Therapy for Tuberculosis: M. vaccae Inclusion into Routine Treatment

Tuberculosis (TB) – an infectious airborne disease –is a re-emerging major global health problem. Each year, there are around nine million new cases of TB, and close to two million deaths among 14 million persons with active clinical disease. All countries are affected, but 85% of cases occur in Africa (30%) and Asia (55%), of which India and China alone represent 35% (World Health Organization, 2011).

This adaptation creates the basis of clinical latency in tuberculosis.Although these sequestered, semidormant bacilli have been much investigated, their paucity makes direct studies in vivo problematic, and multiple researches on this question have been performed such as in vitro oxygen deprivation or intracellular growth in macrophages (Wallis, R. S., 2005).
M. tuberculosis is an atypical member of its genus (Stanford, J. L.;Bahr, G. M.;Rook, G. A. W.;Shaaban, M. A.;Chugh, T.D.;Gabriel, M.;Al-Shimali, B.;Siddiqui, Z.;Ghardanis, F.;Shahin, A. & Behbehani, K., 1990).Apparently the capacity of M. tuberculosis to cause illness is due not only to the severity of the damage it causes to the host tissue but also to its aptitude to alter the immune response, to one that is inappropriate.It is evident that new alternative and improved treatment options are needed.In consequence, more efficient resources were considered crucial to improve the employed chemotherapy.Significant efforts have been directed at finding new drugs and vaccines against TB.(Small, P. M., 2009).Thus, the immunomodulatory effects of a heat killed Mycobacterium vaccae (M.vaccae, Mv) preparation have been investigated by Stanford, J. et. al. during the 1970´s.It has been stated that the variation of disease expressions and severity was entirely inherent in the host and his surroundings, disease depending on human genetic control of the immunological response in interaction with environmental factors rather than to bacterial features.In the environment a free-living mycobacterium, the potentially beneficial M. vaccae was recognized as an important source for influencing the human immune response (Stanford, J.L. & Paul, R. C., 1973;Stanford, J. L. & Rook, G. A.W., 1983).

Clinical trials of adjunctive immunotherapy
The concept of immunotherapy in tuberculosis is not new and many immune based interventions have been investigated as adjuncts to convenional chemotherapy.It is evident that the modulation of immune reactivity can be of great therapeutic value.

IFN-
As IFN- is central to antimycobacterial host defenses; it has been used in several clinical trials of adjunctive immunotherapy.In mice, IFN-enhances the mycobactericidal capacity of macrophages by increasing the production of reactive nitrogen intermediates, such as nitric oxide.Condos et al. reported in 1997 the first study of therapeutic IFN- in patients with tuberculosis without evident defects on IFN- production or responsiveness.In this investigation 500 g of IFN- was administered 3 times per week by aerosol to 5 patients with MDR tuberculosis together with their previous therapy.The study found that sputum smear results became negative and the number of colony-forming units (CFU) tended to fall.Three similar successive studies performed by other investigators showed that differed in IFN- type, dose, and route of administration were not successful in inducing any hopeful results.The only randomized, placebo-controlled, multicenter trial of inhaled adjunctive IFN- for MDR tuberculosis was done by InterMune in 2000, and the trial was stopped because of a lack of efficacy and the data obtained have never been published.Subsequent investigations have indicated that IFN--induced genes, such as IP-10 and iNOS, are already upregulated in the lung in patients with tuberculosis and that therapeutic aerosol IFN- has a relatively minor additional effect.These findings indicate that the fairly A.; Phillips, N. B.; Phillips, M.; Averill, L. E.; Daniel, T. M. & Ellner, J. J., 1996; Wallis, R. S., 2005).Levels subsequently decrease as the bacillary burden is diminished by treatment (Ribeiro-Rodrigues, R.; Resende Co, T.; Johnson, J. L.; Ribeiro, F.; Palaci, M.; Sá, R. T.; Maciel, E. L.; Pereira Lima, F. E.; Dettoni, V.; Toossi, Z.; Boom, W. H.; Dietze, R.; Ellner, J. J. & Hirsch, C. S., 2002;Wallis, R. S., 2005).It was shown in experimental animals that neutralization of TNF- interferes with the early recruitment of inflammatory cells to the site of M. tuberculosis infection and inhibits granulomas formation (Kindler, V.; Sappino, A. P.; Grau, G. E.; Piguet, P. F. & Vassalli, P., 1989; Algood, H. M.; Lin, P. L.; Yankura, D.; Jones, A.;Chan, J. & Flynn, J. L., 2004;Wallis, R. S., 2005), and TNF- blockade also reduces the microbicidal activity of macrophages and natural killer (NK) cells (Roach, D. R.;Bean, A. G.;Demangel, C.;France, M. P.;Briscoe, H. & Britton, W. J., 2002;Hirsch, C. S.;Ellner, J. J., Russell, D. G. & Rich, E. A., 1994;Wallis, R. S., 2005).
The effects of potent immunosuppressive and/or anti-TNF- therapies on microbiologic outcomes in tuberculosis have been investigated in two controlled clinical trials.Both were conducted with HIV-1-infected patients who had relatively well -preserved tuberculosis immune responses (based on the presence of high CD4 cell sum and cavitary lung disease).The studies shared a single placebo control arm (for tuberculosis therapy only).Their major aim was to examine the role of TNF- in the HIV disease progression due to tuberculosis; as such, their main end points were CD4 cell count and plasma HIV RNA load.Nevertheless, both studies prospectively accrue clinical and microbiologic data as indicators of safety.

High-dose methylprednisolone:
In a comparative study was reported (Mayanja-Kizza, H.; Jones-Lopez, E.; Okwera, A.; Wallis, R. S.; Ellner, J. J.; Mugerwa, R. D.; Whalen, C. C. & Uganda-Case Western Research Collaboration, 2005;Wallis, R. S., 2005) in which 189 subjects received either prednisolone (2.75 mg/kg/day) or placebo during the first month of conventional anti-TB therapy.The prednisolone dosage was selected on the basis of a phase I study indicating that it reduced the rate of tuberculosis-stimulated TNF- production ex vivo by one-half.During the second month, the daily dose was reduced to 0 mg/kg; the average subject received a cumulative dose of 16500 mg.Though there is extensive experience with the use of corticosteroids to diminish tuberculosis symptoms, no previous studies have examined the microbiologic effects of doses of this magnitude.Unexpectedly, one-half of prednisolone-treated subjects had conversion of sputum culture results to negative after 1 month of treatment, compared with 10% of subjects in the placebo arm (P<0.001).This effect was bigger than that observed in the landmark study in which the addition of rifampin to a 6-month regimen of streptomycin and isoniazid reduced the relapse rate from 29% to 2% and increased the 2-month sputum culture conversion rate from 49% to 69% (East African-British Medical Research Councils, 1974;Wallis, R. S., 2005).The effect of prednisolone therapy was not due to reduced sputum production, which decreased similarly during treatment in both study arms.There were no serious opportunistic infections.However, prednisolone-treated subjects were more likely to experience other early serious adverse events, including edema, hyperglycemia, electrolyte disturbances, and severe hypertension.
There have been no reports of deleterious effects of corticosteroids on microbiologic outcomes in patients withTB.
Early studies of immunotherapy for TB were those of Robert Koch who used injections of "old tuberculin" during the last 10 years of the 19 th century (Koch, R., (a) 1890; Koch, R., (b) 1890).
In the early 20 th century, Charles Stevens developed "Stevens cure" based on a root called Umckaloabo from South Africa (Sechehaye, A., 1920), recently shown to have potent antimycobacterial activity (Seidel, V. & Taylor, P. W., 2004; Kim, C. E.; Griffiths, W. J. & Taylor, P. W., 2009) and particularly to act as a TNF- antagonist.In 1904 Friedrich Friedmann developed a turtle tubercle suspension of live Mycobacterium chelonae, which he later called "Anningzochin" which was available until recently from Laves-Arzneimittel GmbH, Barbarastr.14, A-30952, Ronnenberg, Germany (Friedmann, F., 1904;Hart, C. A.;Beeching, N. J. & Duerden, B. I., 1996;Rosenau, M. J.& Anderson, J., 1915).Although Friedman investigated this mycobacterium species and showed that it was able of confer immunity against tuberculosis, he never considered that it might cause a limited tuberculous process.In the 1920s and 30s, Henry Spahlinger developed a serum from horses immunized with various extracts of tubercle bacilli (Spahlinger, H.;Macassey, L. & Saleeby, C. W., 1934).Even though many investigations supported the success of these different preparations in the treatment of tuberculosis, until very recently immunotherapy has not contributed significantly to its treatment (Sechehaye, A., 1920).

Immunomodulatory therapy in tuberculosis
Two problems confronted the early attempts of immunotherapy for tuberculosis.First, in the absence of drugs, the immunotherapy was directed towards the total destruction of the tubercle bacillus in the host.Secondly it was then thought that the triggering of immune reactivity in tuberculosis was synonymous with protection.The concept of immune reactivity in mycobacterial infections embraces both protective immunity and also tissue destruction.Distinguishing between them has been a controversial topic for many years (Stanford, J. L. & Rook, G. A.W., 1983).During the last decades it was resolved by the demonstration of two functional subpopulations of helper T cells -TH1 and TH2 (Flynn, J. L. & Ernst, J. D., 2000).
Immunotherapy, is directed to replace an inadequate immune reaction by an appropriate one.The keys to reaching success for immunotherapy arise from the evidence of the considerable variation in the efficacy of vaccination with BCG from one country to another.This is due to prior contact with environmental mycobacteria, which, depending on species, could provide some degree of protection or the antagonistic reaction of tissue necrosis.
Although the search for new vaccines and immunotherapies should continue, investigation of those already available to us is important and is the purpose of our investigations.
For many years it has been accepted that variation in clinical presentation and severity entirely rested in the host and his environment, disease depending on an interaction between human genetic control of the immunological response influenced by environmental factors.In the environment are the free-living mycobacteria and it was from amongst them that the potentially beneficial M. vaccae and the deleterious M. scrofulaceum were identified as important factors influencing the human immune response (Stanford, J. L. & Paul, R. C., 1973;Stanford, J. L. & Rook, G. A.W., 1983).It is now established that genetic diversity within Mtb, expressing significant phenotypic differences between clinical isolates, may also be important (Flynn, J. L. & Ernst, J. D., 2000).
BCG is commonly referred to as a vaccine but its effects are very different from those of other vaccines and it is better designated as an immune modulator influencing susceptibility to leprosy (Truoc, L. V.; Ly, H. M.; Thuy, N. K.; Trach, D. D.; Stanford, C. A. & Stanford, J. L., 2001) and malignant melanoma (Grange, J. M.; Stanford, J. L.; Stanford, C. A. & Kölmel, K. F., 2003) as well as tuberculosis.Indeed the concept of a vaccine in its commonly used sense against tuberculosis is a difficult one as illustrated by the difficulty in interpreting the Tuberculin test.A positive Tuberculin test can signify protection, susceptibility and the presence of disease (Stanford, J. L. & Lemma, E., 1983), thus attempting to vaccinate using the species-specific, group iv antigens of Mtb (Stanford, J.;Stanford, C.;Stansby, G.;Bottasso, O.;Bahr, G. & Grange, J., 2009) is unlikely to be successful.

Mycobacterium vaccae -a part of our environment
The idea of using a saprophytic mycobacterium that causes no harm, has few side effects and is unable to induce adverse reactions in patients, as a potential immunotherapeutic or vaccine has only been considered during the last few years.Mycobacterium vaccae (NCTC 11,659), is a rapidly growing scotocromogenic organism.
First isolated in Germany from the surroundings of cattle, the potential and the importance of the species was first appreciated from field studies in Uganda.A killed suspension of this strain was first added to BCG and investigated as a combined vaccine.Later it was recognized as an immunotherapeutic agent.Immunotherapy with M. vaccae improves immune recognition of common mycobacterial antigens and also regulates immune reactions away from necrotic processes.The re-introduction of cellular responsivness to common mycobacterial antigens indicates that M. vaccae should induce protective immunity and suppress antagonostic responses.Looked at in the opposite way, failure to make a response to common mycobacterial antigens is an attribute of diseases that should be responsive to treatment with heat-killed M. vaccae.

M. vaccae, its adjuvants
The cell walls of all mycobacteria possess potent adjuvant activity attributed to structural lipids and glyco-lipids.
The actions of these adjuvants vary between species.Thus BCG and most species of mycobacteria enhance the type of immune response for which the recipient is already primed, whereas M. vaccae and probably a small number of other Actinomycetales enhance the most beneficial cellular immune responses.

M. vaccae, its antigens
M. vaccae possesses the group i antigens shared by all mycobacteria and most other aerobic genera of the Actinomycetales.Some of these antigens are partially cross-reactive with those expressed by mitochondria, when stressed, in animal tissues.
M. vaccae lacks the groups ii and iii antigens, and the group iv antigens of pathogenic mycobacterial species.
All the information obtained from several studies performed in countries around the world, from minor investigations to those made using a placebo control and a properly randomized trial, show that increased cure rates in newly diagnosed TB patients receiving M. vaccae is only associated with minimal side effects.Studies of immunotherapy with M. vaccae in drug-resitant, relapsed and chronic TB Patients have shown that it is also favorable under these conditions.The effects are more readily seen when specific chemotherapy is difficult to establish or ineffective because of low patient compliance, or resistance to multiple drugs.
Progress was suggested from the early work with irradiation-killed organisms in leprosy to the study in London of modulation of tuberculin skin-test responses, and the first comparative trials in The Gambia and Kuwait.In these successive investigations the dose of 10 9 heat-killed organisms, equivalent to 1 mg wet-weight of bacilli, has been used as a standard dose.A series of small trials in Argentina, India, Nigeria, Romania, South Africa, Uganda and Vietnam have shown that the method can be effective across wide-ranging geographic variability, with South Africa as the only country where almost no effects were recorded (Dlugovitzky, D.;Stanford, C. & Stanford, J., 2011).
Despite this wide geographical efficacy, it is likely that the schedule of treatment with M. vaccae should change with different environments.Thus single doses were effective in the Gambia, Nigeria, Kuwait, Romania and the UK, but further South in Africa the environment may necessitate multiple doses, just as some diseases such as cancer require repeated doses to overcome the drive towards Th2 exerted by the tumour.
Numerous studies have shown that certain patterns of cellular immunity are associated with active disease and others are associated with health and recovery from disease.Modulating the immune response from the one to the other is now possible with M. vaccae and this chapter records its successful achievement (Ottenhoff,

Our initial studies on immune response against M. tuberculosis
The purpose of the early series of studies that we have carried out to investigate the immune response of patients with pulmonary tuberculosis has been to make steps towards immunotherapy as an effective addition to standard short-course chemotherapy and to identify proper in vitro alternative markers of successful treatment for its evaluation.A good deal of the immunological work on TB has been done on murine models -animals that have www.intechopen.comUnderstanding Tuberculosis -Analyzing the Origin of Mycobacterium Tuberculosis Pathogenicity 482 short lives and do not normally suffer from this disease.And in consequence we wanted to make use of appropriate methods for and related to human patients.Initial studies in our laboratory in Rosario, Argentina, have shown that the changes in cellular immune response in pulmonary tuberculosis patients are related to the severity of disease and to the administration of tuberculosis chemotherapy.We showed that increased levels of IL-8 in the pleural exudates of patients with pulmonary tuberculosis, in comparison with those patients with pneumonia-associated pleural effusions, was associated with different levels of expression of CD3, CD4, CD19, CD25 and CD68 markers on their cells (Dlugovitzky, D.; Rateni, L.; Torres-Morales, A.; Ruiz-Silva, J.; Piñesky, R.; Canosa, B.; Molteni, O. & Bottasso, O., 1997; Caruso, A. M.; Serbina, N.; Klein, E.; Triebold, K.; Bloom, B. R. & Flynn, J. L., 1999).This data suggested that increased IL-8 levels in pleural effusions plays a key role in initiation and maintenance of inflammatory reactions.
Patients with moderate to severe pulmonary tuberculosis showed a marked and significant decrease in their circulating levels of cells bearing these phenotypes when compared with those of healthy persons, with patients with pneumonia-associated pleural effusions or with patients with mild pulmonary tuberculosis.Differences between the levels of these cell markers on pleural and peripheral T-cells from pulmonary tuberculosis patients may be the consequence of an incursion of T-lymphocytes from the circulatory system to the pleural cavity, probably linked to the presence of chemokines within the pleural fluid including IL-8 (Fulton, S.A.; Reba, S. M.; Martin, T.D. & Boom, W. H., 2002).
In other assays in pulmonary tuberculosis, circulating immune complexes and the main peripheral blood T-cell subsets were evaluated (Dlugovitzky, D.; Luchesi, S.; Torres-Morales, A.; Ruiz-Silva, J.; Canosa, B.; Valentini, E. & Bottasso, O., 1995).This showed that immune complex levels in cases with severe disease are significantly higher, and expression of CD4 on T lymphocytes significantly lower than in cases of mild disease (Fiorenza, G.; Farroni, M. A.; Bogué, C.; Selenscig, D.; Martinel Lamas, D. & Dlugovitzky, D., 2007).Diverse studies of our group helped to explain the effective cellular immune response detected in less severe tuberculosis cases and simultaneously, the impaired cell-mediated immune response in severe cases.Several immune mechanisms within cell-mediated immunity generate a multifaceted response involving activated macrophages, T cells, and cytokines directed to manage mycobacterial infection.Other cell populations also take part in the immune response against mycobacteria and may be important in the development of the disease ( Polymorphonuclear neutrophils (PMN) are the professional phagocytes first at the site of bacterial invasion and are able to play a protective role in opposition to M. tuberculosis in the early phase of infection controlled by T lymphocytes.Although recruitment of neutrophils to bronchoalveolar spaces has been described during active human tuberculosis and associated with local chemokine expression, it has not been clarified whether neutrophils have direct bactericidal or immunologic functions.In vitro studies suggest that human neutrophils are mycobacteriocidal and are activated by soluble mycobacterial antigens (Fiorenza, G.;Bottasso, O. A.;Rateni, L.;Farroni, M. A. & Dlugovitzky, D., 2003).
Several mechanisms including phagocytosis of bacteria and the subsequent generation of reactive oxygen intermediates during oxidative bursts are considered important instruments for destruction of mycobacteria (Jones, G. S.; Amirault, H. J. & Andersen, B. R., 1990).Several findings demonstrated a significant alteration in PMN functions in pulmonary tuberculosis.Production of reactive oxygen intermediates was reduced in severe disease and was significantly increased by antituberculosis chemotherapy (Denis, M. J., 1991).Recognition of Mtb by phagocytic cells leads to cell activation and production of cytokines, which in itself leads to further activation and cytokine production in a complex process of regulation and cross-regulation (Denis, M. J., 1991).Thus phagocytic cells are thought to contribute to the control of infection through the production of chemokines (Appelberg, R.; Castro, A. G.; Gomes, S.; Pedrosa, J. & Silva, M. T., 1995), the induction of granuloma formation (Riedel, D. D. & Kaufmann, S. H., 1997) and the transference of their own microbicidal molecules to infected macrophages (Ehlers, S., 2003).Levels of circulating cytokines correlate significantly with the severity of the disease, antibody concentration and the reduction of Th1 activities.We evaluated plasma cytokines of type-1 and type-2 in relation to humoral and cell-mediated responses in patients with different amounts of lung damage and with different clinical symptoms of tuberculosis.We found that patients with pulmonary tuberculosis of different levels of severity have higher serum levels of IFN-, IL-2, IL-4 and IL-10 when compared with those of healthy controls.Mean titers of IFN-, and IL-2, in mild and moderate patients were found to be greater than in those with severe disease, whereas moderate and advanced patients showed higher levels of IL-4 in comparison with mild cases.Raised levels of interleukin-10 were more prevalent in advanced disease, and statistically different from those in patients with mild disease.This cytokine pattern would explain the effective cellular immune responses found in patients with less severe tuberculosis in comparison with those of patients with advanced disease in whom cellular immunity is seriously damaged (Dlugovitzky, D.; Luchesi, S.; Torres-Morales, A.; Ruiz-Silva, J.; Canosa, B.; Valentini, E. & Bottasso, O., 1995).
We investigated the relationship between the competence of lymphocytes to proliferate and induce cytokine synthesis in vitro, in response to stimulation with antigens, and the amount of pulmonary involvement in tuberculosis patients.Higher levels of IFN- compared with IL-4 in culture supernatants of Peripheral Blood Monuclear Cells (PBMC) stimulated with Mtb antigens were observed in patients with mild tuberculosis (Bay, M. L.; Dlugovitzky, D.; Urízar, L., 1997).To amplify these results we assessed in vitro the synthesis of the cytokinestransforming growth factor beta (TGF-) and IL-1.Reduced concentrations of IFN- and IL-4 and an increased synthesis of TGF- were observed in patients with moderate tuberculosis in comparison with those with mild disease.

www.intechopen.com
The immune system generally responds in a regulated way to microbes and eliminates them, but it does not respond to self-antigens unless regulatory mechanisms are impaired and unresponsiveness or tolerance to self-antigens is not maintained (Van Parijs, L. & Abbas, A. K., 1998).Such a disharmonic immune response may result in several autoimmune diseases.The altered Th1 and Th2 expression found in severe tuberculosis patients may lay them open to such diseases.To investigate this we inquired into the incidence of arthritic manifestations (Poncet's disease) in such patients.The kinds and distribution of T cell subsets in these cases and the presence of several auto-antibodies were also investigated.In the detected arthritic cases an augmented number of CD4+ Tcells was observed in comparison with CD8+ T cells and autoantibodies were detected.However, we could not rule out the presence of unknown factors that might be partly responsible for the reactive arthritis.(Dlugovitzky, D.; , 2000).
Several studies have established that continuous IL-12 production is necessary for maintenance of the pulmonary Th1 cells required for host control of persistent Mtb infection and suggest that breakdown of this mechanism could be a contributing factor in the reactivation of disease (Feng, C. G.;Jankovic, D.;Kullberg, M.;Cheever, A.;Scanga, C. A.;Hieny, S.;Caspar, P.;Yap, G. S. & Sher, A., 2005).
Our results suggest that the synthesis of nitric oxide by the host is not always associated with a favourable evolution since higher levels are synthesized in cases with severe tuberculosis.Other authors propose that this event may be related to the interaction of several cytokines and/or eicosanoids through disease related induction of immune reactions (Tunçtan, B.;Okur, H.;Calişir, C. H.;Abacioğlu, H.;Cakici, I.;Kanzik, I. & Abacioğlu, N., 1998).It has also been shown that an inverse correlation exists between TNF-, TGF- and NO concentrations in serum, behavior that could be a predominantly TGF- effect (Fiorenza, G.;Rateni, L.;Farroni, M A.;Bogué, C. & Dlugovitzky, D. G., 2005).
The production of NO, TNF- and IL-12 by the peripheral blood monocytes of patients suffering from MDR-TB has been investigated by others and NO production was found to be significantly depressed.A sub-cellular fraction of Mtb whole cell lysate, culture filtrate protein or lipoarabinomannan induced higher concentrations of NO to be released by peripheral blood monocytes from newly diagnosed tuberculosis patients in comparison with those from MDR-TB patients (Sharma, S.; Sharma, M.; Roy, S.; Kumar, P. & Bose, M., 2004).

Respiratory diseases treated with M. vaccae to date have been:
Pulmonary tuberculosis, Bronchial aspects of hay-fever, Bronchial asthma, Lung cancer Related conditions under investigation Chronic obstructive pulmonary disease (COPD) in man and recurrent airway obstruction (RAO) in horses.
Arterial disease.Myocarditis.(These are being investigated with related bacterial immuno-modulators)

Salient results of immunotherapy studies in treatment of tuberculosis
In a preliminary study conducted some years ago in Carrasco Hospital, 14 pulmonary tuberculosis patients receiving heat-killed, borate-buffered M. vaccae (SRL172) had a better outcome than did 7 patients who received placebo (Vacirca, A.; Dominino, J. C.; Valentín, E; Bottasso, O. & Stanford, J., 1993.Subsequently we have carried out three small studies of this immunotherapy.All were performed in newly diagnosed, moderate to severe, pulmonary tuberculosis patients.In the first of these, the effects of a single dose given by intradermal injection was monitored to evaluate the potential of the approach and assess the value of the selected investigations.Levels of IFN- rose and TNF- fell, with decreases also in levels of IL-4, IL-10 and anti-hsp 70 kDa (Dlugovitzky, D.; Bottasso, O.; Dominino, J. C.; Valentini, E.; Hartopp, R.; Singh, M.; Stanford, C. & Stanford, J., 1999) (Table 1).From subsequent researches performed by our group, we concluded that immunotherapy with M. vaccae promotes changes in the immune response and improves patient recovery.Respiratory Burst expression (Fig. 3) increased in the successives samples of intradermal and oral M. vaccae treated TBP, and it was higher in those patients receiving oral M. vaccae then in those receiving intradermal Mv in relation to placebo recipients.
IFN-(Fig. 4), TNF-(Fig.5), IL-6 and IL-10 (Fig. 6) levels in PBMC and PMN culture supernatants.and IL-6, IL-10 (Fig. 7) and TNF- values in plasma (Fig. 2) also increased more in those receiving oral M. vaccae than in intradermal M. vaccae recipients.The immunomodulatory effect of both oral M. vaccae and intradermal.M. vaccae treatments was shown both by Respiratory Burst expression and cytokine increase in culture supernatants and plasma, with oral therapy the more effective.The data obtained with all 3 immunotherapy regimens produced significantly better results than those achieved with chemotherapy alone.

Respiratory burst of PBMNC
The data show that the addition of oral capsules of M. vaccae to a DOTS program in the treatment of drug-sensitive tuberculosis would have the clinical advantages of hastening sputum negativity and recovery from the disease.Such a strategy would reduce new infections, both among contacts and in the community at large and might allow shortening of the treatment period.& Rook, G. A., 2008) act to direct the modulated response to the sites of expression of host-cell stress proteins (Matzinger, P., 1994).This may be especially to those stress proteins of mitochondrial origin showing homologies with the common antigens of mycobacteria (  1996) of the intestine play a part analogous to that of the dermal dendritic cells in the skin.

IL-10 levels in plasma
In addition to the reported immunological results, the bacteriological findings indicated that the conversion to negative of both sputum smear and culture was significantly enhanced by injected or oral immunotherapy with M. vaccae above that achieved by chemotherapyalone.
Although this study deals with drug-sensitive tuberculosis, the reported immunological changes, which are paralleled in both injected and oral studies, allow confidence that the oral formulation will prove of similar efficacy in patients infected with drug-resistant bacilli (Stanford, J.  & Etemadi, A., 2001), where excellent clinical results have already been obtained in the treatment of MDR-TB.As the immunological data obtained in the oral study, albeit with a more intensive schedule, paralleled that of the intradermal study it is logical to suppose that MDR-TB could also be treated successfully with oral M. vaccae.The properly functioning immune system recognizes and regulates the appropriate response to disease and would be capable of destroying both drug-sensitive and drug-resistant organisms quite impartially.
This approach to treatment at the outset would allow initial resistance to be treated early and at the same time discourage secondary resistance due to treatment inadequacy.As an example, at the chest hospital in Ho Chi Minh City, Vietnam, 12 patients accepted for immigration into the USA were subsequently found to be infected with highly drugresistant organisms.They failed to be cured with the latest drugs provided from the USA, but following up to twelve injections of M. vaccae (administered on the initiative of the staff of the Chest Hospital) all were cured and allowed into the USA.Similar results have been obtained in several countries (Stanford, J. L.; Stanford, C. A.; Grange, J. M.; Lan, N. N. & Etemadi, A., 2001). www.intechopen.com

Conclusions of the 3 studies
The inclusion of immunotherapy with SRL-172 improved the results of DOTS chemotherapy and it led us to the conclusion that this therapy might allow a reduced period of chemotherapy without loss of efficacy and help to prevent the development of multi-drugresistance.The three small studies of immunotherapy with heat-killed, borate-buffered, M. vaccae for drug-susceptible pulmonary TB developed in the department in Medicine Faculty of Rosario have produced successful results.It was demonstrated that the transformation of a Th2 response, towards Th1, is accompanied by clinical, bacteriological and radiological improvement in the immunotherapy recipients.
The results showed that three injected doses of M. vaccae were more effective than a single dose, and that ten oral doses scattered throughout the period of chemotherapy, were as effective, or more so, than was the injected preparation.The reagent deserves formal field trials, particularly in patients infected with highly drug-resistant strains of tubercle bacilli.
In conclusion, we have found that immunotherapy with M. vaccae in TB, whether by injection or by the oral route, hastens recovery, bacteriologically, clinically and radiologically, as well as returning immune responses towards those of healthy persons.

Fig. 3 .Fig. 4 .TNFFig. 5 .ILFig. 6 .
Fig. 3. Respiratory index for polymorphonuclear cells and for mononuclear cells, calculated by dividing the mean fluorescence value for H37Rv-stimulated cells by the mean fluorescence value for unstimulated cells.