periods of time.
The extended duration of therapy, coupled with the side effects, often results in poor patient adherence, treatment failure, and the emergence of drug resistance. It is agreed that immune-based therapies are urgently needed to complement tuberculosis drug disco-very (Achkar et al., 2007; Kaufmann, 2006; Tomioka, 2004). We also believe that the immunotherapy is the indispensable part of therapeutic strategies against TB (Pylypchuk, 2003). Many effective immune interventions are available against bacteria, protozoa, fungi and vi-ruses (Ershov, 2003). While effective the mechanism of most immunomodulators is poorly understood. This draw-back should be balanced against clinically confirmed benefits.
Some medicinal herbs were shown to modulate the immune response to TB (Tomioka, 2004), while others exerted direct antimycobacterial activity (Newton et al., 2000). From the review of available to us medical literature it is apparent that very few medicinal plants have demonstrated TB-curing properties.
Recent story, describing how the Zulu's traditional herbal remedy became European phytomedicine, further highlights the difficulties of finding and introducing an effective TB drug from a botanical source (Bladt and Wagner, 2007). It is quite unlikely that herbal immunomodulators used in our study act as tuberculostatic agents since in vitro growth of M. tuberculosis laboratory strains, H32 and H37Rv, was not affected directly by
Dzherelo or  Svitanok (Melnik et al., 1999). The same rate of response to treatment regardless whether patients were drug resistant, re-treated, or had HIV also suggests that the combination of herbs used by us does not interfere with replication pathways of mycobacteria. Dramatic weight gain observed in our patients indicates that their mechanism of action differs from ATT since tuberculosis drugs seldom produce significant weight gain (Paton et al., 2004).
This is further supported by the fact that diseases etio-logically unrelated to M. tuberculosis were responsive to the therapy with  
Dzherelo and  Lizorm  (Bodnar et al., 2002).
Tuberculosis remains an enormous global health problem. There are 8 million new cases and 2 million deaths from TB annually (Reid et al., 2006). Despite the overwhelming burden of disease, no new treatment regimens were developed since 1960's and current strains of TB are becoming gradually resistant to existing drugs. The emergence of XDR-TB with high mortality rate within very short period of time raises grave concerns of a future epidemic of virtually untreatable TB (Gandhi et al., 2006).
Our study provides the preliminary evidence of the efficacy of ATT in combination with
Dzherelo,  Svitanok and  Lizorm against XDR-TB. These herbal immunomodulators were recommended in Ukraine as an immune adjunct to TB therapy (Melnik et al., 1999). The major advantage associated with our intervention is twice-reduced time to negative culture conversion and at least three-fold shortened duration of treatment. These benefits along with other significant improvements such as weight gain, lack of hepatotoxicity, reduced anemia and inflammation, impressive radiological and clinical recovery are all in favor of adjunctive immune approach to ATT.
Additional studies need to be conducted to develop better understanding of this immunomodulating combination against drug-resistant TB and to enlarge the current arsenal of TB drugs.
ACKNOWLEDGEMENTS
We thank all participants who volunteered in this study. The generosity of Ekomed company in supplying phyto-concentrates is appreciated very much. The enthusiastic support of clinical staff and technicians who contributed to this study has been of tremendous help to bring this study to conclusion. We are grateful to other investigators of herbal immunomodulators for sharing their insight and providing helpful suggestions.
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Synthetic and Natural Immunomodulators Acting as Interferon Inducers

Dmytro S. Silin1*, Oksana V. Lyubomska1, Feliks I Ershov2, Valeriy M. Frolov3 and Galyna A. Kutsyna3,+
Laboratory of Molecular Virology, Medical and Biology Center, School of Biomedical Sciences, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK; 2Gamaleya Institute of Epidemiology and Microbiology, 18 Gamaleya Str., Moscow, 123098, Russia and ^Department of Infectious Diseases and Epidemilology State Medical Univerity, 50-Years of Defence of LuhanskStr., Luhansk 91045, Ukraine
Abstract: Interferons are first immunomodulatory molecules that have been shown to display a wide range of applications due to their antiviral, antibacterial, antitumor, and inflammatory activities. Natural and recombinant interferons are among most common biologic therapeutics worldwide. Interferon inducers, however, are less known and have been mostly developed and used in former socialist countries. Despite the fact that they are virtually unknown to the Western world, they represent a substantial market share of modern pharmacopoeia in former socialist republics. This review provides a brief description of most popular interferon inducers including Amyxin, Amizon, Anandin, Arbidol, Blasten, Cy-cloferon, Galavit, Groprinosine, Hepon, Immunoxel, Dzherelo, Kagocel, Larifan, Ligfol, Likopid, Mebavin, MIGI-KLP, V-5 Immunitor, SCV-07, Milife, Neovir, Poludan, Ragocin, Ridostin, Thymogen and Savratz, some of which were in use for several decades for the same clinical indications as for interferons. The variety and choice offered by the pharmaceutical industry behind the former "iron curtain" certainly deserves the appreciation, familiarity and application prospects for medical and research investigators worldwide.
INTRODUCTION
Immune system is the main regulatory system controlling homeostasis of the body and participates virtually in all (processes) cycles of the life from birth to death. The incompetence of the immune system opens door to infectious, malignant, autoimmune, and inflammatory diseases. There are many modern interventions directed to stimulation, modulation or suppression of the immunity by various routes.
Interferons are extremely important category of protein therapeutics aiding defense against infections and malignancies carrying foreign for host genetic information. Interferons are intra- and inter-cellular signaling proteins of three classes - alpha, beta, and gamma, which differ by their activity, cell origin and cell targets. Natural and recombinant interferons are widely used in the modern therapy of acute and chronic infectious and oncological diseases and some immune disorders. Alpha interferons such as Laferon, Intron A, Welferon, Reaferon, Viferon, Viaferon, Roferon A as well as beta interferons - Betaferon, Feron, Fron, Rebif, and others represent the type I interferons which express high antiviral activity and widely applied in a complex antiviral therapy [19]. Gamma interferons such as Iimmukin, Interferonlagen, mega-D-interferon and others represent the type II interferons which increase MHC II level on antigen-presenting cells and regulates the level of inflammatory and immune responses. Gamma interferons were successfully applied for
*Address correspondence to these authors at the Laboratory of Molecular Virology, Medical and Biology Center, School of Biomedical Sciences, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK; E-mail: d.silin@queens-belfast.ac.uk and Department of Infectious Diseases and Epidemilology State Medical Univerity, 50-Years of Defence of Luhansk Str., Luhansk 91045, Ukraine; E-mail: kutsyna@list.ru
therapy of viral, malignant, and autoimmune diseases [10-14].
However, interferons are species-specific and for the replacement therapy species-specific proteins are necessary, which is a limiting factor for their using in the veterinary and animal experimentations. On the other hand, administration of interferons could activate negative reverse loop of regulation, inhibiting endogenous interferon production and it could be undesirable side effect, particularly in the chronic cases of diseases. To overcome both of the above restricting factors, interferonogens could be successfully applied, as they are not species-specific and stimulate endogenous production of interferon.
Generally, interferonogens have significant advantages comparing to native or recombinant interferons: single administration of interferonogens increases interferons to therapeutic level for up to several days, whilst interferons administration should be multiple and in high dosage as their semi-life is about 20-40 minutes; such high-rate administration of interferons could turn on regulatory machinery of their endogenous synthesis and severe side effects [15,16]; overdosing of interferonogens (and according side effects) is practically impossible as interferon synthesis is still controlled by organism; and, finally, interferonogens are mostly not antigenic and could be used long period repeatedly.
The first successful clinical application of experimental viral interferonogen IVS (inactivated Semliki Forest virus) in therapy of viral ocular infection was performed in the USSR by A.A.Kasparov and colleagues in 1966 [17]. Starting from that several interferonogenes were discovered and investigated, most of them by scientific groups of the former USSR [18].
Among contemporary immunomodulators with interfer-onogenic activity the physical, chemical and biological agents could be designated. Physical influence on the immune system by low-intensity laser, ultrasound, low-frequency magnetic field etc. could normalize various immunity subsystems activity, particularly phagocytosis, cellular and humoral immune response by both interferon-dependent and independent pathways [19].
As a result of many years of screening several promising interferonogenes were revealed among various kinds of natural and synthetic compounds (fluorenones, acridanones, gossypol derivatives, polynucleotides, ds-RNAs etc.) They have quite high chemotherapeutic index and could be useful for prophylaxis and treatment of viral and other diseases. By chemical synthesis and biotechnology means low molecular weight substances were obtained such as Neovir, Cy-cloferon, Kagocel, Amixin, as well as high molecular weight substances such as Poludan, Ridostin, Larifan, and others. Others preparations with interferonogenic properties were discovered from natural biological sources, for example, Milife - from fungi; "MIGI-KLP"- from mussel; Immunomax and Immunoxel (Dzherelo) - from medicinal plants; V5 Immunitor - from pooled blood.
In the next chapters we concentrate on some scientifically proven and industrially manufactured interferonogenes and review their properties related to clinical uses.
Hepon. Manufacturer: "Immapharma", Russia
Hepon is synthetic immunomodulator based on tetrade-capeptide: Thr-Glu-Lys-Lys-Arg-Arg-Glu-Thr-Val-Glu-Arg-Glu-Lys-Glu, induces alpha and beta-interferons, inhibits inflammatory cytokines, stimulates humoral immunity. Experimentally demonstrated inhibition activity of Hepon on hepatitis C virus replication in human cell cultures [20], antiviral activity of Hepon was also demonstrated for rabies with dose-dependant protection of up to 40% mice [21], Herpes simplex viruses types 1 and 2 with one hundred fold reduction of viral titer in vitro and 36% protection after 10 LD50 dose challenge [22]. Hepon-treatment intensifies antibody production against HIV1-antigens [23] and increases concentration of CD4 and NK cells, functionality of neutro-phils and CD8 T-cells, and decreases virus load in the blood of HIV-infected patients [24].
Stimulation of activity of intestinal mucosal immunity was demonstrated in several clinical trials [25,26]. In the experimental and clinical studies was proved efficiency of therapy with Hepon and Immunomax (another immunocor-rector, developed by the same group) during acute purulent surgical infections [27]. There were no noted contraindications and adverse reactions associated with Hepon.
Cycloferon. Manufacturer: "Polysan", Russia
Cycloferon is a synthetic analogue of Cytrus Grandis alkaloid, stimulates B-cells, macrophages and other cells and tissues to produce almost pure type 1 interferons. It was reported to have up to 100-fold upregulation of beta-interferon gene and 10-fold upregulation of alpha-interferon gene in the human blood samples after administration of Cycloferon without affecting essentially the activity of other genes of blood cells [28]. In the placebo controlled multicentered study on totally 16,000 children and adolescents Cycloferon demonstrated clear epidemiological benefit in the prophy-laxys of the influenza and other acute respiratory viral infections with 1.5-2.9 -fold decreased morbidity and 41-90% protection index [29]. Its efficiency was demonstrated in chronic infections of upper respiratory tract too [30]. Specific antiviral activity of Cycloferon against adenovirus type 6 in vitro [31] and herpes virus on experimental herpetic infection was demonstrated [32]. The author's (D.S.) personal observations in the veterinary hospital have revealed antiviral efficiency of Cycloferon in cases of canine distemper and parvoviral gastroenteritis. The duration of the disease, commonly, decreases for 3-4 days when standard complex therapy was supported by Cycloferon. In the animals with normal immune status Cycloferon induced the formation of the serum interferon in high titers (up to 20,000) with the peak achieved 4-8 hours after the injection and increased survival rate in generalized herpes infection by 30-100% in comparison with the controls. Under immunosuppression caused by gamma-radiation or cyclophosphamide the titers of serum interferon were 4-8 times lower and the protective effect of this preparation was considerably milder [32]. However, in HIV-infected patients the remission period of herpes simplex virus 1 and 2 infections is prolonged after combination of antiviral treatment with Cycloferon [33]. The antibacterial activity of cycloferon was demonstrated for various pathogenic and opportunistic species [34], and correction of the immune status after anti-tumor therapy was also observed [35]. Anti-apoptotic activity of Cycloferon was seen in the hypothalamic neurosecretory centers [36].
Amyxin (Amixine). Manufacturer: "Lancepharm", "Dalhimpharm", "Masterlek", Russia; and Odessa Physico-Chemical Institute, Ukraine
Amyxin (Tilorone) induces alpha, beta, and gamma interferons by intestinal epithelium, hepatocytes, and granulo-cytes.In the animal models, 4-24 hours after oral administration, maximum levels of interferon are reached in the intestine, liver and blood, resulting in efficient prevention and therapy of chronic enteritis and hepatitis [37]. Besides potent interferonogenic activity, Amyxin causes activation of NK and phagocytes in peripheral blood [38]. Interestingly, linkage of RNA-Amyxin complex to bead carriers improves in-terferonogenic properties and proves that mechanism of such activity requires the contact between the effector and the cell surface without its penetration into the cell [38]. Antiviral properties of Amyxin are well documented on a range of viruses. Thus, experimental Haemorrhagic fever studies reveals 52% protection of animals by combined Amyxin-Virosole therapy which was superior to the effect of their monotherapy [40], although some regimens of Amyxin only provided protection up to 61% with oral administration and up to 65% with subcutaneous injection [39]. Preventive Amyxin therapy in population groups with high hemorrhagic fever with renal syndrome (HFRS) risk prevents development of HFRS and acute respiratory viral infection [41]. In the same study it was shown that Amyxin in chronic viral hepatitis (CVH) improved general condition of the patients, removed jaundice of the skin and sclera, normalized activity of aminotransferases and blood bilirubin level. Virus replication was stopped in 25% cases of chronic HBV and in 1.6% cases of chronic HCV infection [39]. The 33% lethality reduction by Amyxin was demonstrated in experimental West Nile Fewer in vivo [40], while antiviral effect of RNA-Amyxin molecular complex was registered in vitro for three virus-cell systems: vesicular stomatitis virus (VSV) - murine fibroblast L929 cells, Venezuelan equine encephalitis virus (VEEV) - swine embryo kidney (SEK) cells and encephalo-myocarditis virus (EMCV) - established piglet testicular (EPT) cells [42]. The administration of Amyxin simultaneously with polyvalent vaccination of pups in context of emergency prophylaxis, seemed to reduce cases of vaccination failure, although efficiency of Amyxin in cases of developed canine distemper and parvovirosis was insignificant even at early stages of diseases. The efficacy of Amyxin for flu and acute respiratory viral infections' prophylaxis and treatment was demonstrated in a controlled trial of the risk group of medical personnel [43]. Amyxin in combination with herpes vaccination was highly efficient (87.9-90.9%) for the treatment of herpetic keratitis and prevented the relapse of the disease [44].
Arbidol. Manufacturer: "Dalchimpharm", "Masterlek", Russia
Arbidol (ethyl- 6 - bromo - 4- [(dimethylamino)methyl]- 5 - hydroxy - 1 - methyl 2 - [(phenylthio)methyl] - indole - 3-carboxylate hydrochloride monohydrate) stimulates humoral and cellular immunity, posses interferonogenic and antioxi-dant activity. Arbidol was shown to have effects on nonspecific defense factors, on capacity to induce interferon and to activate phagocytes in particular. Arbidole-treated patients with lower baseline immunity showed improvement in im-munological parameters (in the counts of CD4 and CD8 lymphocytes, B lymphocytes, in the levels of serum immu-noglobulins). Arbidol produces a high preventive and therapeutical effects in influenza A and B and other acute respiratory viral infections, prevents postinfluenza complications, reduces the incidence of exacerbations of chronic diseases in postinfluenza patients [45]. In the randomized, double-blinded, placebo controlled trial was revealed that the median duration of naturally acquired influenza was 72.0 hours in Arbidol group and 96.0 hours in placebo group. The median area under the curve (AUC) of decreased total score were significantly higher in Arbidol group than in placebo group, thus Arbidol was effective and well tolerated in the treatment of early naturally acquired influenza [46]. Specifically, reproduction of human IVA antigenic strains H1N1, H2N2, H3N2, remantadin-sensitive and remantadin-resistant strains of influenza virus as well as pathogenic for humans strains of avian influenza virus H5N1 and H9N2, were inhibited in vitro by Arbidol [47]. Efficiency of arbidol against bird flu virus H5N1 isolated from wild birds and poultry in Russia was proved in vitro [48, 49], and in the treatment of humans during avian influenza outbreak [50]. The wide spectrum of antiviral activity against respiratory viruses has led to the assessment of its efficiency on hepatitis C virus in cell culture. Long-term Arabidol treatment of Huh7 cells chronically replicating a genomic length genotype 1b repli-con resulted in sustained reduction of viral RNA and protein expression, and eventually cured HCV infected cells. Besides, pre-treatment of human hepatoma Huh7.5.1 cells with 15 microM ARB for 24 to 48 hours inhibited acute infection with JFH-1 virus by up to 1000-fold [51].
Amizon. Manufacturer: "Pharmac", Ukraine
Amizon (N-methyl-4-benzylcarbamidopyridinium jodide) - derivative of isonicotinic acid belongs to analgetic group and among anti-inflammatory, antifever and analgetic properties expresses interferonogenic activity increasing 3-4 fold endogenous interferon in plasma, enhancing humoral and cellular immunity. Antiviral and immunomodulatory activity of amizon was clinically demonstrated on patients with hepatitis B and C with renal lesions [52] and chronic toxic hepatitis [53]. Clear clinical improvement was detected in 149 patients with Mumps treated by complex therapy with amison in comparison to 177 patients obtaining the same conventional treatment without amizon [54]. Antiinflamma-tory activity of amizon enhance its positive interferonogenic influence on patients with acute infectious inflammation [55].
Neovir. Manufacturer: "Pharmavit", Russia, "Pharm-synthez", Russia
Neovir (for veterinary use Camedon. Manufacturer: MEDITER, Russia) - sodium 10-methylencarboxylate-9-acridone - induses high titres of endogenous interferon, particularly alpha-interferon with peak interferonogenic activity at few hours after intramuscular injection prolonging up to 16-20 hours. Antiviral activity of Neovir was demonstrated on patients with chronic viral hepatitis B and C [56], and individual therapy programs for such patients were developed [57]. Very powerful interferonogenic activity of Neovir allowed to use it successfully on the spectrum of bacterial diseases [58,59]. Some positive effect of Neovir on steroid hormones receptors in uterus cancerous tissues was shown [60]. Moreover, Neovir exerted the direct cytotoxic action on HT-29 and K-562 cells, intact and transfected with mdr1 gene. Preliminary incubation of cells with Neovir for 24 h efficiently increased the cytotoxic effect of doxorubicin and vincristine. The enhancement of toxic action of doxorubicin for HT-29 cells had, as a rule, additive character, while for HT-29 MDR1 cells the interaction was synergistic (CD(50) was decreased by 2.85- and 8.67-fold respectively). The effect of vincristine toxicity enhancement didn't depend on mdr1 gene expression and had synergistic character. Neovir enhanced the cytotoxic effect of doxorubicin in relation to K-562 and K-562 MDR1 cells by 3.18-fold and more than by 100-fold respectively. Preincubation of HT-29 cells with Neovir has resulted in 2000-fold decrease of 5-fluorouracil
CD(50) and in 36.6-fold for HT-29 MDR1 cells. Thus, the effect of Neovir seems to have no relation to the action on the mechanisms of multiple drug resistance and may be mediated through some other pathways [61]
Kagocel. Manufacturer: "Nearmedic plus", Russia
Kagocel - is a potent inducer of so-called "late interferons', a mixture of alpha and beta interferons, produced by Tand B-lymphocytes, macrophages, granulocytes, fibroblasts, endothelial other cells after oral administration of one dose of Kagocel the peak titer of interferon is registered in the intestine in four hours, although peak titer in blood registered in 48 hours, and interferonogenic response lasts up to 5 days. In vitro kagocel indused production of alpha and gamma interferons and interleukin 2 by human long-term cell cultures of different origin: J-96 and J-41 (monocytic leukemia), SW-13 (adenocarcinoma), and MT-4 (T-cell leukemia) [62]. The antiviral effect of Kagocel on the reproduction of Herpes simplex virus including its mutant strains resistant to basic antiherpetic medicine Acyclovir was demonstrated. Kagocel inhibited reproduction of Herpes virus type 1 and Herpes virus type 2 in noncytotoxic concentrations. Kagocel was also demonstrated to inhibit the reproduction of Herpes virus type 1, resistant to combination of Acyclovir and phos-phonoacetic acid [63].
Poludan. Manufacturer: "Lens Pharm", Russia
Poludan - complex of polyadenilic and polyuridilic acids in equimolar ratio - induces mostly alpha interferon and some beta and gamma interferons. Subconjunctival injection of poludan increases the level of the interferon in blood and tears more than 10-fold and 7-fold after three hours respectively. The daily injections support elevated level of interferons which, dramatically influenced on ophtalmoherpes [64]. The same group of clinical ophthalmologist developed very promising method of viral and non viral eye lesions treatment. The method of local express auto-cytokine therapy (LEACCT) consists in using an experimentally tested auto-logous complex of cytokines (alpha-, beta-, gamma-inter-ferons, interleukins 2, 8, tumor necrosis factor alpha etc.), which is produced by joining the autoblood of patients with poludan. The administration (subconjunctivally and as instillations) of the autoblood-poludan mixture was effectively used for herpes- and adenovirus keratoconjunctivitis, slow re-epithelization after laser keratectomy and in eye burns (178 patients). Apart from the external LEACCT procedures, a 1-4-time injection of the mentioned mixture into patient's anterior chamber used in endothelial herpetic keratoirido-cyclitis, initial bullous keratopathy, severe keratoconus and in injuries of the anterior lens capsule (117 patients). The clinical-study results (main group -295 patients) show that the increased visual acuity ranging from 0.05 to 1.0 was registered in 85% of cases [65]. The epidemiological effectiveness of poludan for prevention of acute respiratory viral infections was shown on group of (101 students). The placebo group (96 students) received the distilled water. In the students receiving poludan the incidence of acute respiratory diseases was significantly lower than in the control group (p = 0.058), decreasing to two times [66]. Similar data was obtained for prophylactic activity in the cases of the poly-ethiologic group of acute respiratory viral infection during the seasonal peak of the disease, with a coefficient of efficiency of 2.1 and corresponding protection index of 52.7%. Having the same chance of getting infected, individuals protected with these drugs often have the disease in a milder or asymptomatic form [67].
Ridostin. Manufacturer: "Vecterpharm", Russia, "Dia-pharm", Russia
Ridostin - mixture of double - stranded and single -stranded RNA sodium solts - potently induses interferone production and stimulates phagocytosis. Intraperithoneal injection of ridostin to mice induses intensive blood accumulation of interferon with peak at 8 hours, albeit interferone level was low in the respiratory tract and brain. Contrastly, intranasal and aerogenic administration of ridostin induced interferon mainly in the upper respiratory tract and lung [68]. Intracerebral injection of ridostin induced accumulation of interferon in the brain and serum [69]. Combined treatment with killed vaccine and ridostine by the scheme of urgent prophylaxis (3 days before challenge) demonstrated 100% protection of Aujeszky's disease infected minks, 75% protection of foot-and-moth infected pigs, and 50% protection of canine distemper infected dogs. Clinical symptoms of dogs developed canine dictemper was mild and delayed 2325 days post infection [70].
Larifan. Manufacturer: "Pharm", Riga, Latvia
Larifan - double stranded RNA of f2-phage - potently indused interferone after systemic or local administration. Larifan demonstrated high antiviral efficacy against Omsk haemorrhagic fever virus (strain "Ondatra") in experiments with laboratory animals. This drug prevented the death of 65% infected mice and significantly decreased infection severity in rabbits [71]. However, this virus reproduction on cell culture was suppressed mildly whilst human adenovirus serotype 2 wasn't suppressed by larifan in vitro at all [72].
Savratz. Manufacturer: "SRIEM", Russia
Savratz - oxybenzylamine derivative - demonstrated high interferone-inducing capacity with early and late peaks of interferone production (4-8 and 48-96 hours after administration) depending on the route of administration [73]. Savratz showed antiviral activity in vitro against hepatitis C virus on cell cultures SW-13 and MT - 4 [74].
Groprinosine. Manufacturer: "Polfa", Poland
Groprinosine - inosine pranobex - induces interferon, stimulates macrophages activity and lymphocytes proliferation, with specific damage to viral genetic machinery. Antiviral properties of groprinosine were demonstrated in 35 patients with acute virus hepatitis of average severity, who developed, after short-term improvement of general status, a negative dynamics of clinical and laboratory indexes. The 21 patients have received traditional treatment, 14 patients additionally were prescribed groprinosine within 5-10 days. It was shown, that addition of groprinosine to combination therapy positively influenced the disease course, promoted a rapid regress of clinical symptoms, normalization of biochemical indexes of liver function and decreased duration of hospitalization [75].
Milife. Manufacturer: "Vilar", Russia, "Dija", Russia
Milife - biomass of Fusarium sambicium fungi strain VSB-917 - stimulate production of alpha and gamma interferons, normalize humoral, cellular immunity and cytokine homeostasis. Milife administration to mice led to rapid and significant increase in total leukocyte and lymphocyte count in peripheral blood that persisted for at least 3 weeks after a 6 days treatment. Cellularity of lymph nodes, bone marrow and thymus increased significantly at days 4 and 6 of treatment, but returned to pretreatment levels after Milife discontinuation. Though total splenocyte numbers did not change dramatically, there occurred delayed increase in CD4+ cells in the spleen 3 weeks following treatment. Preferential accumulation of CD4+ cells was also found in peripheral blood, with the peak at day 6 of treatment. As a result, CD4/CD8 ratio in blood and spleen was significantly higher in treated than in untreated mice. Splenocytes from treated mice proliferated more vigorously in response to Con A. When added in vitro, Milife also mildly co-stimulated Con A-induced proliferation of splenocytes from intact animals [76].
Mebavin. Ragosin. Manufacturer: "IBC", Uzbekistan
Mebavine and ragosin - soluble gossypol derivatives -possess interferonogenic and inflammation-regulatory activity. Anti-inflammatory activity of mebavin was similar to prednisolone as revealed on patients with adjuvant arthritis [77], without suppression and even with stimulation of immunity [78].
Prodigiosan. Manufacturer: MBRC "Alexis", Georgia
As a polysaccharide extracted from Serratia marcescens and other bacteria Prodigiosan activates enzymatic activity of macrophages and stimulates phagocytic processes.Like other polysaccharides compounds Prodigiosan possesses the direct antibacterial activity and increases efficiency of antibiotics in therapy of infections caused by a wide spectrum drug-resistant bacterial strains [79]. Its interferonogenic properties were demonstrated both in vivo and in vitro [80,81], and its antiviral efficiency was confirmed in complex therapy of viral respiratory diseases [82] and hepatitis B [83]. In the later research efficiency of prodigiosan combined with ibuprofen was more pronounced than monotherapy with reaferon (alpha 2-interferon) in terms of decreasing of total serum IgE levels. Interestingly, another remedy - prodigiosin isolated from the culture broth of Serratia marcescens B-1231 possessed anti-autoimmune properties by suppressing progression of autoimmune diabetes and collagen-induced arthritis [84].
Rusam. Manufacturer: "Bryntsalov A", Russia
Extraction from thermophilic strain C of S. aureus possess antiallergic activity and stimulate cellular immunity and both type of interferon production. Clinical trials in bronchial asthma patients demonstrated high interferonogenic and anti-autoimmune activity [85].
MIGI-K. Developer: "VNIRO", Russia
MIGI-K preparation - a result of acidic hydrolysis of mussels flesh - contains several pharmacologically active compounds: melanoidines, peptides carnosin and taurin, amino acids, polyunsaturated lipids, vitamins and minerals. MIGI-K demonstrated antitumor, immunostimulating, anti-oxidant and radioprotective properties. Preparation secured radioprotection in trials after Chernobyl accident [86] and demonstrated strong antioxidative properties on animal models significantly or completely preventing intensification of lipoperoxidation and depression of antioxidative systems (superoxide dismutase, glutathione peroxidase, nonprotein thiols, lipoantioxidants) in skin and liver of UV-irradiated rats [87]. Interferonogenic activity of MIGI-K allowed recommending it as food addidtive in viral hepatitis and respiratory infections [88].
Blasten. Manufacturer: SIC "Enzypharm", "Enzyme", Ukraine
 Immunomodulatory preparation from cellular walls of Lactobacillus Delbrueckii demonstrated potent immunostimulation of all types of immunity with very wide therapeutic limits. Clinical trials proved efficiency of blasten in complex treatment of oncological diseases [89], respiratory and surgical infections [90]. Very low toxicity and adjuvan-ticity comparable with complete Friend's adjuvant led to recommendation of Blasten to wide use in medical practice by health authorities of Ukraine.
Maxidin. Developer: "Niarmedic-plus", Manufacturer: "Micro-plus", Russia
Maxidin (germanium bis(pyridine-2,6-dicarboxylate)) potently induces interferon and normalizes immunity in secondary immunodeficient conditions. Maxidin is effectively used in immune disorders and viral diseases of animals [91].
Immunoxel (Dzherelo). Manufacturer: "Ekomed" Kiev, Ukraine
This immunomodulator contains the wide spectrum of biologically active substances derived from herbs. The preparation possesses interferonogenic and potent anti-inflammatory activities. Series of clinical trials have demonstrated that Dzherelo induces protective immune response to a broad range of bacterial and viral infections and positive immune activity in autoimmune conditions and cancer as well. Dzherelo has been recommended by the health authorities of Ukraine as an adjunct therapy for TB and seasonal flu [92]. When Dzherelo and anti-tuberculosis therapy (ATT) or antiviral therapy are combined, it improves clinical symptoms and produces higher cure rate than in patients than on chemotherapy alone. It has been shown to achieve faster and superior rate of mycobacterial clearance, reduce HIV burden, accelerate healing of pulmonary lesions, decrease inflammation markers and pro-inflammatory cytokines, liver damage, improve hematology picture, i.e., increased hemoglobin levels, CD4 counts, and enhance significantly quality of life such as weight gain, fever, respiratory function, physical fitness, well-being and better mood. Immunoxel has been shown effective even against multidrug (MDR-TB) and extensively drug-resistant TB (XDR). The details of these beneficial outcomes were published earlier [93-100].
SCV-07. Manufacturer: "Verta", St.Petersburg, Russia. Licensee: "Sciclone", San Mateo, USA
Scv-07 or gamma-D-glutamyl-L-tryptophan, is a synthetic dipeptide with potent immunomodulatory and antimicrobial activity. Verta and SciClone Pharmaceuticals are developing SCV-07, the lead product in a series of immnunostimulants from Verta, for the potential treatment of tuberculosis and hepatitis C virus infection. Phase II clinical trials of the compound are ongoing [101]. SCV-07 has also shown potential in treatment of herpes infection.
Immucor GA-40 and GA-47. Manufacturer: "Alexis", Georgia
Chromatographically purified polypeptide complexes extracted from plants demonstrated antitumor and immunomodulatory activity in all arms of immunity, including stimulation of interferon production.
Likopid. Manufacturer: "Peptek", Moscow, Russia
Likopid or N-acetyl glucosaminyl-1-4-N-acetylmuramyl-L-alanine-D-isoglutamine dipeptide, is a synthetic analogue of the fragment of cell walls of bacteria. It stimulates the functional activity of macrophages and synthesis of cytoki-nes. It is clinically used in adjuvant therapy for chronic immunodeficiency conditions, low current and recurring inflammatory infectious diseases at various sites [102]. Due to broad spectrum activity Likopid is also used for treatment of cytomegalovirus infection and pulmonary tuberculosis [103].
Galavit. Manufacturer "Medikor" Moscow, Russia
Galavit is a monosodium a-luminol or monosodium 5-amino-2-3-dihydro-1-4-phthalazine dione. Galavit inhibits production of inflammatory cytokines such as TNF-alpha, IL-1 through regulation of metabolic activity of macrophages. As such it has been found useful for various clinical indications as follows: gastrointestinal infections of various origins; viral hepatitis; herpes infections; urogenital infections, i.e., chlamidia, endometriosis and other bacterial and fungal infections [104].
V5 Immunitor. Manufacturer: "Monserum", Mongolia
This product is made from hydrolyzed pooled blood of hepatitis B and C carriers by using unique technology. The hepatitis viruses are killed by heat- and chemical inactivation and then formulated into a tablet. The principle for production of V5 is not much different from established principles with old-fashioned killed vaccines, i.e., Hepatitis B vaccine made from pooled plasma. V-5 is available as 850 mg coated pill, ten of which are sealed in a "blister" pallet, with 30 pills per one package. The recommended dose is one-two pills per day. The preparation is stable at ambient temperature for five years. Studies in chronic hepatitis B and C patients have shown nearly 100% efficacy, without any adverse effects, and with positive outcome achievable within one month from treatment initiation [105,106].
Ligfol (Olipifat). Manufacturer: "Ligpharm", Moscow, Russia
Ligfol is obtained as a result of hydrolysis of wood lignin that is reduced to a sterile liquid for injection and has been in veterinary use since 2000. This preparation is quite unusual, bearing in mind its origin and broadness of clinical applications. It has been found useful in the management of stress; as an antioxidant; anti-tumor agent; enhancer of healing; hepatoprotector; hematopoiesis stimulant; inducer of cellmediated immunity and interferon synthesis. These properties may appear unrelated to each other but there are published clinical studies that lend support to these claims [107109].
Anandin. Manufacturer: "Meditere", St. Petersbourg, Russia
Anandin is an injectable and topical preparation of modified sugar, glucosamine-propyl-carbocridone, developed by Travkin and Yakovleva in 1990-1995. It has been used in Russia over the last ten years in humans but predominantly in the veterinary practice without any significant toxicity. Main indications are for acute and chronic viral and bacterial infections; inflammatory conditions; as an enhancer of healing process; and for a variety of immune disorders. In animals it is commonly prescribed for parvovirus enteric infections, pestiviruses, bovine herpes, infectious bovine rhiono-tracheitis (IBR), bovine viral diarrhea (BVD), hepatitis, and many other viral infections of unknown etiology [110,111].
Imunofan. Manufacturer: "Bionox", Moscow, Russia
This immunomodulator consists of a short synthetic pep-tide, (Arg-a-Asp-Lys-Val-Tyr-Arg), which imitates the action of thymopoietin. It is provided as a rectal suppository, injectable solution or intranasal spray. According to Russian studies the pharmacological effect is due to three main modes of action: correction of immune response; restoration of antiodixadant/peroxidation processes; and inhibition of multidrug resistance through interaction with transmembrane pumps responsible for drug resistance. Imunofan is prescribed for a wide range of clinical conditions including as adjunct for cancer therapy; acute and chronic pyogenic infections; opportunistic infections such as Cytomegalovirus; Toxoplasma gondii; Klebsiella pneumonia; Herpes virus; Chlamydiae and Cryptococcus neoformans; HIV; acute and chronic viral hepatitis; diphtheria; as adjuvant for vaccination; and psoriasis. Although it is unlikely, Imunofan may cause inflammatory reactions in certain individuals [112114].
Thymogen. Manufacturer: "Cytomed", Russia
This preparation is perhaps best know interferon inducing immunomodulator. It was originally discovered by Khavin-son et al., and has been sold in Russia since 1991 [115]. It is very simple dipeptide (L-Glu-L-Trp) that is orally available and has been used for innumerable clinical conditions ranging from cancer to infectious diseases and other unrelated uses especially in the neurological or neuroendocrine context. The number of references on PubMed alone is by an order of magnitude higher than for any other of above reviewed substances. Thymogen is fully synthetic but since it has been discovered by screening other preparations of thymic extract, Thymalin and Vilon, it appears to affect various immune responses by mimicking the function of the thymus.
CONCLUSIONS
There are several dozen clinically deployed immunomodulators in Russia and former Soviet block countries. Most popular ones are listed in this review. They have been used with various success rates in a large number of patients, but are practically unknown in the English-language medical literature. We hope that this review provides a glimpse into current situation and perhaps will stimulate further research in this exciting area.
Table 1.    Summarized Data on Immunomodulators as Compiled from Available Literature Sources, Authors Own Clinical Observations, and Personal Communications
Category of PreparationsCommercial NameClinical Indications
Synthetic, low-molecular

Anandin

Amyxin

Arbidol

Amizon

Cycloferon

Hepon

Galavit

Groprinosine

Imunofan

Likopid

Maxidin

Neovir

Thymogen

Rabies, hepatitis A, B, C virus, TB, herpes simplex virus type 1 and 2, HIV, influenza, acute and chronic respiratory viral infections, adenovirus type 6, mumps, canine distemper, parvovirus, panleukopenia, viral hemorrhagic fever, West Nile fever, vesicular stomatitis virus, Venezuelan equine encephalitis virus, encephalo-myocarditis virus, chronic enteritis, surgical infections, keratoconjunctivitis, rhinitis, secondary immunodeficiencies, malignant diseases.
Synthetic, high-molecularPoludanOphathalmoherpes, influenza, acute and chronic respiratory viral infections, viral hepatitis B, rabies, HIV.
Natural, low-molecular

Kagocel

Ligfol  Mebavi

Ragocin 

Savratz

Herpes simplex, influenza, acute and chronic respiratory viral infections, hepatitis C, rabies, enteroviruses
Natural, high-molecular

Ridostin Larifan

Prodigiosan

Influenza, acute and chronic respiratory viral infections, Aujeszky's disease, foot-and-mouth disease, canine distemper, rabies, Omsk haemorrhagic fever, herpes
virus
Natural, complex

Blasten

Dzherelo(Immunoxel) Milife

Rusam

V5 Immunitor MIGI-K

Influenza, TB, acute and chronic respiratory viral infections, oncological and autoimmune diseases, malignancies, viral hepatitis, purulent wounds
 
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Cytokine profiles of HIV patients with pulmonary tuberculosis resulting from adjunct immunotherapy with herbal phytoconcentrates Dzherelo and Anemin

Contents lists available at ScienceDirect
CYTOKINE
journal homepage: www.elsevier.com/locate/issn/10434666
Lyudmila G. Nikolaeva a'*, Tatiana V. Maystata, Volodymyr S. Pylypchukb, Yuri L. Volyanskiic, Galyna A. Kutsyna d'*
a Kharkov Regional AIDS Prophylaxis and Prevention Center, Kharkov Medical Academy of Postgraduate Education, 6 Bor'by £$reet, Kharkov 61044, Ukraine
b Ekomed^LC, 80-A Prospect Pravdy, Kiev 04208, Ukraine
c I.I. Mechnikov Institute of Microbiology and Immunology, 14 Pushkinskaya street, Kharkov 61057, Ukraine   
d Luhansk Regional AIDS Center and Luhansk State Medical University, p0-Years of Defense ofLuhansk Street, Luhansk 91045, Ukraine
ARTICLE    INFO                                               ABSTRACT              

Article history:

Received 31 August 2008
Received in revised form 4 October 2008
Accepted 10 October 2008
Available online xxxx

Keywords:
AIDS
Botanical
Herbal
Immunotherapy
XDR
MDR
Mycobacterium tuberculosis               
Phytoconcentrate
Phytomedicine
Phytotherapy
Ukraine   
                   
Dzherelo and Anemin when combined with standard anti-tuberculosis therapy (ATT) were shown to produce better clinical outcome than chemotherapy alone. Sixty HIV-positive patients with active pulmonary TB were equally divided into three matched groups to receive either ATT, ATT + Dzherelo, or ATT + Dzherelo + Anemin. Peripheral blood samples were measured by ELISA for plasma levels of IL-2, IL-6, TNF-a, IFN-y, and IFN-a. After 6 months of follow-up Dzherelo and Dzherelo + Anemin combinations produced 61% (P = 0.005) and 44.4% (P =0.06) higher levels of IL-2, whereas in ATT group they were 33.1%_(P = 0.002). The levels of IL-6 increased by 17% (P = 0.15) in ATT group, but declined in both immune intervention groups by 26.2% (P = 0.007) and 21.3% (P = 0.22). TNF-a was suppressed in two immunotherapy groups by 19.1% £P = 0.06) and 76.3% (P = CJX)2)' respectively, but had risen by 14%(P = 0.42) in ATT patients. The pattern of production of IFN-y was opposite to that of TNF-a, but statistical significance was stronger in patients receiving ATT and Dzherelo + Anemin than in Dzherelo group: ^34% (P =^.004), +31.9% (P = 0.008), and +17.3% (P =0.33), respectively. Moderately decreased levels of IFN-a were observed in all treatment arms (range (j.9-16.6%) but differences were not significant. Despite considerable intra-group variation in cytokine production, the baseline inter-group averages were not statistically different indicating that the results were not biased by sample heterogeneity. Immunomodulators used in this study possibly act by enhancing natural immune response against TB. Expanded study of other cytokines and correlates relevant to control and protection from TB and HIV is needed in order to identify biomarkers of favorable treatment outcome, which may aid design of better immune interventions and vaccines.
                                    © 2008 Published by Elsevier Ltd.                 
 

1. Introduction

Mycobacterium tuberculosis is a ubiquitous pathogen with roughly one-third of the world's population carrying the bacillus. However, the vast majority of persons infected with the mycobacte-rium will never develop clinical symptoms of the infection [1].ltis agreed that the balance between Th1 and Th2 cytokine expression influences the course of disease in individuals with pulmonary TB [1-3]. It is generally understood that severity of TB is associated with diminished production of Th1 type cytokines such as lFN-y, TNF-a and lL-2. Cytokines of Th2 type such as lL-4, lL-5, IL-6, lL-10,1L12, TGF-p, and lL-13 have been also described as being involved in mycobacterial immunopathogenesis. Nonetheless, conflicting opinions still exist in the literature about importance of particular cytokines of either Th types in the immune response against TB [1-6]. The host response to M. tuberculosis is a complex interplay between multiple pro- and anti-inflammatory cytokines. This complexity presents a considerable challenge in distinguishing those that play more influential role in protective immunity.
Dzherelo is a multi-herbal, oral immunomodulator recommended by the health authorities of Ukraine as an adjunct therapy for TB [7]. Anemin is another phytoconcentrate used in Ukraine— mainly for correcting anemia—a condition common in TB patients. We and others have conducted several clinical trials involving TB patients the results of which indicated that when Dzherelo and anti-tuberculosis therapy (ATT) are combined, it improves clinical symptoms and produces higher cure rate than in TB patients on ATT alone. It has been shown to achieve faster and superior rate of mycobacterial clearance, reduce HIV burden, accelerate healing of pulmonary lesions, decrease inflammation markers and liver damage, improve hematology picture, i.e., increased hemoglobin levels, CD4 counts, and enhance significantly quality of life such as  weight gain, fever, respiratory function, physical fitness, well-being and better mood. The details of these beneficial outcomes were published earlier [7-12]. The patient population presented in this study has been investigated earlier for changes in peripheral blood cell subsets and viral load as a result of Dzherelo intervention [11,12]. In this study, we evaluated whether changes in cytokine production profiles resulting from our immune-based intervention may reveal specific immune correlates associated with positive clinical outcome.

2. Materials and methods
2.1. Patients
Male patients, aged 20-49 years, have been selected and divided into three matching groups, each consisting of 20 individuals by exclusion/inclusion criteria such as age bracket, gender, TB infection type, HIV disease stage, alcohol/drug abuse, prior treatment, and disease stage. Another inclusion criterion was the lack of any form of anti-retroviral therapy prior to and during the trial. Active pulmonary tuberculosis was certified by a medical history and clinical findings compatible with pulmonary tuberculosis, a chest X-ray showing lung involvement, and positive sputum smear for acid-fast bacilli or the culture of M. tuberculosis. The participation in this trial was voluntary and patients were enrolled only after signing the written consent indicating that they were free to withdraw from the study at any time. The conduct of the trial was approved by the Ethics board of Kharkov Medical Academy of Postgraduate Education, Kharkov, Ukraine.
2.2. Treatment regimens
All patients received standard anti-tuberculosis therapy (ATT) administered under DOTS schedule which consisted of once daily dose of Isoniazid (H) 300 mg, Rimfapicin (R) 600 mg, Pyrazinamide (Z) _2000/mg, Streptomycin (S) ^000mg and jEthambutol (E)
 ^200 mg. All anti-TB drugs were procured through the centralized national supply system of Ukraine. Before initiation of herbal therapy the patients received anti-tuberculous drugs for 6 months. The first group, which served as a control, continued receiving ATT. The second group of patients received, in addition to HRZSE, twice per day dose of Dzherelo which was given as 50 drops diluted in 100 ml of water. The third group received in addition to Dzherelo a same dose of another herbal supplement, Anemin. First two groups of patients were evaluated by us earlier on our comparative study assessing changes in peripheral blood cells and viral load [11,12]. The over-the-counter phytoconcentrates were generously supplied by Ekomed company. Dzherelo contains concentrated aqueous-alcohol extract from medicinal plants such as aloe (Aloe arborescens), common knotgrass (Polygonum aviculare), yarrow (Achillea millefolium), purple coneflower (Echinacea purpurea), St.
John's wort (Hypericum perforatum), centaury (Centaurium erythraea), snowball tree berries (Viburnum opulus), nettle (Urtica dioica), dandelion (Taraxacum officinale), sweet-sedge (Acorus calamus), oregano (Oreganum majorana), marigold (Calendula nofficinalis),    seabuckthorn   berries   (Hippophae   rhamnoides), elecampane (Inula helenium), tormentil (Potentilla erecta), greater plantain (Plantago major), wormwood (Artemisia sp.), Siberian golden root (Rhodiola rosea), cudweed (Gnaphalium uliginosum), licorice (Glycyrrhiza glabra), fennel (Foeniculum vulgare), chaga (Inonotus   obliquus),   thyme   (Thymus   vulgaris),   three-lobe beggarticks (Bidens tripartite), sage (Salvia officinalis), dog rose (Rosa carina), and juniper berries (Juniperus communis).
Anemin is recommended for anemic patients and contains aqueous alcohol extract of succulent aloe leaves (Aloe arborescens), marsh buckbean leaves (Menyanthes trifoliate), nettle leaves (Urtica dioica), common knotgrass (Polygonum aviculare), St. John's wort (Hypericum perforatum), and oregano (Oreganum majorana). Except marsh buckbean other herbs in Anemin preparation are also found in Dzherelo. Both products were approved in 1997 by the Ministry of Health of Ukraine as a dietary herbal supplements. In 1999 Dzherelo was recommended by the Ministry of Health as an immune adjunct to the therapy of pulmonary tuberculosis [7]. In 2006 they received status of a functional food—special category of supplements which can carry medical claims substantiated by clinical evidence.
2.3. Cytokine measurement
Supernatants from peripheral blood of patients were harvested and stored at ^20 °C until enzyme-linked immunosorbent assay (ELISA) measurement. Samples were taken at baseline and two times after, with three-month intervals between. ELISA kits for measuring cytokines 1L-2, IL-6, IFN-y, IFN-a, and TNF-a were purchased from a commercial supplier (ProCon, St. Petersburg, Russia). Assays were carried out according to the manufacturer's protocol.
2.4. Statistical analysis
The obtained results were analyzed with the aid of statistical software STATMOST (Datamost, South Sandy, UT). The baseline values relative to 3rd and 6th months of follow-up were evaluated by paired or unpaired Student t-test. The statistical difference between three sets of data obtained before, during, and at the end of TB therapy was analyzed by repeated measure, two-way ANO- VA. The Kruskal-Wallis and Friedman nonparametric tests were used to test ranked differences between responses among three groups. Wilcoxon signed-rank test was used to compare median values. All statistical analyses were two sided and probability values were considered as significant at the cut-off levels of P 6 0.05.
3. Results
A total of 60 patients were enrolled in this study and equally divided into three matched groups which received either ATT, ATT + Dzherelo or ATT  Dzherelo + Anemin. Blood samples were taken at study initiation and after three and six months. Plasma samples were analyzed by ELISA for the presence of IL-2, IL-6, TNF-a, IFN-y, and IFN-a. The Table 1 shows the dynamics of absolute values (mean ± SD) of these cytokines as expressed in pg/ml. The statistical significance of data presented in this table was evaluated by repeated measure, two-way ANOVA and Friedman's non- parametric ranking test. To better visualize observed changes we have transformed obtained mean values into percentage values in relation to pre-treatment levels considered as zero (Fig. 1). After 6 months of follow-up Dzherelo and Dzherelo + Anemin combination produced significantly higher absolute and relative levels of IL-2, whereas the levels of the same cytokine in ATT group were reduced by one-third (P < 0.002) in comparison to baseline (Table 1 and Fig. 1). The difference between study end results of three groups was statistically significant by Kruskal-Wallis test (P = 0.0004). The levels of IL-6 were increased in ATT group but reduced in both immune intervention groups. However, the statistical difference was observed only in Dzherelo group (P = 0.007). Nevertheless the disparity between inter-group IL-6 levels was significant^P = 0.04). TNF-a was strongly suppressed in two immunotherapy groups with probability values, P = 0.06 and P = 0.02, respectively, but its increase in ATT-treated patients appeared insignificant by ANOVAJ73 = 0.42) while Friedman test revealed significance (P = 0.03).
Table 1 Absolute values of pre- and post-treatment cytokine levels from three TB treatment regimens.

 The independence of results from three groups at 6 months was slightly above cut-off level(P = 0.07). The pattern of production ofIFN-y was opposite to that of TNF-a, but statistical significance was stronger in patients receiving ATT or Dzherelo + Anemin than in Dzherelo group: P < 0.004 and P = 0.008, respectively. Results from Dzherelo were affected by a slight drop in cytokine levels at end of three month (P = 0.33) which had also affected ranking test results (P = 0.07). The output of IFN-a was unremarkable, decreased levels (range 0.9%-16.6%) were observed in all three treatment groups, but these were not statistically significant at any given time.
While there was a considerable intra-group individual variation in levels of cytokines, particularly with TNF-a, observed differences in cytokine production resulting from three different treatment modalities were not due to population heterogeneity at study entry. Unpaired Student t-test comparing independent inter-group averages for every cytokine in each individual group has not shown any statistical difference at baseline. The P values for ATT vs. ATT+ Dzherelovs. ATT + DzhereloAnemin were 0.9080 vs. 0.9186 vs. 0.8351, respectively. Kruskal-Wallis as well as Wilcoxon signed-rank nonparametric tests also failed to reveal any bias in distribution of patients among three matched groups.
4. Discussion
Immunotherapy of TB merits more attention than it has previously received [3,13,14]. However, a caution is required so that protective and not harmful aspects of immunity are induced. Various immunotherapeutic approaches as an adjunct to TB.
chemotherapy have been tested including the modulation of cyto-kine levels, administration of environmental Mycobacterium vac-cae, and antibody therapy in order to modulate the host immune response to Mycobacterium tuberculosis [14]. Pro- and anti-inflammatory cytokines play a critical role in protection from mycobacte-rial infection and changes in their production pattern may predict clinical outcome. The immune intervention which we employed in this study appeared to shift cytokine production in a direction that was opposite to cytokine output resulting from chemotherapy in all evaluated cytokines except IFN-a.
Two groups of patients, i.e., those who received ATT and ATT + Dzherelo, were investigated by us earlier for changes in select subsets of peripheral blood leukocytes and viral load at months 1 and 2 post-therapy [11,12]. The results of that study have shown that after 2 months of follow-up the total CD3+ T lymphocytes increased in Dzherelo recipients, whereas in the control group they decreased. The population of CD4 T-cells expanded in Dzherelo arm but declined in ATT group. The CD8 cells fluctuated slightly upward in both groups but changes were not significant. The ratio between CD4/CD8 cells deteriorated in ATT arm but improved in Dzherelo arm. The percent of CD3+HLA-DR+ activated lymphocytes had fallen in ATT group but rose in Dzherelo recipients. The changes in CD20+ B lymphocytes were insignificant in both arms. No difference was seen in the amount of CD3-CD16+CD56+ natural killer (NK) cells in ATT arm, while in Dzherelo recipients they declined significantly. The viral load, measured by plasma RNA-PCR, decreased in 17 out of 20 Dzherelo recipients, but increased in the same number of patients on ATT. Dzherelo thus appeared to have favorable effect on surrogate immune markers and viral burden in HIV/TB patients when given as the immunomodulating adjunct to ATT. In this study we present changes in cytokine production at 6 and 9 months post-therapy. Due to delay in getting cytokine kits we were, unfortunately, unable to conduct this study within the same time period as the cell subset phenotype and viral load studies. For this reason results from this investigation are not directly correlated with prior findings.
At the end of 6 months of treatment Dzherelo and Dzherelo + Anemin combinations produced 61% and 44.4% more of IL-2, whereas the levels of this cytokine in ATT alone group were reduced by 33.1% (Fig. 1). Incidentally, Toossi et^l. reported that patients with pulmonary TB had 81.2% lower output of tuberculin-induced IL-2 as compared with healthy tuberculin reactors. Defective IL-2 production was associated with more extensive disease on chest X-ray implying that administration of this cytokine could be beneficial to patients with TB [15]. The first initial trial of IL-2 had shown encouraging results. However, randomized, placebo-controlled trial of therapeutic interleukin-2 demonstrated significant delays in clearance and conversion rate of M. tuberculosis in sputum culture. This clinical study revealed potential antagonism between TB drugs and immunotherapy and suggested that exogenous supplementation with cytokines will not necessarily fulfill expectations based on mechanistic studies [14]. By the same token, it is unclear whether higher levels of IL-2, originally described as the T-cell growth factor, are responsible for increased CD3+ T lymphocyte levels observed in Dzherelo recipients [12].
Many studies in TB patients and non-TB controls deal with analysis of cytokine production in response to mitogens or mycobacte-rial antigens such as PPD or tuberculin. IL-6 was often found elevated in such assays [16]. Nagabhushanam et^l. reported that excess of IL-6 secreted by mycobacterium-infected macrophages may contribute to the inability of the cellular immune response to eradicate infection viajFN-y dependent pathway [17]. In addition to many other functions IL-6 is considered to be non-specific inflammatory cytokine shown to exacerbate deleterious immune restoration syndrome in tuberculosis and HIV co-infected patients [5,18]. Our results indicate that production of IL-6 was suppressed by Dzherelo (P = 0.007) or Dzherelo + Anemin(P = 0.2) but appears to be increased by ATTj(P = 0.15). As Dzherelo has been found equally effective in treating both TB and HIV our findings support prior studies suggesting that the downregulation ofIL-6 dependent inflammatory process may be beneficial to the host.
IFN-a is a multifunctional cytokine with established activity in many infectious diseases. IFN-a synergizes with IL-12 and plays an important role in inhibiting Th2 cytokines through antagonizing effect on IL-4, IL-10, or other immunosuppressive cellular factors and by promoting Th1 response via induction of IL-2 and IFN-y [18]. Administration of aerosolized IFN-a to TB patients had shown more favorable effects than in those given chemotherapy alone [19]. In our study, IFN-a was the only cytokine that behaved similarly in all three treatment groups. While tendency to decrease was observed it was not statistically significant. For this reason it is not clear whether this cytokine has contributed to observed increase in IL-2 and IFN-y levels or it plays any role in outcome from therapy. Thus, in case of IFN-a we do not know how to interpret our observation without being biased by an erroneous a priori attribution of cause and effect.
TNF-a is a monocyte-activating cytokine which stimulates anti-mycobacterial activity and helps to maintain the integrity of tuberculous granulomas in which M. tuberculosis is contained [1]. Our results indicate that both Dzherelo and Dzherelo + Anemin combinations cause significant decrease in baseline TNF-a levels by as much as 19.1% (P = 0.02) and 76.3%fP = 0.06) respectively. This effect is opposite to the trend in ATT-treated patients where TNF-a secretion continued to escalate. Since most studies agree that higher levels of TNF-a were associated with favorable clinical prognosis as the immunomodulating adjunct to ATT. In this study we present changes in cytokine production at 6 and 9 months post-therapy. Due to delay in getting cytokine kits we were, unfortunately, unable to conduct this study within the same time period as the cell subset phenotype and viral load studies. For this reason results from this investigation are not directly correlated with prior findings.
At the end of 6 months of treatment Dzherelo and Dzherelo + Anemin combinations produced 61% and 44.4% more of IL-2, whereas the levels of this cytokine in ATT alone group were reduced by 33.1% (Fig. 1). Incidentally, Toossi et^l. reported that patients with pulmonary TB had 81.2% lower output of tuberculin-induced IL-2 as compared with healthy tuberculin reactors. Defective IL-2 production was associated with more extensive disease on chest X-ray implying that administration of this cytokine could be beneficial to patients with TB [15]. The first initial trial of IL-2 had shown encouraging results. However, randomized, placebo-controlled trial of therapeutic interleukin-2 demonstrated significant delays in clearance and conversion rate of M. tuberculosis in sputum culture. This clinical study revealed potential antagonism between TB drugs and immunotherapy and suggested that exogenous supplementation with cytokines will not necessarily fulfill expectations based on mechanistic studies [14]. By the same token, it is unclear whether higher levels of IL-2, originally described as the T-cell growth factor, are responsible for increased CD3+ T lymphocyte levels observed in Dzherelo recipients [12].
Many studies in TB patients and non-TB controls deal with analysis of cytokine production in response to mitogens or mycobacte-rial antigens such as PPD or tuberculin. IL-6 was often found elevated in such assays [16]. Nagabhushanam et^l. reported that excess of IL-6 secreted by mycobacterium-infected macrophages may contribute to the inability of the cellular immune response to eradicate infection viajFN-y dependent pathway [17]. In addition to many other functions IL-6 is considered to be non-specific inflammatory cytokine shown to exacerbate deleterious immune restoration syndrome in tuberculosis and HIV co-infected patients [5,18]. Our results indicate that production of IL-6 was suppressed by Dzherelo (P = 0.007) or Dzherelo + Anemin (P = 0.2) but appears to be increased by ATTj(P = 0.15). As Dzherelo has been found equally effective in treating both TB and HIV our findings support prior studies suggesting that the downregulation ofIL-6 dependent inflammatory process may be beneficial to the host.
IFN-a is a multifunctional cytokine with established activity in many infectious diseases. IFN-a synergizes with IL-12 and plays an important role in inhibiting Th2 cytokines through antagonizing effect on IL-4, IL-10, or other immunosuppressive cellular factors and by promoting Th1 response via induction of IL-2 and IFN-y [18]. Administration of aerosolized IFN-a to TB patients had shown more favorable effects than in those given chemotherapy alone [19]. In our study, IFN-a was the only cytokine that behaved similarly in all three treatment groups. While tendency to decrease was observed it was not statistically significant. For this reason it is not clear whether this cytokine has contributed to observed increase in IL-2 and IFN-y levels or it plays any role in outcome from therapy. Thus, in case of IFN-a we do not know how to interpret our observation without being biased by an erroneous a priori attribution of cause and effect.
TNF-a is a monocyte-activating cytokine which stimulates anti-mycobacterial activity and helps to maintain the integrity of tuberculous granulomas in which M. tuberculosis is contained [1]. Our results indicate that both Dzherelo and Dzherelo + Anemin combinations cause significant decrease in baseline TNF-a levels by as much as 19.1% (P = 0.02) and 76.3%fP = 0.06) respectively. This effect is opposite to the trend in ATT-treated patients where TNF-a secretion continued to escalate. Since most studies agree that higher levels of TNF-a were associated with favorable clinical prognosis our findings may appear to contradict common wisdom. On the other hand, the risk of developing TB was 20-fold higher in non-TB patients who were treated with TNF antagonists, e.g., monoclonal anti-TNF antibodies and soluble TNF receptor [20]. Since TNF is a major inflammatory molecule that exacerbates symptoms of AIDS, clinical studies were conducted in which treatment modalities with anti-TNF activity were evaluated in HIV-positive patients co-infected with TB. Interestingly enough a positive trend toward superior resolution of lung infiltrates, closure of lung cavities, improvement in mycobacterial clearance score, and weight gain was observed [14]. Our findings appear to support the opinion that therapies directed at suppression of TNF-a might be beneficial for TB patients, even though in non-TB cases this strategy was associated with higher risk of re-activation of latent tuberculosis.
IFN-y is considered to be most important cytokine in the protective response against TB [21]. JFN-y is an essential component of the activation cascade of other cytokines such as GM-CSF, IL-2, and IL-4 which act as cofactors with anti-TB activity [22]. IFN-gam-ma production is strongly depressed during active TB, correlates inversely with disease severity, and increases during therapy. After 6 months of treatment Dzherelo and Dzherelo + Anemin increased IFN-y production by 17.3% (P = 0.33) and 31.9% (p = ^008), respectively, while in ATT recipients the reverse trend was observed (34%; P = 0.004). Our findings are in line with results of several clinical trials of inhaled or injected IFN-y which have shown positive clinical outcome [14,23,24]. Our findings are also in line with observations that in active-TB patients M. tuberculosis-induced IFN-y and TNF-a production are driven in opposite directions
[1,25,26].
An understanding of cytokine responses that are crucial for control of M. tuberculosis has major implications for the development of immune-based prophylactic and therapeutic interventions [13]. Our investigation of cytokines is limited in scope mostly to Th1 axis components and does not fully explain the mechanism of action of immune modulators used in this study. Th2 response, which is considered equally important, has not been evaluated by us [27]. Other surrogate markers of disease progression and prognosis such as for example, beta2-microglobulin, neopterin, tumor necrosis factor receptor II (TNFRII), CD8/CD38 cells ratio, soluble urokinase plasminogen activator receptor (suPAR), and CXCL10 (IP-10) need to be evaluated as well [28].
Nevertheless, judging from what we have observed so far, our immunomodulators appear to enhance naturally-occurring immune response against TB, perhaps by contributing to the down-regulation of inflammatory reaction [29]. We have shown before that patients receiving conventional chemotherapy with Dzherelo and related botanical supplements had significantly higher and faster cure rate than those given chemotherapy alone [7-12]. Dzherelo induces higher number of CD4 lymphocytes and reduced viral burden—surrogate markers associated with favorable course of HIV infection [11,12]. Expanded study is now required to identify the role of other immune correlates associated with control and protection against M. tuberculosis infection. The results may reveal not only biomarkers related to better clinical outcome but also may help designing better immunotherapies and vaccines [30].
Acknowledgments
We thank all participants who volunteered in this study. The support of clinical staff and technicians who contributed to this study has been of tremendous help to us. We are grateful to other colleagues who shared their insight and provided helpful suggestions based on their own experience with phytoconcentrates used in present study. This work was presented in part at the Keystone Symposia on HIV Pathogenesis and HIV Vaccines, March 27-Apr 1, 2008, Banff, Alberta, Canada, through a grant from Bill and Melinda. Gates Foundation's Global Health Travel Award, which is gratefully  acknowledged.
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Adjunct Immunotherapy of Tuberculosis in Drug-Resistant TB and TB/HIV Co-Infected Patients

Nathalia D. Prihoda1 • Olga V. Arjanova1 • Lyudmila V. Yurchenko1 • Nina I. Sokolenko1 • Lyudmila A. Vihrova2 • Volodymyr S. Pylypchuk3 • Galyna A. Kutsyna4*
Lisichansk Tuberculosis Dispensary, Lisichansk, Ukraine 2, Lisichansk Regional Hospital, Lisichansk, Ukraine 3, Ekomed LLC, Kiev, Ukraine, Luhansk Regional AIDS Center and Luhansk State Medical University, 50-years of Defense of Luhansk street, Luhansk 91045, Ukraine, Corresponding author: * kutsyna@list.ru
ABSTRACT
Open-label, salvage anti-tuberculosis therapy (ATT) combined with DZHERELO (IMMUNOXEL), Svitanok, and LIZORM - over-the-counter immunomodulators from medicinal plants - was conducted in 20 Ukrainian patients, comprising seven who had HIV co-infection. Except five patients with HIV, all other individuals had multidrug-resistant TB (MDR-TB) including 7 (35%) patients with XDR-TB. Patients hospitalized in our TB dispensary were treated under directly observed therapy (DOT) until repeated negative culture conversion and recuperation from radiological and clinical symptoms. The average duration of therapy was 16.2 ± 5.2 weeks (range 10.630.3; median 16). The mean time to bacterial clearance was 4.4 ± 1.8 weeks (range 1.3-8.9, median 4.3). All patients (95%), except one, gained weight, ranging between 3-17 kg with average 8.7 kg (P=0.000009). The liver function tests revealed that the level of total bilirubin had decreased from 15.5 to 11.6 umol/L (P=0.009). Alanine transaminase (ALT) declined from elevated 53.1 IU/L to normal 30.4 IU/L level (P=0.001). Hemoglobin levels increased from 103.2 to 117.3 g/L (P=0.00005). Inflammation-associated, elevated leukocyte counts returned back to normal from 8.9 to 6.9 x 109 cells/L (P=0.003). Patients improved clinically and radiologically and were hence discharged from the hospital. These findings support prior trials indicating clinical benefit of adding immunomodulators to TB treatment regimens. The combination of ATT with botanical preparations enhances the clinical efficacy of DOT and is safe and beneficial even to patients with poor prognosis due to drug resistance and/or co-infection with HIV.
Keywords: AIDS, botanical, drug resistance, Ekomed, herbal, HIV, immunotherapy, MDR, Mycobacterium tuberculosis, phytoconcen-trate, phytomedicine, phytotherapy, Ukraine, XDR.
INTRODUCTION
The tuberculosis (TB) epidemic is on the rise in many countries, including Ukraine. If left untreated the active form of TB will kill two of every three people (Karachun-skii 2000). This problem is further compounded by HIV co-infection, since one-third of AIDS-related deaths results from TB. Ukraine has the highest prevalence of TB/HIV co-infection in Eastern Europe (van der Werf et al. 2006). The effectiveness of TB therapy is significantly lower among patients with HIV/AIDS (Karachunskii 2000). The
World Health Organization (WHO) estimates that a person with both HIV and TB infection is thirty times more likely to become ill with TB than a person with Mycobacterium tuberculosis infection alone (Reid et al. 2006). The rate of relapse and mortality are consistently higher even when TB/HIV patients are treated with anti-tuberculosis therapy (ATT) under directly observed treatment program (DOT) (Khauadamova et al. 2001). Drug resistance along with HIV-accompanying immunodeficiency is the main cause of treatment failure. Recently published survey of Nikolayev-skyy et al. (2007) indicates that in Ukraine the multi-drug resistant form of TB (MDR-TB) was found in 27.3% of TB patients but was twice higher (54.8%) among formerly incarcerated individuals.
The first line of TB drugs includes isoniazid, rifampicin, ethambutol, pyrazinamide, and streptomycin. There are six classes of second-line TB drugs including fluoroquinolones: e.g., ciprofloxacin, moxifloxacin; aminoglycosides: e.g., amikacin, kanamycin; polypeptides: e.g., capreomycin, vio-mycin, enviomycin; thioamides: e.g. ethionamide, prothionamide; cycloserine; and para-aminosalicylic acid (Zaitzeva 2004). Other TB drugs, which are not on the WHO list, include: rifabutin; macrolides: e.g., clarithromycin; linezolid; thioacetazone; thioridazine; arginine; and vitamin D. MDR-TB is diagnosed when M. tuberculosis are resistant to at least isoniazid (H) and rifampicin (R), two most commonly-used, first-line drugs. The extensively resistant TB (XDR-TB), in addition to lack of sensitivity to H and R, is also resistant to any one of fluoroquinolones, and at least one of second-line injectable drugs, e.g., capreomycin, kanamycin, and amikacin (Migliori et al. 2007). This emerging form of TB caused worldwide concern after recently reported outbreak in Kwazulu Natal province of South Africa where 52 of 53 patients with XDR tuberculosis and HIV co-infection had died within 2 weeks from the time of diagnosis (Gandhi et al. 2006).
Immunomodulators Dzherelo , Svitanok and LIZORM are made from a proprietary combination of medicinal plants and are commonly used in Ukraine for the management of TB and HIV infections, including patients with dual infection (Arjanova et al. 2006; Prihoda et al. 2006; Zaitzeva 2006; Chkhetiany et al. 2007; Nikolaeva et al. 2008). They have been approved in 1997 by the Ministry of Health of Ukraine as functional supplements with therapeutic indications. In 1999 Dzherelo and Svitanok combination were specifically recommended as an immune adjunct to the therapy of pulmonary tuberculosis (Melnik et al. 1999). So far, the phytoconcentrates we have used in this study have been taken by several hundred thousand individuals for various indications including chronic bacterial and viral infections such as TB and HIV, autoimmune diseases,and malignancy (Chkhetiany et al. 2007).
Published studies have demonstrated that Dzherelo can significantly shorten the duration of treatment and helps to achieve higher response rate even in those who are HIV co-infected or have MDR forms of TB (Arjanova et al. 2006; Prihoda et al. 2006; Chkhetiany et al. 2007). Dzherelo has also been found to decrease the hepatotoxicity associated with ATT (Zaitzeva 2006). Svitanok is commonly used for counteracting liver-damaging effect of chemotherapy and in hepatitis therapy. LIZORM is routinely used for alleviating symptoms of autoimmune disorders (Bodnar et al. 2002). Our study was aimed at evaluating the combined effect of Dzherelo , Svitanok, and LIZORM in a representative sample of hospitalized patients who received the anti-TB therapy under DOT. Patients were selected among those who had particularly poor prognosis due to drug resistance and/or HIV co-infection.
MATERIALS AND METHODS
Patients
Twenty patients with active TB and poor prognosis due to resistant TB and/or HIV co-infection were selected to be given in addition to standard ATT the over-the-counter, immunomodulating phyto-preparations manufactured by Ekomed company. The age of patients ranged between 24 and 58 years with mean/median age of 39.7/39.5 years. The female/male ratio was 4/16. Eleven patients presented with first-diagnosed TB and nine patients had previously treated, relapsed, i.e., chronic TB. Fifteen patients had drug-resistant TB, including seven with XDR-TB, and five patients in TB/ HIV subgroup had drug-sensitive TB. All study patients presented with acute symptoms of pulmonary TB that required hospitaliza-tion. Most common symptoms were prolonged heavy cough, pain in the chest, high fever, profuse night sweats, fatigue, and loss of weight and appetite. Active pulmonary tuberculosis was certified by a medical history and clinical findings compatible with tuberculosis, a chest X-ray showing lung involvement, and positive sputum smear for acid-fast bacilli (AFB) and the culture of M. tuberculosis. The diagnosis of HIV infection was established by standard ELISA test further confirmed by Western blot. All HIVpositive patients were in advanced stage III of HIV infection. None of the patients received anti-retroviral therapy prior to and during follow-up. The conduct of the study was approved by the internal review board (IRB) of Lisichansk TB dispensary. The participation in this study was voluntary and patients were eligible to enroll only after signing the written consent.
Treatment regimen
Individualized, first- and second-line anti-TB drugs were administered to hospitalized patients based on physician's decision prior to or after results of drug susceptibility tests. Drugs were administered under DOT schedule. In addition to ATT, patients received a daily dose of Dzherelo which was given as 30 drops diluted in a half-glass of water 30 minutes before breakfast. Some patients received DZHERELO-PI - slightly modified form of Dzherelo .
The same dose, 30 drops, of LIZORM and Svitanok were given before lunch and supper respectively. Sputum smear and culture examinations for AFB were performed at monthly intervals. The decision to discharge was based on at least twice-repeated negative culture outcome and satisfactory clinical and radiological findings.
Anti-tuberculosis drugs and phytopreparations
All anti-TB drugs were procured through the centralized national supply system of Ukraine. The over-the-counter phytoconcentrates,
Dzherelo , Svitanok, and LIZORM, were generously supplied by Ekomed company. DZHERELO (IMMUNOXEL) contains concentrated aqueous-alcohol extract from medicinal plants such as aloe (Aloe arborescens), common knotgrass (Polygonum aviculare), yarrow (Achillea millefolium), purple coneflower (Echinacea purpurea), centaury (Centaurium erythraea), snowball tree berries (Viburnum opulus), nettle (Urtica dioica), dandelion (Taraxacum officinale), sweet-sedge (Acorus calamus), oregano (Oreganum majorana), marigold (Calendula officinalis), seabuck-thorn berries (Hippophae rhamnoides), elecampane (Inula hele-nium), tormentil (Potentilla erecta), greater plantain (Plantago major), wormwood (Artemisia sp.), Siberian golden root (Rhodi-ola rosea), cudweed (Gnaphalium uliginosum), licorice (Glycyr-rhiza glabra), fennel (Foeniculum vulgare), chaga (Inonotus obli-quus), thyme (Thymus vulgaris), three-lobe beggarticks (Bidens tripartite), sage (Salvia officinalis), dog rose (Rosa canina), and juniper berries (Juniperus communis). Svitanok contains flowers of immortelle (Helichrysi arenarii), barberry roots (Ber-beris vulgaris), chicory roots (Cichorium intybus), coriander seeds (Coriandrum sativum), marigold (Calendula officinalis), wormwood, and maize cores with stigmas (Zea mays). LIZORM contains concentrated aqueous-alcohol extract from barberry roots (Berberis vulgaris), aronia berries (Aronia melanocarpa), St. John's Wort (Hypericum perforatum), centaury, nettle, common knotgrass, wild strawberry leaves (Fragaria vesca), greater celandine (Chelidonium majus), and immortelle. All phytopreparations were approved in 1997 by the Ministry of Health of Ukraine as dietary supplements. In 2006 they received the status of a functional food - superior category of herbal supplements which can carry medical claims substantiated by clinical evidence.
TB drug resistance testing
The drug resistance profile to first- and second-line TB drugs was tested with a commercially supplied kit (Tulip Diagnostics, Goa, India). The cultures of M. tuberculosis derived from sputum of each patient were inoculated into ready-to-use tubes containing TB drugs incorporated at manufacturer-predetermined concentrations into standard Lowenstein-Jensen agar slants. The inoculae were incubated at 37°C and checked periodically until appearance of colonies in control tubes without drugs. The calculation of the proportion of resistant bacilli was done by comparing counts on drug free and drug-containing Lowenstein-Jensen medium, essentially as described by Laszlo et al. (1997).
Statistical analysis
The obtained results were analyzed with the aid of statistical software STATMOST (Datamost, South Sandy, UT). All statistical analyses were done on intent-to-treat basis, involving the total number of patients without subgrouping them into responders and non-responders. Simple statistical calculations such as determination of standard deviation, mean and median, were performed with the same software. Where available the baseline values relative to the end of study values were evaluated by paired or unpaired Student t-test. When required the stratification analysis of patients was conducted to reveal the difference between distinct clinical categories. The probability values were considered as significant at P<0.05 cut-off value.
RESULTS
The duration of DOT ranged between 10.6-30.3 weeks with average/median 16.2/16 weeks (Table 1A, 1B). The treatment lasted until patients were discharged from the dispensary upon twice-repeated negative culture findings and clinical and radiological improvements. The time to negative culture conversion ranged between 9-62 days with mean/ median 30.6/30 days. Mycobacterial clearance was confirmed by repeated cultures at monthly intervals.
There was no difference between chronic, previously treated TB versus first-diagnosed TB cases in terms of days to discharge, i.e., 111.6 vs. 114.8 (P=0.42) or days to myco-bacterial clearance, 33.7 vs. 28 (P=0.16). A similar stratification analysis comparing TB/HIV with TB alone patients reveals that patients with dual infection appear to require longer treatment, i.e., 127.9 vs. 105.5 days, but the difference was not statistically significant (P=0.15). Similarly, negative culture conversion occurred about nine days later in TB/HIV individuals than in TB patients, i.e., 36.1 vs. 27.5 days, but the difference was not statistically reliable (P=0.08). The comparison of treatment outcomes between 15 drug-resistant and 5 drug-sensitive cases also failed to reveal statistical difference. Time to negative culture was 30.3 vs. 31.4 days and time to discharge 106.2 vs. 134.8 days with probability values P=0.4 and P=0.18, respectively.
Table 1A Baseline and outcome characteristics of TB patients treated with ATT in combination with Dzherelo , Svitanok, and LIZORM.

*Criteria for definition of XDR are as per WHO recommendation. ATT drugs are abbreviated as follows: Isoniazid (H), Rimfapicin (R), Pyrazinamide (Z), Ethambutol (E), Streptomycin (S), Levofloxacin (L), Ofloxacin (O), Ciprofloxacin (CPX), Pefloxacin (PFX), Kanamycin (K), Amikacin (A), Para-aminosalicylic acid (PAS), Rifabutin (RFB), Ethionamide (ETH), Prothionamide (Prothio)
Table 1B Baseline and outcome characteristics of TB patients treated with ATT in combination with Dzherelo , Svitanok, and LIZORM.

15 drug-resistant and 5 drug-sensitive cases also failed to reveal statistical difference. Time to negative culture was 30.3 vs. 31.4 days and time to discharge 106.2 vs. 134.8 days with probability values P=0.4 and P=0.18, respectively.
At the end of study almost every patient had gained substantial lean body mass - an effect that was evident within one month from initiation of the therapy. Except one TB/HIV patient (#19) who lost 10 kg, all other patients gained weight, ranging between 3 and 17 kg. The average accrual in lean body mass was 8.7 kg (median 9.5 kg), which was statistically highly significant as evidenced by a paired Student's t-test (P=0.0000009).
The potential hepatotoxicity of ATT combination with herbal preparations was monitored by quantitative liver function tests. Surprisingly, despite intensive chemotherapy patients have shown signs of better liver function. The level of total bilirubin had decreased from mean 15.5 to 11.6 Limol/L - a favorable change that was statistically significant (P=0.009). Similarly, the values of alanine transaminase (ALT), another marker of liver damage, have declined from abnormally high (53.1 IU/L) to normal levels (30.4 IU/L) - a change that was also statistically significant
(P=0.01).
Another phenomenon observed during therapy is a reversal of baseline anemic state and pro-inflammatory condition - symptoms very common in TB and HIV. Most patients at study entry displayed signs of anemia and had abnormally elevated leukocyte counts. At the end of treatment these parameters were improved in a statistically significant manner. The levels of hemoglobin had risen from 103.2 to 117.3 g/L (P=0.00005), whereas leukocyte counts returned back to normal levels from 8.9 to 6.9 x 109 cells/L (P=0.003).
Flow cytometry measurements of T lymphocyte counts conducted at study entry and at the end of follow-up were available in 6 of 7 TB/HIV patients (Table 2).
Table 2 Changes in absolute CD4+ and CD8+ T-lymphocyte counts among TB/HIV patients   
 * The sequence of patients' numbers corresponds to that shown in Table 1.  
The helper CD4+ cells declined in two patients, while remaining patients had displayed an increase in their lymphocyte numbers. From average 371 cells/ul at baseline they have risen to 566 cells/ul - an increase equal to 52% (P=0.07). The absolute numbers of CD8+ T-lymphocytes appeared to decline but no statistical significance has been reached (P=0.1). The increase in CD4 cells and decline in CD8 cells resulted in almost doubled ratio of CD4/CD8 cells, i.e., from baseline 0.475 to 0.848 at the end of study (P=0.03).   
 DISCUSSION             
Tuberculosis remains an enormous global health problem. There are 8-9 million new cases and 1.5-2 million deaths from TB annually. Despite the overwhelming burden of disease, no new compounds were developed in last 40 years and current strains of TB are becoming resistant to existing drugs. The emergence of XDR-TB raises a serious concern of epidemic of virtually untreatable TB. It is clear that currently available chemotherapies for the treatment of TB are not perfect - they require multiple tuberculosis drugs to be taken for long periods of time. The duration of therapy, coupled with the side effects, often results in poor patient adherence, treatment failure, and the emergence of drug resistance (Zaitzeva 2004). The results of this small-scale study, consisting of representative group of drug-resistant patients from our dispensary, indicate that when tuberculosis drugs are combined with immunomodulating herbal preparations, Dzherelo , Svitanok, and LIZORM, they are able to produce rapid clinical and radiological improvements and disappearance of M. tuberculosis from a sputum culture within one month from treatment initiation.
Our findings support prior clinical studies of ATT that were conducted mostly with Dzherelo and occasionally with Dzherelo and Svitanok combination (Melnik et al. 1999; Arjanova et al. 2006; Prihoda et al. 2006; Zaitzeva 2006; Chkhetiany et al. 2007). Our results indicate that the combination of three phytoconcentrates is even more effective in enhancing the efficacy of ATT and reducing the duration of treatment. Conversion of sputum mycobacterial culture from positive to negative is considered the critical interim indicator of the efficacy of anti-TB intervention (Holtz et al. 2006). We observed culture conversion at median 32 days (range 10-62 days). This is twice shorter than reported median 60 days culture conversion time (range 4462 days) among drug-resistant TB patients in Latvia who were treated with first- and second-line TB drugs (Holtz et al. 2006). Same time to culture conversion, i.e., 2.1 months, was reported by Yew et al. (2003) in their retrospective MDR-TB study in Hong Kong. However, their mean duration of successful chemotherapy was 14.5 months as opposed to 3.9 months in our study. Similar range of therapy duration for drug-resistant TB, i.e., 11-24 months, was reported by Japanese investigators (Yoshiyama et al. 2007). If these studies are representative of best success rates in MDR-TB therapy then our immunomodulatory intervention affords twofold reduction in culture conversion time and shortens treatment duration by at least three-fold.
Numerous published studies have shown that patients with dual infection are less susceptible to TB treatment and had very poor prognosis despite best available standard of care (Karachunskii 2000; Khauadamova et al. 2001; Dean et al. 2002; Reid et al. 2006). Our patients with TB/HIV appear to require more time to achieve culture conversion and were prone to remain in the hospital longer than individuals without co-infection. However, due to limited sampling we were not able to confirm this impression by statistical analysis. We would need larger cohort of patients to determine whether TB/HIV patients are more refractory to the im-munotherapy. Paradoxically, we did not see any difference in duration of treatment between TB patients with primary and chronic infections. Similarly, no meaningful pattern appeared when we compared drug-sensitive and drug-resistant patients for difference in time to culture conversion and duration of DOT. At least, theoretically, those with previously failed treatment or MDR-TB would require longer time to clear the infection (Sacks et al. 2001). These observations suggest that, unlike ATT, our phytoconcentrates do not have direct effect on M. tuberculosis growth and appear to act through modulation of the immune response (Melnik et al. 1999; Pylypchuk 2003).
No adverse effects attributable to the use of phytocon-centrates were observed at any time during therapy. Contrary, as a result of combination treatment our patients enjoyed better quality of life and were tolerating ATT much better than those who received ATT without phytotherapy. Despite liver damaging ATT regimen, our patients had shown amelioration of the liver function as evidenced by normalization of ALT and bilirubin. This illustrates that herbal supplementation with Dzherelo, Svitanok, and LIZORM is safe and can neutralize or even reverse the hepatotoxicity of anti-tuberculosis drugs. The levels of hemoglobin improved markedly indicating that anemia was no longer of concern to our patients. This finding is highly relevant to the fact that anemia is an independent factor associated with an increased risk of mortality (O'Brien et al. 2005). TB-associated inflammation is another factor associated with poor prognosis (Feshchenko et al. 1997; Breen et al. 2004). Elevated leukocyte counts, indicative of ongoing inflammatory reaction, became normal as well. These beneficial effects were observed in all three categories of TB patients, i.e., MDR-TB, XDR-TB, and patients with HIV co-infection.
It is well know that the expansion of helper T-cell population and increased ratio of CD4/CD8 cells are associated with better prognosis in TB and HIV patients (Rodrigues et al. 2002). In TB/HIV group of patients we had 6 individuals out of 7 for whom we had data on such immune parameters. The measurement of CD4+ T-lymphocyte counts revealed that, except two individuals, four other patients had significantly higher number of cells at the end of follow-up. The ratio of CD4 to CD8 cells had almost doubled which was due to both an increase in CD4 and decrease in CD8 numbers (P=0.03). However, the P values for CD4 (P=0.07) and CD8 counts (P=0.1) were above <0.05 significance threshold. This may have been due to small sample size. In prior studies with larger number of patients the positive changes in absolute and relative T lymphocyte numbers as well as CD4/CD8 ratio were always highly significant (Chkhetiany et al. 2007; Nikolaeva et al. 2008).
Tuberculosis is a wasting disease (Schwenk and Macallan 2000). Many patients with TB and/or HIV, especially in advanced disease stage, suffer from cachexia. This condition is poorly manageable and is one of the leading factors contributing to higher morbidity and mortality (Edwards et al. 1971; Villamor et al. 2006). Khan et al. (2006) reported that patients with underweight problem had higher risk of TB relapse and that changes in weight observed during early stages of treatment were an independent predictor of disease progression. The outstanding feature associated with our therapy is a dramatic body weight gain. The results show that 19 out of 20 (95%) patients had gained lean body mass equal to almost 13% of baseline weight (P=0.0002). TB drugs seldom enhance body weight (Paton et al. 2004). The only known to us report of significant weight gain in TB patients has been described by Donald et al. (1997). In their placebo-controlled study the increase in body mass, i.e., mean gain 8.9 kg, of magnitude similar to ours, has been described when TB patients were administered plant-originated p-sitosterol and sitosterolin. Unfortunately, these phytosterols had no effect on the rate of mycobacterial clearance. Other interventions which enhance weight but without effect on TB are corticosteroids and nutritional supplements (Smego and Ahmed 2003; Paton et al. 2004). The remarkable property of herbal immunomodulators Dzherelo, Svitanok, and LIZORM in reversing weight loss along with substantiated adjunct effect on both TB and HIV will be particularly advantageous to those who live in resource-poor countries, where malnutrition is a common occurrence and deaths are more prevalent due to this cause (Farmer et al. 1991; Yew and Leung 2006).
Our study is unique since for the first time it reports treatment of Ukrainian patients with the XDR form of TB. Despite very poor prognosis we were able to achieve bacterial clearance within 4 weeks and attain major clinical and radiological improvements. As a result these patients were discharged from the dispensary after 110 (median 117) days. Our results contrast to two available clinical studies dealing with treatment of XDR-TB. In a report from South Africa 52 out of 53 (98%) patients had died within two weeks from diagnosis (Gandhi et al. 2006). However this extreme mortality rate might not be representative of the situation when more advanced clinical care is available. The retrospective study published by Kim et al. (2007) indicates that in South Korea the failure rate due to XDR-TB was 44.2%, whereas 27.4% patients with MDR did not respond to the therapy. Our experience is limited since we had only seven patients with XDR-TB and we need to confirm our findings in a larger cohort. According to published surveys on global prevalence of XDR-TB, the occurrence of XDR patients among MDR-TB cases is in 10-20% range (CDC 2006; Migliori et al. 2007; Shah et al. 2007). If these estimates are correct we will be able to find and recruit sufficiently large XDR population for an expanded study in the future. It is agreed that immune-based therapies are urgently needed to complement TB drug discovery (Johnson et al. 2003; Kaufmann 2006; Achkar et al. 2007). We also believe that the immunotherapy must be the indispensable part of therapeutic interventions against tuberculosis (Pylypchuk 2003). Many potent immunomodulators are available against bacteria, protozoa, fungi and viruses (Ershov 2003). While often effective their mechanism is poorly understood in most cases. This drawback should be balanced against clinically confirmed benefits. From the review of available to us medical literature it is apparent that very few medicinal plants have shown scientifically confirmed TB-curing properties. Recently reported story, describing how the Zulu's traditional herbal medicine became a candidate TB drug, further highlights the difficulties of finding and implementing an effective TB remedy from botanical sources (Bladt and Wagner 2007). Nonetheless, some medicinal herbs were shown to modulate the immune response to TB (Tomioka 2004), while others exerted direct or indirect antimycobac-terial activity (Klun and Youmans 1973; Newton et al.2000). Unfortunately, we do not know which active ingredients in our multi-herb preparations are responsible for the observed clinical effect. It is unlikely that they act as tuber-culostatic agents since in vitro growth of M. tuberculosis reference strains H32 and H37Rv was not affected directly either by Dzherelo or Svitanok (Melnik et al. 1999) and diseases etiologically unrelated to TB were responsive to these preparations (Bodnar et al. 2002). It is clear that this issue must be further investigated by experts in medicinal plants.
Our study provides further evidence of safety and efficacy of Dzherelo, Svitanok and LIZORM combination, former two of which were recommended in Ukraine as an immune adjunct to TB therapy (Melnik et al. 1999). In conclusion, we must emphasize that in this small study all patients were amenable to the therapy. The multi-country analysis of MDR-TB treatment outcomes has shown that cure rates were 52 and 29% for newly-diagnosed and retreated TB cases, respectively (Espinal et al. 2001). Thus, it is possible that when larger group of patients is evaluated then a certain number of treatment failures may emerge. Thus, additional studies need to be conducted in order to develop better understanding of this unique combination and to increase treatment options for TB patients with poor prognosis.
ACKNOWLEDGEMENTS
We thank all participants who volunteered in this study. The enthusiastic support of clinical staff and technicians who contributed their effort to this study has been of tremendous help to us. We are grateful to other colleagues who shared their insight and provided helpful suggestions based on their own experience with phytocon-centrates used in present study. The final stage of this study was supported by compassionate financial support graciously provided by MAPI Research Trust, Lyon, France - a non-profit organization that advances the art and the use of scientific approaches to patient-reported outcome measures. This work was presented in part at the Keystone Symposia on HIV Pathogenesis and HIV Vaccines, March 27 - Apr 1, 2008, Banff, Alberta, Canada, through a grant from Bill and Melinda Gates Foundation's Global Health Travel Award, which is gratefully acknowledged.
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Changes in CD4+ T-cells and HIV RNA resulting from combination of anti-TB therapy with Dzherelo in TB/HIV dually infected patients

Lyudmila G Nikolaeva1 Tatyana V Maystat1 Lilia A Masyuk2 Volodymyr S Pylypchuk3 Yuri L Volyanskii4 Galyna A Kutsyna5
'Kharkov Regional AIDS Prophylaxis and Prevention Center, Kharkov Medical Academy of Postgraduate Education, Kharkov, Ukraine; 2Jovtnevsky Correctional Colony No.17, State Department of the Penitentiary of Ukraine in Kharkov Region, Kharkov, Ukraine; 3Ekomed LLC., Kiev, Ukraine; 4I.I. Mechnikov Institute of Microbiology and Immunology, Kharkov, Ukraine; 5Luhansk Regional AIDS Center and Luhansk State Medical University, Luhansk, Ukraine
Correspondence: Lyudmila G Nikolaeva Kharkov Regional AIDS Prophylaxis and Prevention Center, Kharkov Medical Academy of Postgraduate Education, 6 Bor'by St., Kharkov, Ukraine Tel +380508093822 Fax +3804630631 Email kutsyna@list.ru
Abstract: The open-label, phase II clinical trial of antituberculosis therapy (ATT) with or without oral immunomodulator Dzherelo (Immunoxel) was conducted in TB/HIV coinfected, antiretroviral therapy naïve patients to evaluate the effect on CD4 T-lymphocyte counts and viral load. The arm A (n = 20) received isoniazid (H); rimfapicin (R); pyrazinamide (Z); streptomycin (S); and ethambutol (E), and arm B (n = 20) received 50 drops of Dzherelo twice per day in addition to HRZSE. After 2 months in 90% of  Dzherelo patients the population of absolute CD4 T-cells expanded by an average of 71.2% (from 174 to 283 cells/|ll; P = 0.00003), but declined in ATT-alone patients (182 to 174; P = 0.34). The ratio between CD4/CD8 cells deteriorated in 80% of individuals in arm A (1.213 > 0.943; P = 0.002), but improved in the same proportion of patients in arm B (1.244 > 1.536; P = 0.007). The number of total CD3+ lymphocytes rose from 728 to 921 cells in arm B (P = 0.025) whereas it fell from 650 to 585 cells in arm A (P = 0.25). The viral load, as measured by plasma RNA-PCR, decreased in 70% of  Dzherelo recipients (2.174 > 1.558 copies/ml; P = 0.002), but increased in 70% of HRZSE only receivers (1.907 > 2.076 copies/ml; P = 0.03).  Dzherelo has a favorable effect on the immune status and viral burden in TB/HIV patients when given as an immunomodulating adjunct to ATT. Keywords: dual infection, herbal, immunotherapy, MDR-TB, multi-drug resistant, Mycobacterium tuberculosis, phytotherapy, Ukraine, XDR-TB
Introduction
In developing countries, tuberculosis (TB) coinfection is common among human immunodeficiency virus (HIV)-positive individuals with probability of death 2- to 3-fold higher than among acquired immunodeficiency syndrome (AIDS) patients without active TB (Gupta et al 2007). The World Health Organization estimates that a person with both HIV and TB infection is thirty times more likely to become ill with TB than a person with Mycobacterium tuberculosis infection alone (Reid et al 2006). Ukraine has the highest rate of TB/HIV coinfection in Eastern Europe (van der Werf et al 2006). It is recognized that the effectiveness of TB therapy is significantly lower among patients with HIV/AIDS. The occurrence of mortality and relapse are consistently higher even when TB/HIV patients are treated with antituberculosis therapy (ATT) under directly observed therapy (DOT) (Khauadamova et al 2001). Drug resistance accompanied by HIV-associated immunodeficiency is the main cause of treatment failure. The recent survey of Nikolayevskyy and colleagues (2007) had shown that in Southern Ukraine the multi-drug resistance (MDR) rates were significantly higher among former prison inmates compared with nonprisoners (54.8% vs. 27.3%).
The oral immunomodulator  Dzherelo has been successfully used in Ukraine for the management of TB and HIV infections, including patients with dual infection. Published studies by us and others have demonstrated that  Dzherelo can significantly shorten the duration of treatment and helps to achieve higher response rate even in those who are HIV coinfected, have MDR or extensively-drug resistant (XDR) forms of TB (Chkhetiany et al 2007; Prihoda et al 2007; Nikolaeva et al 2008).  Dzherelo has also been found to decrease the hepatotoxicity resulting from TB chemotherapy (Zaitzeva 2006).  Dzherelo was approved in 1997 by the Ministry of Health of Ukraine as an immunomodulating supplement. In 1999,  Dzherelo was recommended as an immune adjunct to the conventional therapy of pulmonary tuberculosis (Melnik et al 1999).  Dzherelo contains aqueous-alcohol extract from various medicinal plants. So far,  Dzherelo has been used by over 150,000 individuals for various indications including chronic bacterial and viral infections such as TB and HIV, autoimmune diseases, and malignancy.
Several studies have shown that, similarly to HIV, the immune control of M. tuberculosis is dependent from CD4+ T-lymphocytes (Serbina et al 2001; Morris et al 2003; Manas et al 2004; Kalou et al 2005). The M. tuberculosis and HIV infections are associated with pronounced deterioration of the immune system as evidenced by higher depletion rate of helper CD4+ cells and enhanced HIV replication. The immunotherapeutic approaches toward TB began receiving more attention recently (Tomioka 2004; Kaufmann 2006; Achkar et al 2007). There are many types of immune modulators that have been used clinically for viral infections, but for TB the choice of immune interventions is limited (Ershov 2003). Our study was aimed at evaluating the effect of Dzherelo on T-lymphocyte populations and viral load among TB/HIV patients in comparison with control population, which received the ATT only.
Materials and methods
Patients

Male patients, aged 19^2 years, have been randomized into arms A and B, each consisting of 20 patients. The majority of patients in our study were correctional facility inmates in advanced clinical stage III of HIV-1 infection with average baseline CD4+ T-cell counts below 200 cells/microliter (cells/|jl). The diagnosis of HIV infection was established by standard ELISA test and further confirmed by Western blot. Active pulmonary tuberculosis was certified by a medical history and clinical findings compatible with pulmonary tuberculosis, a chest X-ray showing lung involvement, and positive sputum smear for acid-fast bacilli or the culture of M. tuberculosis. The conduct of the trial was approved by the State Department of the Penitentiary of Kharkov region,Ukraine. The participation in this trial was voluntary and patients were eligible to enroll only after signing the written consent.
Treatment regimens
None of the patients received antiretroviral therapy prior to and during 2-months of follow-up. All patients received standard antituberculosis therapy administered under DOT schedule which consisted of once daily dose of isoniazid (H) 300 mg; rimfapicin (R) 600 mg; pyrazinamide (Z) 2,000 mg; streptomycin (S) 1,000 mg; and ethambutol (E) 1,200 mg. The arm B received, in addition to HRZSE, twice per day dose of Dzherelo which was given as 50 drops diluted in a half-glass of water. Dzherelo contains concentrated aqueous-alcohol extract from medicinal plants such as Aloe (Aloe arborescens), Common knotgrass (Polygonum aviculare), Yarrow (Achillea millefolium), Purple coneflower (Echinacea purpurea), St. John's Wort (Hypericum perforatum), Centaury (Centaurium erythraea), Snowball tree berries (Viburnum opulus), Nettle (Urtica dioica), Dandelion (Taraxacum officinale), Sweet-sedge (Acorus calamus), Oregano (Oreganum majorana), Marigold (Calendula officinalis), Seabuckthorn berries (Hippophae rhamnoides), Elecampane (Inula helenium), Tormentil (Potentilla erecta), Greater plantain (Plantago major), Wormwood (Artemisia sp.), Siberian golden root (Rhodiola rosea), Cudweed (Gnapha-lium uliginosum), Licorice (Glycyrrhiza glabra), Fennel (Foeniculum vulgare), Chaga (Inonotus obliquus), Thyme (Thymus vulgaris), Three-lobe Beggarticks (Bidens tripartite), Sage (Salvia officinalis), Dog rose (Rosa canina), and Juniper berries (Juniperus communis). The over-the-counter phytoconcentrate  Dzherelo was generously supplied by Ekomed company (Kiev, Ukraine).
Immunophenotyping of lymphocyte subsets
The peripheral blood of patients with TB/HIV was analyzed with Clonospectr panel of monoclonal antibodies against surface antigens of T-lymphocytes (MedBioSpectr, Moscow, Russia). The absolute and relative (%) values of total CD3+ lymphocytes, helper T lymphocytes (CD3+CD4+) and cytotoxic T lymphocytes (CD3+CD8+) were assessed in a blinded fashion by fluorescent microscopy at baseline and after 1 and 2 months on the therapy. In addition the changes in the ratio between CD4 and CD8 cells were evaluated as a part of assessment of the immune status of patients. The samples of the blood from 19 healthy blood donors were analyzed as a reference for normal values.
PCR analysis
Stored frozen samples of plasma were processed in bulk with commercially available PCR kit (AmpliSense HIV-1, Central Research Institute of Epidemiology, Moscow, Russia) designed for quantitative analysis of HIV-RNA copies. Tests were carried out at baseline and after two months of the therapy.
Statistical analysis
The obtained results were analyzed with the aid of statistical software STATMOST (Datamost, South Sandy, UT). The baseline cell numbers relative to 1st and 2nd months post-therapy were evaluated by paired Student t-test. Unpaired t-test was used to compare data from TB/HIV patients to normal blood donors. The nonparametric values of viral load were evaluated by Wilcoxon signed-rank test. Spearman rank-order test was used for correlation analysis, which was carried out on absolute and relative numbers of T-cells and PCR results. The correlation analysis was also conducted on differential values of these parameters, ie, differences between baseline and 2-month results. All statistical calculations were per intent-to-treat basis or the total number of patients without subgrouping them into responders and nonresponders. The probability values were considered as significant at P < 0.05.
Results
After one month of therapy there was a clear distinction in measured T-cell counts between recipients of ATT-alone and those who received ATT with Dzherelo. This disparity became even more evident at the end of the second month of therapy (Figure 1). The changes in viral load among TB/HIV patients of both groups have also reached statistical significance even though none of the patients have ever received the antiretroviral therapy (Table 1). These findings are described in detail as follows.
CD3+ total T-lymphocytes
After one month on ATT-alone arm, the absolute and percent (%) values of total CD3+ lymphocytes per microliter of blood have not changed appreciably, ie, 650 (36.8%) vs. 634 (37.2%); P = 0.38 (P = 0.23), as analyzed by paired Student t-test. However, in the HRZSE group, which received Dzherelo, there was a significant difference in total CD3+ values as early as one month post-therapy, ie, 728 (37.5%) vs. 902 (40.4%); P = 0.015 (P = 0.04). After 2 months, the number of total CD3+ lymphocytes increased further to 921 in arm B (P = 0.025) whereas in the control arm it decreased to 585 cells (P = 0.25). The difference in treatment outcomes between two groups, ie, 921 vs. 585 was significant (P = 0.004), while baseline values were not statistically different (P = 0.15). Compared with baseline there was a 26% increase and 11.2% decrease in absolute number of total lymphocytes in arms B and A respectively (Figure 1). In arm B, 15 out of 20 (80%) patients were responders displaying the increase in absolute CD3 lymphocytes, while in arm A, less than half of patients (45%) had a positive response.
Abbreviations: H, Isoniazid; R, Rifampicin; Z, Pyrazinamide; S, Streptomycin; E, Ethambutol.
CD4+ T-lymphocytes
No significant changes were seen after one month in ATT-alone arm, ie, from 182 (24.2%) to 203 (24.5%) cells/^l; P = 0.063 (P = 0.28), whereas in the Dzherelo arm, the number of helper cells have risen significantly from 174 (23.3%) to 257 (27.3%) cells; P = 0.00003 (P = 0.0004). At the end of the second month, CD4 lymphocytes in arm B have risen further to 283 (31%) P = 0.0000004. However, the changes in arm A were insignificant 174 (25.3%) P = 0.13. When study completion results of  Dzherelo recipients were compared in terms of accrual of CD4+ lymphocytes with the entry levels, there was a highly significant increase of 34% and 71% in relative and absolute values. In arm A, relative and absolute CD4 cell numbers fell by 4.5% and 5.5%, respectively. The absolute and relative CD4 numbers were indistinguishable at baseline with P = 0.4 (P = 0.2), but at study conclusion the differences became highly significant with P = 0.001 and P = 0.003 for absolute and relative values, respectively. Out of 20 patients on Dzherelo 18 (90%) had seen their T-cells rise, while among those who received HRZSE-alone only 40% of patients had shown the increase in absolute CD4 lymphocytes (Figure 1).
CD8+ T-lymphocytes
After one month in the HRZSE-alone group, CD8+ cells increased in a significant manner from 159 (21.7%) to 199 (24.3%); P = 0.01 (P = 0.02), while in the  Dzherelo group,the changes were insignificant, ie, from 159 (20.8%) to 190.2 (19.8%); P = 0.21 (P = 0.05). At the end of the second month, the CTL population in the ATT-alone group was still above baseline, ie, 180 cells (27.7%), an accrual that was statistically significant in percent terms (P = 0.000009), but not significant when calculated in absolute numbers (P = 0.17). On the contrary, in the second month, numbers of CD8 cells among  Dzherelo recipients had not increased in any significant manner, from 159 (20.8%) to 183 (20.7%), with P values being 0.13 and 0.47 for absolute and percent figures, respectively (Figure 1). The absolute and relative CD8 numbers were almost identical between two arms at baseline 159 (21.7%) vs. 159 (20.8%) at P = 0.5 (P = 0.3), but at study conclusion they reached P = 0.4 and P = 0.00002 for absolute and relative values, respectively. In the HRZSE arm, 70% and 85% of patients had shown an increase in absolute and relative numbers of CD8 lymphocytes, while in the  Dzherelo group, 65% and 50% of patients had a similar response.
CD4/CD8 ratio
The differential changes in CD4 and CD8 lymphocyte numbers had affected the CD4/CD8 ratio in patients on the HRZSE-alone regimen as early as one month after treatment initiation. Their ratio had declined from 1.213 baseline value to 1.06 (P = 0.009). In contrast, the CD4/CD8 ratio among Dzherelo recipients increased from 1.244 to 1.416, which was, however, slightly above the cut-off value (P = 0.06). The disparity between CD4 and CD8 lymphocytes had progressed even further by the end of the second month. Among HRZSE-alone patients the ratio had declined to 0.943 (P = 0.002), while in the  Dzherelo group the ratio had risen to a level that is commonly considered to be as a normal, ie, 1.536 (P = 0.007). This ratio was statistically indistinguishable from the ratio of normal blood donors, ie, 1.54 vs. 1.76 (P = 0.09). The proportion of patients who experienced increase in their CD4/CD8 ratio was 80% and 20% for arms B and A, respectively.
Lymphocyte subsets in normal blood donors
Samples of the peripheral blood of 19 healthy individuals were analyzed to obtain the normal distribution values of lymphocyte subsets. The average number of absolute and relative (%) CD3 lymphocytes were 1,370 ± 169 cells/^l (52.9 ± 6.8). The values of CD4 and CD8 lymphocytes were 622 ± 89 (35.9 ± 4.3) and 349 ± 42 (19.9 ± 2.1) respectively, with ratio being 1.76 ± 0.19. The absolute numbers of CD3 and CD8 cells were approximately two-fold and CD4 cells three-fold higher than in patients with TB/HIV.
Viral load
The viral load, as measured by plasma RNA-PCR at baseline and at the end of the second month, increased in the ATT group (1907 to 2076 copies/ml; P = 0.025, by Wilcoxon signed rank test), but decreased in the  Dzherelo group (2174 to 1558 copies; P = 0.002). About two-thirds of the patients (14/20) on HRZSE-alone had shown the increase in viral load, while the same proportion of patients on Dzherelo (70%) had a reduction in their number of viral copies (Table 1).
Correlation of viral load with T-cells
The Spearman rank-order analysis has revealed that at study conclusion the differential accrual in HIV copies in arm A patients seem to be inversely dependent on differential of absolute CD4 numbers (r = -0.4124; P = 0.07) but directly correlated with relative CD8 counts at two months (r = 0.42; P = 0.07). However, the corresponding correlation values in  Dzherelo recipients were r = -0.01 (P = 0.96) and r = -0.03 (P = 0.91) for CD4 and CD8 cells, respectively. The only significant correlation found in the Dzherelo arm was an inverse relation between differentials of relative CD8 counts and viral load (r = -0.46; P = 0.04). The viral load was inversely dependent from CD4/CD8 ratio in arm A (r = -0.49; P = 0.03), but not in the  Dzherelo arm (r = 0.24; P = 0.3). No correlation was found between relative and absolute CD3 cell numbers and HIV RNA levels in arm A, but in arm B higher differential counts were significantly correlated with lower viral load (r = -0.49; P = 0.03).
Discussion
Our results indicate that when  Dzherelo is added to ATT, it can produce a significant increase in CD3+ and CD4+lymphocytes, and a better CD4/CD8 ratio, but no significant change in CD8-bearing lymphocytes. Furthermore, Dzherelo produces statistically significant lower viral load; an effect that we have shown in HIV-infected individuals without concomitant TB (Nikolaeva et al 2008).
Our 2-month DOT study conducted in a population consisting mostly of incarcerated individuals reveals that when  Dzherelo is added to ATT the absolute and relative numbers of CD4+ T-cells increase by 71% and 34%, respectively. Thus, in our hands  Dzherelo produced the same outcome as reported earlier (Chkhetiany et al 2007; Prihoda et al 2007; Nikolaeva et al 2008). For example, AIDS patients who received  Dzherelo with or without standard antiretroviral therapy gained between 38%-93.5% more CD4 lymphocytes (Chkhetiany et al 2007; Nikolaeva et al 2008). It is well established that elevated CD3 and CD4 counts and higher CD4/CD8 ratio are associated with better prognosis in patients with HIV as well as TB (Serbina et al 2001; Morris et al 2003; Manas et al 2004; Kalou et al 2005).  Dzherelo appears to normalize the lymphocyte homeostasis since at the end of study the CD4/CD8 ratio of treated patients became indiscernible from the ratio of normal healthy individuals, ie, 1.54 vs. 1.76 (P = 0.09). Thus,  Dzherelo is likely to influence positively the outcome of treatment and disease progression in our study population.
Treatment of TB in HIV-infected individuals is a daunting task when compared with TB in HIV-negative persons (Khauadamova et al 2001; Reid et al 2006; Gupta et al 2007). This task is particularly challenging when one has to treat inmate populations, which as a rule have higher prevalence of drug-resistant TB and more advanced HIV disease (Nikolayevskyy et al 2007). Due to frequent failure rate of TB therapy in this particular population of patients the immune intervention with  Dzherelo has been sought to evaluate its benefit. In the prior studies Dzherelo has been shown to increase by two- to three-fold the success rate of ATT and shorten significantly the duration of treatment even among those who had MDR-TB or XDR-TB (Prihoda et al 2007). It had also reduced the toxic side effects of ATT, the hepatotoxicity in particular. Elevated liver aminotransferase ALT and AST levels caused by ATT have been shown to return to normal levels after addition of  Dzherelo (Zaitzeva 2003). Furthermore, cachexic patients who received  Dzherelo were shown to gain weight (Chkhetiany et al 2007; Prihoda et al 2007). However, these studies have not dealt with the effect of  Dzherelo on viral load among dually infected TB/HIV patients.
The viral load is a predictor of HIV disease progression, its persistent elevation in TB/HIV coinfected patients is indicative of poor prognosis (Morris et al 2003; Kalou et al 2005). While there were earlier indications that  Dzherelo reduces viral burden (Chkhetiany et al 2007; Nikolaeva et al 2008), our study is the first to report this phenomenon in TB/HIV patients. Despite the fact that the HIV RNA levels had decreased by less than a log the difference between baseline and outcome levels was highly significant. 70% of patients in arm A experienced the increase in viral load, while the same proportion of patients in arm B had reduced viral burden. This observation is encouraging since successful ATT regimens, some which reported to restore the immunity in TB/HIV patients, were not affecting the viral load (Newton et al 2000; Morris et al 2003; Kizza et al 2005). Thus, our observation is quite unique. Except, perhaps, thalidomide, prednisone, and likopid, we are not aware of any other immunity-restoring preparation that would affect both the TB and HIV (Bekker et al 2000; Svistunova et al 2002; Mayanja-Kizza et al 2005). However, several experimental immunotherapeutic regimens were tried with various degrees of success in TB patients including for example interferon, tumor necrosis factor antagonists, cytokines, thymus-derived Russian immunomodulators such as thymalin, thymogen, T-activin, and vilosen (Khudzik et al 1998; Wallis 2005).
Many studies have been conducted aimed at identifying the immune cells controlling TB and TB/HIV coinfections. While there is a consensus that cellular immune responses play a critical role in disease progression, much more has to be learned in order to understand the relationship. The correlation analysis has revealed a certain degree of relation between viral load and CD4 or CD8 cells in arm A, but they were not statistically significant except CD4/CD8 ratio.  Dzherelo does not influence directly HIV replication and it is likely that the observed effect on viral load is mediated via immune cells (Melnik et al 1999).
Surprisingly, in arm B, the viral load was not dependent on changes in CD4 cells although significant inverse correlation was found with CD3 and CD8 cell numbers. However, this arm is characterized by a significant gain in total and CD4 T-lymphocytes but not changes in CD8-bearing population. Thus, we do not know how and which cells are involved in Dzherelo-influenced viral control. It is possible that the viral replication is regulated by a subpopulation of T lymphocytes which cannot be reliably identified by surface markers we have used. The protective immune response also needs to be separated from undesired inflammatory response (Achkar et al 2007). The possibility that CD8 T-cells are involved in upregulation instead of suppression of viral replication is suggested by correlation analysis in arm A. On the other hand, in arm B the inverse relation between differentials of relative CD8 counts and viral load suggests the opposite effect. The full significance of elevated CD3, CD4, CD4/CD8, but unchanged CD8 cells resulting from Dzherelo administration is yet to be understood. It is clear that the understanding of the immune mechanism controlling M. tuberculosis may aid in design of better vaccines and immunotherapies.  Dzherelo confers cellular responses suggestive of immunity-restoring action, which perhaps explains the higher success rate of the therapy when it is combined with ATT.
Currently available chemotherapy for the treatment of TB are not perfect, they require multiple anti-TB drugs to be taken in combination for long periods of time (Gupta et al 2007; Reid et al 2006). This can cause side effects, poor drug adherence, treatment failure, and the emergence of drug resistance with major social and economic consequences, especially in low-income countries. It is agreed that novel immune-based therapies are urgently needed to complement antitubercular drug discovery (Khudzik et al 1998; Tomioka 2004; Wallis 2005; Kaufmann 2006; Achkar et al 2007). We also believe that the immunotherapy is the indispensable part of therapeutic strategies against TB (Pylypchuk 2003). Many effective immunomodulators are available against bacteria, protozoa, fungi and viruses (Ershov 2003). While clinically effective their mechanism is not well understood in most cases. This drawback should be balanced against therapeutic benefits. Some medicinal herbs were shown to modulate the immune response to TB (Tomioka 2004), while others exerted direct antimycobacterial activity (Newton et al 2000). But it is unlikely that  Dzherelo is tuberculostatic since it does not affect mycobacterial growth in vitro and diseases or pathogens etiologically unrelated to M. tuberculosis were responsive to the therapy (Melnik et al 1999; Pylypchuk 2003).
Our study provides preliminary evidence into the putative mechanism of action of  Dzherelo, which has been recommended in Ukraine as an immune adjunct to TB therapy. Further studies are required to develop better understanding of the potential of Dzherelo and to enlarge the arsenal of tuberculosis drugs.
Acknowledgments
We thank all participants who volunteered in this study. The generosity of Ekomed in supplying  Dzherelo is appreciated. The enthusiastic support of clinical and technical staff who contributed to this study has been critical to successfully conclude this study. The discussions with other investigators of  Dzherelo who shared their insight and provided helpful suggestions have guided our study and we are thankful to all of them. This work was presented at the Keystone Symposia on HIV Pathogenesis and HIV Vaccines, March 27 - Apr 1, 2008, Fairmont Banff Springs, Banff, Alberta, Canada, with support from Bill and Melinda Gates Foundation's Global Health Travel Award, which is gratefully acknowledged.
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Impact of Adjunct Immunotherapy with Multi-herbal Supplement Dzherelo (Immunoxel) on Treatment Outcomes in End-stage TB/HIV Patients

Impact of Adjunct Immunotherapy with Multi-herbal Supplement Dzherelo (Immunoxel) on Treatment Outcomes in End-stage TB/HIV Patients

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Olga V. Arjanova1, Nathalia D. Prihoda1, Larisa V. Yurchenko1, Nina I. Sokolenko1, Lyudmila A. Vihrova2, Volodymyr S. Pylypchuk3, Valery M. Frolov4, Galyna A. Kutsyna4*

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4Luhansk Regional AIDS Center and Luhansk State Medical University, Luhansk, Ukraine                                                                                                       

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Cain KP, Kanara N, Laserson KF, Vannarith C, Sameourn K, et al. (2007) The epidemiology of HIV-associated tuberculosis in rural Cambodia. Int J Tuberc Lung Dis 11: 1008-13. »  CrossRef  »  PubMed  »  Google Scholar

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Received September12, 2009; Accepted December 30, 2009; Published December 30, 2009

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Citation: Arjanova OV, Prihoda ND, Yurchenko LV, Sokolenko NI, Vihrova LA, et al. (2009) Impact of Adjunct Immunotherapy with Multi-herbal Supplement Dzherelo (Immunoxel) on Treatment Outcomes in End-stage TB/HIV Patients. Journal of Antivirals and Antiretrovirals 1: 086-088. doi:10.4172/jaa.1000013

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Abstract

        •

Prognosis for TB/HIV co-infection is very unfavourable. In terminally-ill patients treatment options are often limited to palliative care. In our salvage, 2-month therapy of 40 late-stage TB/HIV patients we administered to half of the patients TB drugs along with over-the-counter botanical immunomodulator Dzherelo (Immunoxel). Despite best possible care 6 patients had died. Remaining 14 patients experienced marked clinical improvements and one patient was discharged due to full recovery. Among 20 matched subjects on conventional TB regimen, 12 died and only one was slightly better-off. These results indicate that Dzherelo might reduce mortality (P=0.055) and improve significantly the quality of life (P=0.00002). Improvement in quality of life is also supported by substantial weight gain (mean/median 3.3/4 kg) in much higher proportion of patients than among those who received TB drugs only, i.e., 16 vs. 1 (P=0.000001). At the end of two months 13 (65%) patients became sputum smear negative versus only one individual (5%) in ATT group (P=0.00007). These results suggest that adjuvant immunotherapy improves significantly therapy outcome and reduces mortality. Larger study is warranted to confirm the benefit of Dzherelo.

      • Perspectives

Keywords

         •

AIDS; End-stage; Herbal; Mycobacterium; Mortality; Quality of life; Salvage therapy; Survival; Terminal disease                  

         •

Introduction

                 

         •

HIV-positive individuals with tuberculosis are particularly vulnerable since standard anti-TB (ATT) and anti-HIV drugs (ART) are not very effective in this category of patients and prognosis is worse than for two infections separately (Kang’ombe et al., 2004). In the terminal stage of TB/HIV treatment options are severely restricted and it is usually too late to seek any meaningful therapy. Patients with TB/HIV are often from the low social strata and cannot afford expensive antiretroviral therapy, which could potentially prolong their life. Ukrainian health authorities do provide free TB drugs, but supplies of ART are limited and terminal TB/HIV patients are not on the priority list and thus seldom have a chance to receive them. Due to these unfortunate circumstances most patients are left without ART option. These patients are often ostracized, denied medical care at specialized AIDS clinics due to fear of airborne spread of tuberculosis, and usually end up in TB dispensaries where their only treatment option is antibiotics. At this stage the quality of life is very poor and mortality is high.

         •

Dzherelo is a multi-herbal oral immunomodulator, recommended but not approved by the health authorities of Ukraine as an adjunct therapy for TB (Chechitiany,et al. 2007). We and others have conducted several clinical trials involving TB/HIV-positive volunteers, which have shown that when Dzherelo and ATT are combined the quality of life and TB cure rates are drastically improved (Chechitiany et al., 2007; Prihoda et al., 2007; Nikolaeva et al., 2008; Nikolaeva et al., 2008; Prihoda et al., 2008). Adjunct immunotherapy has been shown to achieve faster and higher score of mycobacterial clearance, reduce HIV burden, accelerate healing of pulmonary lesions, decrease inflammation and liver damage, improve hematology picture, e.g., higher hemoglobin levels and CD4 counts, and enhance significantly quality of life (QOL) such as weight gain, fever, respiratory function, physical fitness, emotional well-being and better mood. In end-stage stage disease, however, the death is a critical endpoint, which defines unequivocally the merit of therapeutic intervention. In this study we investigated whether in addition to previously demonstrated positive clinical and QOL outcomes Dzherelo can reduce mortality rate.

           •

Materials and Methods

                    

           •

Patient population and intervention

                    

           •

This project started as a salvage therapy for patients with extremely poor prognosis (Prihoda et al., 2008). At any given time our TB dispensary has 80-100 end-stage hospitalized TB patients of which about 2/3 are HIV infected. The turnover is quite fast - an average upper life expectancy is about 5-6 months. Since dramatic differences were observed when we used Dzherelo in non-terminal TB and HIV cases, we thought that terminally-ill patients may also benefit from this intervention. We thus gave Dzherelo to those without any reasonable prospect of survival, but surprisingly the outcome was startling and much better than we initially anticipated. Such results were clearly unprecedented but a control was needed to advance our findings beyond anecdotal level. After long deliberation our Ethics board had decided that our salvage therapy can evolve into a clinical trial with matching arms provided that patients in ATT alone arm gave their informed consent. For us, it was and still is a difficult ethical issue as to which patient will receive potentially life-saving intervention and which one will not. In May 2008 the MAPI Trust graciously gave us a small contribution, which we used to start this project. Twenty end-stage TB/HIV patients received individualized TB drugs regimen while matching control group of patients had same TB drugs supplemented with 50 drops of Dzherelo given in a half-a-glass of water twice-a-day.

Follow-up procedures and statistics

At study entry all patients with confirmed HIV-positive test had a physical examination, chest X-ray, and sputum analysis for the presence of Mycobacterium tuberculosis. Patients were given best palliative care and followed on a daily basis. Those who remained alive were analyzed again two months later. The statistical difference between two groups was measured by Fisher’s exact 2x2 test with P threshold value set at =0.05.

Results

Forty terminally-ill TB/HIV volunteers entered into this comparative study. At the end of 2 months of follow-up 6 patients in Dzherelo group had died despite best possible palliative care (Table 1). In remaining 14 patients marked clinical improvement was observed and one patient was discharged due to full recovery from TB symptoms. By comparison, among 20 terminally- ill, matched group of patients receiving conventional TB drugs, 12 patients died and only one person had experienced a limited clinical improvement. These results indicate that when Dzherelo is co-administered with chemotherapy to end-stage TB/ HIV patients it might reduce mortality (P=0.055) and improve significantly the quality of life (P=0.00002). Improvement in quality of life among Dzherelo recipients is also supported by substantial weight gain (mean/median 3.3/4 kg; range 1-7 kg) in much higher proportion of patients than among those who received TB drugs only, i.e., 16 vs. 1 (P=0.000001). Positive changes as evidenced by sputum smear clearance and radiological improvements were equally impressive. At the end of two months 13 (65%) patients became sputum smear negative versus only one individual (5%) in ATT group (P=0.00007). In opinion of the treating physician one patient in Dzherelo group had fully recovered and was hence discharged from the dispensary. However this was a single case and statistically not significant (P=0.5) even though none of the patients on TB drugs alone had recovered to that extent.
Table 1: Preliminary findings in 40 end-stage TB/HIV patients treated for 2 months.

Discussion

In developing countries TB is the primary cause of AIDS-related deaths and it is agreed that available treatment options are not satisfactory (Kang’ombe et al., 2004; Cain et al., 2007; Ngo et al.,2007; Swaminathan et al., 2008; Saraceni et al., 2008). Our preliminary findings in 40 terminally-ill TB/HIV patients indicate that botanical immunomodulator Dzherelo can significantly improve the quality of life and reduce mortality. However, the probability value of better survival compared to conventional TB therapy was marginally significant (P=0.055)indicating that larger population is needed to verify our preliminary observation. To the best of our knowledge there is only one study, which reported positive effect of immunotherapeutic ntervention on survival of terminally-ill AIDS patients (Metadilogkul et al., 2005). In that study approximately one hundred patients were enrolled to show the significance implying that we may need a similar size population.

•    References

Quality of life parameters as judged by weight gain and physician’s assessed clinical improvement were highly significant, P=0.000001 and P=0.0002, respectively. We need, however, develop better methods so that patient reported outcomes (PRO) can be assessed more objectively either by adopting existing questionnaires such as SF-36 or WHOQOL-100. Ideally, we would need to measure outcomes that could assess both HIV and TB associated symptoms – instruments for these two conditions, MOS-HIV and TBscore, were developed by Wu et al., and Wejse et al., respectively (Grossman et al., 2003; Wejse et al., 2008). Such tools will help us to have better understanding of QOL parameters in dually infected patients and will certainly add more substance to our study.
We do have government-supplied free TB drugs for all patients but at terminal stage they are not very effective and many patients default treatment due to adverse effects and lack of progress. Dzherelo is not very expensive but our resources are limited to afford covering every terminally-ill patient. During other clinical trials in non-terminal categories of TB patients we had free supplies of Dzherelo from the manufacturer – Ekomed LLC (Chechitiany et al., 2007; Prihoda et al., 2007; Nikolaeva et al., 2008; Nikolaeva et al., 2008; Prihoda et al., 2008). Even though Ekomed has been quite generous it is difficult to expect that a commercial company will be willing to support all endstage patients hospitalized in the dispensary.
There is an urgent need to expand treatment options for terminally- ill TB patients with or without HIV (Cain et al., 2007; Ngo et al.,2007; Swaminathan et al., 2008; Saraceni et al., 2008; Metadilogkul et al., 2005). Our preliminary findings are encouraging but we need to carry out larger study to confirm them. This may allow us to persuade the public opinion and health authorities that an herbal supplement is worth being considered as an integral part of centralized free TB drugs supplies. As a result lives of many thousands TB patients may be saved in Ukraine and elsewhere. Our dream of helping terminally-ill people to improve quality of life and commute their death sentence will be then materialized.

Acknowledgements


This study was supported by compassionate financial support graciously provided by MAPI Research Trust, Lyon, France - a non-profit organization that advances the art and the use of scientific approaches to patient-reported outcome measures.

References


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Effect of solid formulation of Immunoxel (Dzherelo) as an adjuvant immunotherapy in treatment of ТВ and ТВ/HIV co-infected patients

Olga V. Arjanova -1, Nathalia D. Prihoda -1, Larisa V. Yurchenko -1, Nina I. Sokolenko -1, Lyudmila A. Vihrova - 2, Volodymyr S. Pylypchuk - 3, Valery M. Frolov - 4, Galyna A. Kutsyna - 4*
1-Lisichansk Tuberculosis Dispensary, Lisichansk, Ukraine; 2-Lisichansk Regional Hospital, Lisichansk, Ukraine; 3-Ekomed LLC, Kyiv, Ukraine; *4-Luhansk Regional AIDS Center and Luhansk State Medical University, Luhansk, Ukraine

ТВ and ТВ/HIV co-infected patients received either first-line anti-TB therapy (ATT; Arm A; N=20) or ATT + Immunoxel (Arm B; N=19) - an oral immunomodulator used as an adjunct therapy. In previous studies Immunoxel was formulated as alcohol-water solution of medicinal herbs. In this study a new solid, sublingual formulation of Immunoxel was tested to determine whether it will be as effective as liquid formula. At the end of one-month of follow-up, 17 (89.5%) versus 2 (10.5%) patients on Dzherelo cleared Mycobacterium tuberculosis in sputum samples, whereas only 7 (35%) vs 13 (65%) had conversion (p=0.0006). In ATT arm the body weight at baseline was 62.6±8.3 kg, after one month they have gained 1.1 kg (P=0.03). The mean starting weight in Immunoxel arm В was 54.1±7.5 kg, the immunotherapeutic intervention helped them to gain 2.8kg (p=0.0000004). The blood analysis revealed that hemoglobin (Hb) had increased from 116.1 to 119 (+2.95; P=0.03) in arm A, whereas in those who received Immunoxel Hb rose from 117.3 to 127.7 (+10.4; P=0.00007). Erythrocyte sedimentation rate in arms A and В decreased from 19.1 to 13.1 (P=0.00001) and 20.5 to 10.5 (P=0.0003). The leukocyte count decreased from 10.8 to 9 (P=0.0007) and 9.5 to 5.8 (P=0.00005). Clinical improvement was seen in 7 out 20 and 18 out 19 in arms A and В respectively. In each arm one patient died at the end of follow-up. The arm A and arm В had 7 and 2 patients co-infected with HIV who seemed to respond to therapy at the same rate as ТВ patients. These findings indicate that solid formulation of Dzherelo is equally effective as liquid formulation and contributes positively to the clinical efficacy of tuberculosis drugs.

INTRODUCTION

We and our clinical collaborators in Ukraine have worked on immunotherapy of ТВ over past 10 years.
We have used locally-produced, plant-derived immunomodulator Dzherelo (Immunoxel) and have reported encouraging results and published the data in a dozen of Russian- and English-language medical journals [1-7].
However, in previous studies Immunoxel - an oral immunomodulator used as an adjunct to ТВ therapy - was formulated as alcohol-water extract of medicinal herbs.
In this study a solid, sublingual, sugar-based formulation of Immunoxel was tested to determine whether it will be as effective as liquid formula.

MATERIALS AND METHODS

The study was conducted at the Lisichansk Regional ТВ Dispensary which administers the anti-TB therapy to 600-800 inpatients annually.
The main criteria for the eligibility were an informed consent and positive sputum smear.
Patients were randomly selected among those who were admitted to a special ward in which difficuIt-to-treat cases are managed.

Over half of patients in this facility require individualized treatment rather than WHO-recommended standard ATT regimen.
All patients were bed-ridden and had fever, cough and other common ТВ symptoms such as chest pain, dyspnea, hemoptysis, weight loss and anorexia.
The patients' age, gender, body weight, severity and form of the disease were matched in order to minimize the statistical bias due to population heterogeneity.
The anti-TB drugs were procured free-of-charge from the national supply system administered by the Ministry of Health of Ukraine.
The supplies of Immunoxel and placebo pills were provided by Ekomed company.
Standard ТВ therapy consisted of daily doses of Isoniazid (H) 300 mg; Rifampicin (R) 600 mg; Pyrazinamide (Z) 2,000 mg; Streptomycin (S) 1,000 mg; and Ethambutol (E) 1,200 mg. Individualized therapy comprised first- and second-line anti-TB drugs as decided by a physician prior to or after results of drug susceptibility tests.
The study lasted one month and primary end-point sputum conversion was evaluated at the end of study.

RESULTS

At the end of one-month of follow-up, 2 (10.5%) vs 17 (89.5%) patients had cleared M. tuberculosis in sputum samples and 7 (35%) vs 13 (65%) had culture conversion (P=0.0006) in arms A and В respectively.
In ATT arm the body weight at baseline was 62.6±8.3 kg, after one month the average gain was 1.1 kg (P=0.03).
The mean starting weight in Immunoxel arm was 54.1 ±7.5 kg, the immunotherapeutic intervention helped them to gain 2.8 kg (P=0.0000004).
The hemoglobin (Hb) had increased by 2.95 g/L from 116.1 to 119 (P=0.03) in arm A, whereas those who received Immunoxel had Hb risen by 10.4 g/L from 117.3 to 127.7 (P=0.00007). 
Erythrocyte sedimentation rate in arms A and В decreased
from 19.1 to 13.1 (P=0.00001) and 20.5 to 10.5 (P=0.0003)
respectively.  The leukocyte count decreased from
10.8 to 9 x109 (P=0.0007) and 9.5 to 5.8 x109 (P=0.00005).
Clinical improvement was seen in 7 out 20 and 18 out 19 patients in arms A and B. In each arm one patient died at the end of follow-up.
The arms A and В had 7 and 2 patients co-infected with HIV who seemed to respond to therapy at the same rate as ТВ patients.
Other solid formulations of Immunoxel are now being tested and results will be available in near future.

CONCLUSIONS                                                                      

  One pill of Immunoxel given once-per-day is safe; has not produced any adverse effects.
Administration of Immunoxel with 1st- or 2nd-line ТВ drugs resulted in clearance of M. tuberculosis in sputum smears of 89.5% patients vs. 10.5% among placebo recipients.
*  Sputum conversion occurred very fast - only 1 month treatment was needed.
* No difference was seen when 1st diagnosed ТВ was compared to re-treated ТВ, MDR-TB or HIV-TB - the proportion of converted patients and time to conversion were identical.
* Immunoxel reversed ТВ-associated wasting; average weight gain was 2.8 kg vs. 1.1 kg in placebo.
* Immunoxel eliminated ТВ-associated fever.
* Immunoxel demonstrated anti-inflammatory effect. Erythrocyte sedimentation rate and leukocyte counts reverted back to normal.
* Immunoxel is affordable; easy to administer; stable at room temperature, without need for cold-chains; and is made from readily available natural source.

REFERENCES

1 Prihoda N.D., Arjanova O.V., Yurchenko L.V, Sokolenko N.I., Vihrova L.A., Pylypchuk VS., Kutsyna G.A.: Open label trial of adjuvant immunotherapy with Dzherelo, Svitanok and Lizorm, in MDR-TB, XDR-TB and ТВ/HIV co-infected patients receiving anti-tuberculosis therapy under DOT. J. Med. Plant Res. 1, 117-122 (2007).
2.  Nikolaeva L.G., MaystatT.V, Pylypchuk VS., Volyanskii Yu.L., Masyuk L.A., Kutsyna G.A.: Effect of oral immunomodulator Dzherelo (Immunoxel) in TBI HIV co-infected patients receiving anti-tuberculosis therapy under DOTS. Intl. Immunopharmacol. 8, 845-851 (2008).
3.    Prihoda N.D., Arjanova O.V., Yurchenko L.V, Sokolenko N.I.,
Vihrova L.A., Pylypchuk VS., Kutsyna G.A.: Adjuvant immunotherapy of tuberculosis in drug-resistant ТВ
and ТВ/HIV co-infected patients. Intl. J. Biomed. Pharm. Sci. 2, 59-64 (2008).
4. Arjanova O.V, Prihoda N.D., Yurchenko L.V,
Sokolenko N.I., Vihrova L.A., Pylypchuk VS., Frolov V.M.,
Kutsyna G.A.: Enhancement of efficacy of tuberculosis drugs with Immunoxel (Dzherelo) in HIV-infected patients with active pulmonary tuberculosis. Immunotherapy 1, 549-556 (2009).
5.  Zaitzeva S.I., Matveeva S.L., Gerasimova T.G., Pashkov Yu.N.,
Butov D.A., Pylypchuk VS., Frolov V.M., Kutsyna G.A.: Efficacy and safety of phytoconcentrate Dzherelo (Immunoxel) in treatment of patients with multi-drug resistant
ТВ (MDR-TB) in comparison to standard chemotherapy. Res. J. Med. Sci. 3, 36-41 (2009).
6. Zaitzeva S.I., Matveeva S.L, Gerasimova T.G., Pashkov Yu.N., Butov D.A., Pylypchuk VS., Frolov V.M., Kutsyna G.A.: Treatment of cavitary and infiltrating pulmonary tuberculosis with and without the immunomodulator Dzherelo. Clin. Microbiol. Infect. 15, 1154-1162 (2009).
7.  Arjanova O.V, Prihoda N.D., Sokolenko N.I., Yurchenko L.V,
Vihrova L.A., Pylypchuk VS., Frolov V.M., Kutsyna G.A.: Impact of adjunct immunotherapy with multi-herbal supplement Dzherelo (Immunoxel) on treatment outcomes in end-stage
ТВ/HIV patients. J. Antivir. Antiretrovir. 1, 86-88 (2009).
8.  Prihoda N.D, Arjanova O.V, Yurchenko L.V, Sokolenko N.I., Vihrova L.A., Pylypchuk VS., Frolov V.M., Kutsyna G.A.: Adjuvant immunotherapy of extensively drug-resistant tuberculosis (XDR-TB) in Ukraine. Curr. Res. ТВ 1,1-6 (2009).

ACKNOWLEDGEMENTS

This ongoing study is supported by grant No. UKB1-9017-LK-09 from the U.S. Civilian Research & Development Foundation (CRDF) - a non-profit organization authorized by the U.S. Congress and established in 1995 by the National Science Foundation.
The authors have obtained the Institutional Review Board approval of Lisichansk ТВ Dispensary and have followed the principles of the Declaration of Helsinki for all human or animal experimental investigations.
This study is registered with the Clinical Trials.gov under identifier NCT01061593.

Comparative efficacy of tableted form of Immunoxel vs. control on sputum conversion rate (%) after 1 month of treatment

Immunoxel
80,3
87,5
100
89,5

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