| Abstract|| |
Background : Osteoarticular tuberculosis is coming back with vengeance. Host immunity plays a major role in either containing the disease or allowing the spread. Number of resistant cases and drug defaulters are on the rise. Immunepotentiation (immunomodulation) has shown beneficial response in pulmonary tuberculosis in various studies.
Methods : This study was done to assess immune status of various categories of patients of osteoarticular tuberculosis, to modulate (alter or change) the immune system in non responder patients and to add immunomodulation therapy in some patients from the very beginning of antitubercular chemotherapy and observe their clinical response and objectively assess their immune status.
Prospective study was done in two phases involving 103 patients suffering from osteoarticular tuberculosis. In phase one 61 patients (Two Groups - Group 1 called fresh virgin/responder cases given first line antitubercular drugs (ATT) = 41 patients; Group 2 called Non responders immunomodulated with cycles of oral levamisole, BCG and DPT vaccine as an adjuvant to ATT = 20 patients) assessed for their cellular immune profile. In second phase 42 patients (In three Groups - Group 3A who received only ATT = 15 patients; Group 3B who received ATT and immunomodulation from very beginning = 15 patients; Group 4 who were non responders, put on immunomodulation after minimum three months of ATT =12 patients) were assessed for their interleukin profiles at presentation and after three months of therapy in respective groups. The immune parameters of all above mentioned patients (n=103) were correlated with the type of clinical presentation, course of disease, response to therapy and response to immunomodulation. Follow up in all the groups ranges from 24 - 49 months (mean 27.2 months).
Results : Group1 (n=41): Thirty nine out of 41 patients showed clinicoradiological response at three months of therapy. The CD4 cell counts in these patients rose to a statistically significant (p=. 000) mean of 1140±452.6 cumm indicating that tuberculosis is an important cause of this reversible CD4 T cell lymphopenia. Group 2 (n=20): Thirteen patients showed favorable clinical response to immunomodulation. The CD4 cell counts showed statistically significant (P < .001) improvement after immunomodulation.Group 3 (n=30): After three months of uninterrupted antitubercular chemotherapy in Group 3A some interleukin levels rose ( IL1, IL2, IFN); while other interleukin levels fell (IL10, IL4, IL6, IL12, IL18, TNF). In Group 3B , under the influence of immunomodulation, the rise and fall was quantitatively more than Group 3A. Twenty seven patients responded , by three months, in Group 3. Group 4 (n=12): The initial mean serum IL-1, IL-2 , IFN - r levels were low in these indicating a low beneficial cellular immune status (Th1 cell activity) while initial serum IL-10, IL-4, IL-6, IL-12, IL-18, TNF levels were high in these patients. Under the influence of ATT and immunomodulation, 10 patients showed clinical response and statistically significant improvement in interleukin levels were observed.
Overall, out of total 32 nonresponder patients in the present study, 23 responded to immunomodulation regime. No side effect related to immunomodulation was observed. No patient in this study was diabetic or HIV+ve.
Conclusions : Correlation of above immune parameters with the clinical presentation, the course of disease and the response to therapy reflected various patterns of host immune responses in osteoarticular tuberculosis. If immunomodulating drugs (Immunotherapy) are given along with ATT from very beginning, these beneficial changes are quantitatively more (more marked) indicating robust host immune response to infection, helping control the disease early. Adjuvant immunomodulation has the potential of shortening the total duration of antitubercular chemotherapy, besides inducing beneficial clinical and host immune responses.
Keywords: Host immunity; Osteoarticular tuberculosis; Immunomodulation
|How to cite this article:|
Arora A. Basic science of host immunity in osteoarticular tuberculosis - A clinical study. Indian J Orthop 2006;40:1-15
| Introduction|| |
Tuberculosis continues to be a major health problem despite availability of effective chemotherapy  . There are estimated 88 million new cases every year corresponding to 52 thousand deaths per week or more than 7000 deaths per day  . The steadily declining incidence of tuberculosis has reversed since 1985  . Added to the increasing incidence, is the problem of ominous emergence of drug resistant strains that threaten our capability to control the disease.
Mathematical modeling suggests that 3.2% (or 273,000) of the world's estimated new tuberculosis cases (95% confidence intervals: 185,000 and 414,000) were drug resistance cases in 2000  .
Immuno-pathogenesis  : Mycobacterium Tuberculosis is a facultative intracellular bacterium. Infection and disease are often marked separated in time. The intracellular bacteria that persist at their cellular site may later cause disease, when the sensitive balance, between pathogen and immune response is tipped in favor of infectious agent. Disease occurring in a person previously never exposed to tubercle bacillus is called as "primary tuberculosis". The majority of patients successfully contain the primary infection within 210 weeks and go on to develop a vigorous delayed type hypersensitivity (DTH) response. About 5- 10% develop either post primary disease in the 2 years immediately following infection or possibly reactivation tuberculosis after a latent period.  Post primary tuberculosis develops in previously infected persons either as a result of endogenous reactivation of latent disease or exogenous reinfection. Osteoarticular tuberculosis falls in the category of postprimary tuberculosis.
T Lymphocytes: Lymphocytes are the mediators of specific immunity. There are 2 distinct lymphocyte populations: B cells and T (Thymic) cells. Protective immune reactions in tuberculosis are mainly cell mediated , . Humoral antibodies do not have much significant role to play. Tubercle bacilli are taken up by phagocytic cells once they enter the tissue. If the bacilli are not destroyed, they multiply and kill the cell, leading to a local area of inflammation and invitation to more phagocytes. Some bacilli are transported to regional lymph nodes where they are engulfed by Antigen Presenting Cells (APC). Mycobacteria lying within the APCs cause the CD4 T cells to undergo activation and clonal proliferation. CD4 T cells are generally helper T cells. They produce a variety of cytokines that activate immune cells and certain other host cells and also express cytolytic activities. These CD4 T lymphocytes are central mediators of acquired resistance against intracellular bacteria  . The role of CD8 T lymphocytes is increasingly recognized. These cells produce cytokines and lyse target cells by direct cell contact. The functional activities of CD4 and CD8 cells show remarkable overlap. Hence, the protective immunity against all intracellular bacteria depends on both CD4 and CD8 T cells.
Interleukins (Also called Cytokines or IL): Interleukins are soluble glycoproteins that are produced by and act on various immune and non-immune cells  . Interleukins serve as transmitters between various leucocyte populations and they also promote interactions with other host cells, such as endothelial and epithelial cells. To emphasize interactions between different leucocytes, the term interleukin (IL) was coined for these numerous cytokines. T lymphocytes and Mononuclear Phagocytes (MPs) are major cellular sources of cytokines which, respectively, are termed lymphokines or monokines. CD4 T cells are the most potent producers of lymphokines, but all other T cell populations have also been shown to produce cytokines  .
Monokines: The monokines IL-1 (Interleukin-1), IL-6 (Interleukin-6), and Tumor Necrosis Factor (TNF) are important proinflammatory cytokines, . These 3 monokines cause acute phase responses. IL-1 and TNF act as endogenous pyrogens that stimulate hypothalamic cells to cause fever. TNF is also known as cachectin, because it is responsible for cachexia, the characteristic signs of wasting, for example in tuberculosis. At lower concentrations these three cytokines induce local inflammation by an auto- or paracrine activation mode of action at the site of bacterial growth. When produced in sufficiently high concentrations to enter the vascular system, endocrine effects prevail. IL-8 promotes leucocyte attraction to the site at which microbes lodge  . For these chemotactic cytokines, the term chemokines has been coined. The combined action of proinflammatory cytokines and chemokines is ultimately responsible for the inflammatory response that controls and contains microbial growth at an early stage of infection. IL12 is produced primarily by activated B lymphocytes and Macrophages and various bacterial components are potent inducers of IL-12 secretion  . Early IL-12 production by macrophages infected with intracellular bacteria is a crucial step in the development of a protective T cell response. IL-10 is best characterized as immune inhibitor, although it also activates various immune functions. In intracellular bacterial infections, the immune inhibitory functions of this cytokine prevail. IL-10 is produced by activated T cells, B cells and macrophages. It interferes with the protective immune response against intracellular bacteria by counteracting IFNa and IL-12 effects.
Lymphokines: IL-2 is an important T cell growth and differentiation factor and so is central to various aspects of cell mediated immunity  . Interferon gamma (IFN-a) is the principal cytokine of protective immunity against intracellular bacteria and acts on macrophages in which it stimulates various antimicrobial functions  . IFN-a production further contributes to antibacterial resistance by stimulating Natural Killer (NK) cells, and leucocyte migration. IFN-a is produced not only by T cells, but also by NK cells; it is therefore available during the innate and the acquired immune responses. IL-4 and IL-5 are potent B cell stimulators. IL-4 is a strong antagonist of IFN-a, thus interfering with acquired resistance against intracellular bacteria  .
Problem of non-responsiveness and resistance
Currently, preferred drug therapy for tuberculosis comprises first line antitubercular drugs with an initial intensive two months regime comprising multiple antibiotics - - Rifampicin, Isoniazid, Pyrazinamide (PZA) and Ethambutol or Streptomycin to ensure that mutants resistant to single drug do not emerge. The therapy is then continued in maintenance phase to kill the persisters or dormant bacilli. The increasing clinical unresponsiveness to first line antitubercular drugs (ATT) is alarming. Atypical mycobacterial infection, abuse of broad-spectrum antibiotics and poor compliance of patients has led to development of this clinical situation. The incidence presently varies between 5 and 10 percent , . As the second line antitubercular drugs are too toxic for general use, the focus is now shifting to immune potentiation in patients who do not show adequate response with standard therapy , .
There is no published study in English literature on cytokine profile in patients with osteoarticular tuberculosis. Keeping this fact in view and taking into consideration the role played by Th1, Th2 cells and macrophages in immunity against tuberculosis, problem of unresponsiveness in many patients of osteoarticular tuberculosis and proposed immunostimulatory effect of various agents e.g. Levamisole, BCG and DPT vaccine a prospective study on immune profile and immunomodulation of clinically non-responder patients was carried out.
The aims of present study were to assess immune profile of patients of osteoarticular tuberculosis by assessment of various interleukins and lymphocyte subsets, at presentation and three months after therapy. Moreover immune profile of patients with various types of presentation (acute exudative or chronic), fresh virgin cases and non responders was aimed to be assessed. The effect of immunomodulation on nonresponders in terms of clinical and investigative parameters was analyzed.
| Material and methods|| |
This study was conducted in the Department of Orthopaedics, University College of Medical Sciences (UCMS) & GTB Hospital, Delhi, India in collaboration with Department of Pathology, and Department of Microbiology UCMS between 2001 and 2004 after clearance from internal review board. The study was conducted in two phases: 61 subjects were studied in phase 1. Patients of any age suffering from osteoarticular tuberculosis were subjects of this study. Two groups of patients were undertaken in phase 1 study. Group 1 and 2 patients were studied simultaneously.
Group1(Fresh Virgin cases/Responders): Forty one consecutive patients of freshly diagnosed osteoarticular tuberculosis, on clinicoradiological grounds supplemented by bacteriological and histopathalogical investigations wherever in doubt not suffering from any other clinically detectable major illness were studied. These patients were subjected to following investigations after noting down detailed history and weight along with general physical examination, local examination and relevant systemic examination: Routine Investigations - Hemogram (TLC, DLC, Hb, ESR), Blood Sugar estimation, Serum proteins estimation and CD4 and CD8 cell counts in the peripheral blood , .
The patients were put on first line multidrug antitubercular chemotherapy (ATT) (i.e. INH, Rifampicin, Ethambutol, Pyrazinamide) and the indicated conservative orthopedic management was carried out  .
These patients were initially followed up every 10 to 15 days for first few months to note any untoward reaction to antitubercular therapy. Liver function tests were done after seven days of initiation of antitubercular therapy. In case of any untoward reaction, the chemotherapy was adjusted accordingly. At the end of six weeks clinical response to the treatment was recorded in detail. At three months of the treatment clinical response was noted down, repeat radiographs of the affected part were done, and the patient was subjected to repeat investigations mentioned earlier.
At this stage if the patient showed clinicoradiological improvement, then the ongoing treatment was continued. If the patient did not show anticipated or expected clinicoradiological response then an appropriate change in orthopedic management or chemotherapy was made at any stage of treatment (with minimum of three months of standard chemotherapy).
Group 2: Twenty patients as per criterion laid down by Tuli in 1999 were taken up for study in this group 18 . This group comprised of:
- Patients not showing expected clinicoradiological response after receiving minimum of three months of adequate supervised uninterrupted multidrug chemotherapy or
- Patients who had shown deterioration of disease or spread of disease or appearance of additional lesions anywhere in the body despite being on supervised multidrug chemotherapy for at least three months or
- Patients showing non-healing or breakdown of wound after undergoing surgery under adequate antitubercular cover or repeated reformation of cold abscess/es or
- Recurrence of a previously clinically healed lesion.
These patients were also subjected to routine haematological investigations and CD4 and CD8 cell counts in the peripheral blood at the time of induction in the study.
Group 2 patients were labeled as NON RESPONDERS and in these patients Immunomodulation was attempted as an adjunct to the standard antitubercular therapy as follows:
- BCG (BCG Laboratory, Guindy, Chennai, India) vaccination 0.1 ml by intradermal route on first day of therapy.
- Tab Levamisole 2 mg/kg body weight/day for 3 days, followed by an interval of 7 days (after 3 days of therapy). 6 such cycles of oral levamisole were repeated.
- One month after first BCG vaccination, Injection BCG was repeated i.e. day 30 of therapy.
- One month after second BCG injection, one injection of triple antigen (DPT-diphtheria vaccine 2.5 Lf, Tetanus vaccine 5 Lf., Bordetella pertusiss 20,000 million per 0.5 ml) by intramuscular route i.e. day 60 of therapy.
After four weeks of completion of immunomodulation therapy CD4 and CD8 cell counts were repeated along with clinicoradiological review. Indicated orthopedic management and antitubercular chemotherapy was continued in these patients.
Another 42 patients were studied prospectively in Phase 2. Two main groups of patients formed the subjects for this phase. Group 3 had 30 patients, and group 4 had 12 patients. Group 3 was subdivided into Group 3A and 3B.
Group 3: (Group- 3A = 15 Patients; Group- 3B = 15 Patients) comprised of 30 consecutive patients of freshly diagnosed osteoarticular tuberculosis.
Group- 3A: These patients were put on first line standard ATT regime  .
Group- 3B: These patients were given first line standard antitubercular chemotherapy along with immunomodulation regime "from the start of therapy" i.e. from day one of chemotherapy.
Group 4 : Comprised of 12 consecutive NON-RESPONDER patients as per criterion mentioned in phase 1 study (See Material and Methods of Phase 1 study). These patients were immunomodulated with the regime mentioned in phase 1 of study and ongoing ATT was continued. In recurred cases ATT was restarted along with immunomodula-tion.
Group 3 and Group 4 patients were subjected to routine hematogical (TLC, DLC, Hb, ESR, Blood Sugar estimation, Serum proteins) and following Interleukin assay : IL-1, IL-2, IL-4, 1L-6, IL-10, IL-12, IL-18, Interferon gamma and Tumor Necrosis factor in peripheral blood at the time of presentation and 3 months of the respective therapy. Estimation of these interleukins was done by microwell ELISA test employing commercially available kits using specific monoclonal antibodies for each of the analyte. In all the groups concomitant HIV infection was ruled out by appropriate serological tests. The patients in all 4 groups were followed up for 24-49 months (mean 27.2 months) to look for stability of response / recurrence.
Statistical Analysis was performed using student t test, chi square test and Analysis of variance (ANOVA).
| Results|| |
The mean age was 33 years (range 18 years to 71 years). 24 were males and 27 females. The distribution of disease was as follows: Vertebral - - 13; Osteomyelitis (long / short bone) - 08; Elbow - 05; Wrist - 05; Shoulder - 03; Hip - 03; Soft tissue / lymph node - - 04. Out of 41 patients included in this group only 25 complained of fever at initial presentation. All patients of spinal tuberculosis (Pott's spine) presented with constitutional symptoms while only 12 out of 28 patients of extra spinal tuberculosis presented with the same. Constitutional symptoms resolved in 3 weeks on ATT. Fourteen patients presented with discharging sinuses. Thirteen of these had extra spinal tuberculosis. One patient of spine presented with sinus in the back. Sinuses resolved in all cases on 3 months of therapy in this group. Nine out of 41 patients presented with abscesses. Six patients showed resolution of abscesses on therapy at 3 months. Nine patients with extra spinal disease had localised lymphadenopathy. One patient of Pott's spine had cervical lymphadenopathy also. Thirty nine out of 41 patients showed response at three months of therapy. Investigation data of Group 1 is presented in [Table - 1].
Twenty patients were included in this group. The mean age was 31 (range 15-50 years). 12 were males and 8 were females. 10 patients had vertebral involvement while 4 patients had extravertebral osseous involvement. 2 patients had articular involvement and 4 cases had soft tissue lesions. 11 out of 20 patients complained of fever at time of inclusion (spine-5, articular-2, osteomyelitis-2 and soft tissue abscess2). Fever was low grade and continuous type.16 patients complained of anorexia at time of inclusion in this group. Eight patients presented with discharging sinuses (spine-3, articular-1, osteomyelitis-2 and soft tissue abscess-2). 5 patients had abscess at presentation (spine-2, articular-1, osteomyelitis-0 and soft tissue abscess-2). Both patients of spinal tuberculosis had developed abscesses while on ATT, hence being classified as non-responders. One patient of Pott's spine had concomitant lymphadenopathy in the cervical region FNAC for which was positive for tubercular granulomata. The investigation data is presented in [Table - 2]. None of the patient was found to be diabetic or HIV positive.
Thirteen out of 20 patients responded to immunomodulation [Figure - 1]. The constitutional symptoms resolved within 8 weeks of starting immunomodulation. Sinuses healed in 7 out of 8 patients on completion of immunodomodulation. Of 7 patients who did not respond to immunomodulation one patient of tubercular knee was subjected to arthrodesis of knee. A patient of tubercular calcaneum was subjected to curettage and bone grafting [Figure - 2]. Two patients were referred to another centre for second line drugs. Another 2/7 patients responded to change in chemotherapy. One patient was lost to follow up. No untoward reaction related to immunomodulation was observed. One female patient developed an abscess at the local BCG inoculation site, which bursted out and healed over 12 weeks.
Group 3 (3A and 3B))
There were 30 patients in this group. Sixteen were males and 14 females with mean age of 23 years in Group 3A (range 3-62 years) and 27 [Table - 3]. years in Group 3B (range of 865 years). The distribution of disease is shown in At the time of presentation all patients had pain, 21/30 had anorexia, 16/ 30 had fever, 9/30 had history of weight loss, 9/30 had discharging sinus. After 3 months of therapy, sinus healed in 8 patients. The patient who did not show improvement was surgically intervened later. 23 patients had local abscess or swelling at the time of presentation. Twenty one patients showed reduction in abscess and local swelling at 3 months. Two patients who did not show improvement turned out to be non-responders and had psoas abscess (vertebral involvement). These patients were surgically intervened later. At the time of inclusion in the study 23 patients had restriction in range of motion. Majority of these belonged to articular tuberculosis. At 3 months of follow up 20 patients showed improvement in range of motion. Overall 27/30 patients responded to therapy in this group. Investigative data of Group 3A, 3B is presented in [Table - 4],[Table - 5].
There were 5 males and 7 females with mean age of 27 years (range 4-76 years). Majority belonged to vertebral lesions [Table - 3]. Nine out of 12 patients in Group 4 responded to antitubercular treatment and immunomodulation. One patient of vertebral tuberculosis with psoas abscess, who did not show response to immunomodulation was surgically intervened. The patient then responded to antitubercular treatment. Another patient of sacroiliac tuberculosis was subjected to debrima. The other patient with tubercular lymphadenitis who did not show improvement with standard antitubercular chemotherapy and immunomodulation was put on second line antitubercular drugs. Investigative data this is presented in [Table - 6].
In the present study none of the patient was found to be diabetic or HIV positive.
| Discussion|| |
About 30 million people suffer from tuberculosis throughout the world. About 1.2% of theses patients suffer from osteoarticular tuberculosis , . The basic response to M. tuberculosis is mediated by CD4 T lymphocytes, although some studies indicate the participation of CD8 lymphocytes also ,,,,,,,,, . The present study was conducted to assess the immunological profile of patients suffering from osteoarticular tuberculosis, and any change, if there with treatment.
Cluster differentiation t- lymphocytes, cd4, cd8 cells, and host immunity
CD4 and CD8 Cells in Group 1 (the Fresh Virgin/ Responder Group)
Immunological integrity and lymphocyte function have been found to play important role in tuberculosis. CD4 cells counts in HIV-negative patients with pulmonary tuberculosis have been reported to be normal or low ,,,, , and no clear relationship has been noted between the clinical presentation and the CD4 cell count in some other studies ,, . To clarify this issue in osteoarticular tuberculosis and to delineate changes in the CD4 cell counts during antitubercular therapy, we evaluated the clinical features and CD4 cell counts in patients of osteoarticular tuberculosis.
The CD4 cell counts in the present study ranged from 369 cells/cumm to 1233 cells/cumm with the mean of 801 cells/cumm in these patients at the time of presentation before treatment. The normal value of CD4 cells, was found to be 518-1500/cumm in one study, while it was 928 cells / cumm in an another study, . The latter study found a mean count of 271 cells / cumm in patients with extra - pulmonary tuberculosis. In the present study 25% of the patients had CD4 counts of less than 500 cells/cumm of blood  . The low CD4 cell count at the time of presentation in the presence of absolute lymphocytosis, indicates suboptimal immunological status of these patients. None of our patients in this group was diabetic or HIV positive excluding those with generalized disorder per se as a cause of this finding. In the present study, the serum protein abnormalities were not observed, indicating that chronic infection per se rather than under nutrition was a cause of depressed CD4 cell counts. To identify predictors of low CD4 cell counts, we performed multi variance stepwise linear regression analysis with the predictor variables of total lymphocyte count, serum albumin level, hematocrit level, hematocrit and the extent of disease. The total lymphocyte count was the best predictor of the CD4 cell count (p=. 000) and no other variable added significantly to its predictive value.
All patients showed statistically significant change (p>.001) increase in CD4 cell count after three months of therapy. CD4 cell count ranged from 369 to 2700 cells/cu mm with a mean of 1140 cells/cu mm. These observations attest to the fact that skeletal tuberculosis is one another reversible cause of CD4 cell depletion , . CD8 cells ranged from 304 to 970 cells/cumm in a study by Jones et al  . CD8 T cells are preferentially cytolytic T lymphocytes (CTL) and specifically lyse target cells by direct cell contact, and also produce cytokines  .
M. Tuberculosis being a facultative intracellular bacteria and can survive in the host for years without causing clinical disease. Whenever the host has low immune status, clinical disease can occur  . This fact seems to be attested by the observations of CD4 and CD8 cell counts in this study. Though there are studies, assessing the CD4 and CD8 cells in pulmonary tuberculosis, there is no single study devoted to the subject of profile of these cells in osteoarticular tuberculosis. Ours is first study highlighting reversible sub optimal immunological status of patients with osteoarticular tuberculosis.
CD4 and CD8 Cells in Group 2 (the Non Responder Group)
In the present study 20 patients were labeled as nonresponders as per clinical criteria mentioned in material and methods. Half of the patients had involvement of the spine indicating that quantum of disease material has also some role to play in responsiveness. None of these patients were HIV positive or diabetic. The initial CD4 cell counts in these patients ranged from 320 cell/cumm to 1622 cells/cumm with the mean of 598 cells/cumm. These counts rose to a statistically significant (p=.000) mean of 952 cells/cumm with immunomodulation using BCG and DPT vaccines, and oral levamisole coupled with uninterrupted multidrug antitubercular chemotherapy. The rise in CD4 cell count paralleled improvement in clinical picture. It is a well known that host immunity plays a major role against tuberculosis. It is attested by facts that only 5% of infected individuals develop clinical disease and another about 5% develop post primary tuberculosis later in life. In pre-chemotherapy era 3050% patients with clinical disease (tuberculosis) used to show spontaneous recovery with general measures aimed at improving general health and immunity of the patient  .
Chandra (1983) observed cellular and humoral immunocompetence to be depressed in patients with malnutrition or undernourished patients  . The serum albumin level in our patients ranged form 3.2 gm/dl to 5.0 with the mean of 4.2 gm/dl indicating that nutritional aspect was not responsible for sub optimal immune status in these patients.
Attempts at obtaining cultures of M. tuberculosis on LJ Media failed, possibly because these patients were already on antitubercular treatment when taken up for immunomodulation. Hence we based our inclusion criterion of patients in this group as "clinical nonresponsiveness" rather than "culture and sensitivity" of Mycobacterium. Of 20 patients in this group 13(65%) showed clinical response. Two patients were taken up for surgery, as these two did not show clinical improvement even with immunomodulation. One patient out of these two demonstrated atypical mycobacteria by polymerase chain reaction resistant to INH and Rifampicin.
Interleukins (Th1 and Th2 behavior) and host immunity
Interleukin-1 (IL-1; Inducer of granuloma formation), Interleukin-2 (IL-2; Beneficial immunogenic Th1 activity) and Interferon Gamma (IFN-a; Beneficial immunogenic Th1 activity): IL-1 is produced on stimulation of human monocytes with M. tuberculosis and specific Mycobacterial components  . IL-1 is an endogenous pyrogen and causes acute phase responses  . IL-1 and TNF-a are major inducer of granuloma formation by virtue of their chemotactic properties  . Th1 cells preferentially secrete IFN-g and IL-2 which respectively activate macrophage and cytotoxic lymphocytes (CTL). They are therefore central to cell mediated immunity  . It is also observed that individuals with IFN-a and IFN-a receptor deficiency have disseminated mycobacterial diseases  . IFN-a concentrations are found to be higher at the site of disease in patients with tuberculosis. IFN-y has beneficial immunogenic effects in controlling the disease.
IL-1, IL-2 and Interferon gamma were low at presentation in non-responders (mean 3.83, 5.42 and 10.5 pg / ml respectively) as compared to fresh virgin cases (mean 8.53, 10.6 and 14.67 pg / ml respectively) indicating poor immune status and low beneficial Th1 activity in non-responders. All three interleukins improved with respective therapy in all groups, but the improvement was more marked in patients on immunomodulation (group 3B and group 4), indicating beneficial immunogenic effects of immunomodulation. The improvement in interleukins was paralleled by improvement in clinical picture in all groups.
IL - - 2 is the main interleukin involved in beneficial immunogenic activity. This concomitant rise in serum IL-2 and improvement in clinical feature was in accordance with previous study by Johnson et al that tuberculosis patients receiving recombinant human IL-2 showed a clinical improvement and reduced bacterial loads  . Concentration were found to be high at the site of the disease in patients with pulmonary tuberculosis in one study  . The serum IL-2 level was found to be much lower in patients who presented with acute symptoms than with patients with chronic grumbling disease. This indicates that patients with chronic symptoms have much more Th1 cell activity (cell mediated immunity) than with patients with acute presentation. No such study to the best of our knowledge is available in the literature.
Interleukin-10 (IL-10; Immune inhibitory Interleukin), Interleukin-4 (IL-4; Inhibits immune responses of the host)and Tumor Necrosis Factor (TNF / Cachectin):IL-10 is produced by macrophages and Th2 cells  . IL-10 is best characterized as immune-inhibitor , . IL-4 also has the capacity to inhibit the immune response to M tuberculosis  . Clinical and experimental data in humans and animals suggest that TNF-a contributes both to protection against tuberculosis and to immunopathology. Tuberculosis is characterized by fever, weight loss, a prolonged acute-phase protein response and granuloma formation. These characteristics may partly be due to action of proinflammatory cytokines, e.g. TNF-a, IL-6 and IL-8 ,,, . Excessive local production of TNF may cause marked tissue necrosis that is characteristic of progressive TB and may result in TNF-a release into the circulation contributing to systemic manifestations of TB such as fever and cachexia. In contrast, a physiological concentration of TNF-a contributes to antimycobacterial defense, and local production leads to granuloma formation, control of infection and mycobacterial elimination ,,, .
In the present study non-responder patients had almost two times the levels of IL-10 (161.5 pg/ml), IL-4 (67.33 pg/ml) and TNF (338.08 pg/ml) as compared to fresh virgin cases (IL-10 105.47 pg/ml; IL-4 34.67 pg/ml and TNF 228.29 pg/ml) indicating overwhelming inhibitory interleukin milieu in nonresponders. By therapy in respective groups, the levels of above three interleukins decreased, many times more in patients with immunomodulation, indicating the beneficial response of the latter. Between groups 3A and 3B (all fresh virgin cases) the response was better in immunomodulated group (3B) [Table - 4],[Table - 5],[Table - 6]. Jacobs et al in 2000 demonstrated that IL-10 deficient mice eliminate Mycobacterium bovis Calmette-Guerin bacillus faster than the wild-type mice. The granulomas in the former were significantly larger as compared to latter  . In the present study patients with widespread disease and acute exudative presentation had very high levels of Tumor Necrosis Factor.
Interleukin-6 (IL-6), Interleukin-12 (IL-12)Interleukin18 (IL-18) : The levels of these cytokines were found to be elevated in all three groups (more so in Non-responder groups) which decreased with therapy. These cytokines do not play a major role in Th1 and Th2 dichotomy. However , the reduction in these cytokines was quantitatively more in patients with Immunomodulation, once again underlining the beneficial response of Immunomodulation [Table - 4],[Table - 5],[Table - 6].
Th1 and Th2 Dichotomy: The plethora of cytokines produced by CD4 helper T cells imposes the central control function in immunity on this population. The enormous diversity of biological activities regulated by CD4 T cells, some of them counterproductive, makes this task impossible for a single cell type. Accordingly, there is further division within the CD4 T cell population. According to their cytokine profiles, so-called Th1 and Th2 cells, both derived from a Th0 precursor cell, can be distinguished  . Hence, CD4 cells (T helper) mature into two distinct cell types, ThI and Th2, those secrete different & mutually antagonistic cytokines  .
The Th1 cells preferentially secrete IFN-5 And IL-2 , . In contrast, Th2 cells preferentially produce IL-4, IL-5 and IL10. IL-4 is also the central cytokine in B cell activation. Other cytokines are produced by both Th types. Differentiation of Th1 and Th2 cells is controlled at two major stages. Soon after antigen encounter, cells of the innate immune system produce cytokines that promote preferential differentiation of either Th type. Rapid production of IL-12 and IFN-a, respectively, by macrophages or NK cells at the outset of infection with bacterial pathogens ensures preferential Th1 cell development  . By contrast, early IL-4 production promotes Th2 cell development with further support by IL10 from macrophages. IL-10 "down-regulates" an active immune response in tuberculosis, including deactivation of macrophages, inhibition of T cell proliferation and suppression of cytokine production by T lymphocytes  . At the stage of an established immune response, Th1 and Th2 cells counter-regulate each other with IFN-a from Th1 cells and IL-4 from Th2 cells  . In most cases, Th1 and Th2 cells are not activated exclusively in response to a given antigenic stimulus, but rather there are quantitative differences.
Thus, in infections Th1 cells can be viewed as beneficial and Th2 cells as detrimental. In general, however, Th1 cells act as inducers, and Th2 cells as terminators, of cell mediated immunity against intracellular pathogens. Hence, both T cell subsets seem to be necessary for the successful combat of microbial invaders without enduring damage to the host. This can only be achieved by the activation of Th1 and Th2 cells in a highly co-ordinated and sequential manner  .
Building on this key discovery, Bretscher demonstrated that contact with antigens of a pathogen such as tubercle bacillus even in small quantity induces a detectable immune response  . This response is able to imprint the immune system with information that determines whether subsequent contact with the tubercle bacillus induces a Th1 or Th2 mediated reaction. By this means the immune system is programmed to respond to Tubercle bacillus in an unified & coherent manner. The blood levels of various Interleukins reflected the status and milieu of the host immunity. The levels of various Interleukins, also gave information regarding the "type" of host immune response. In tuberculosis, there can be immunogenic response (trying to contain the infection) or inflammatory response with overwhelming tissue necrotising reaction, which tend to cause large abscesses or marked tissue destruction. In general, in the present series, patients with contained and localised lesions had high levels of immunogenic interleukins (e.g. IL-2, IFN-gamma), patients with large abscesses and widespread lesions had high levels of proinflammatory cytokines (e.g. TNF, IL-1, IL-6, IL-12) whereas non-responders had high levels of immune inhibitory cytokines (IL-10, IL-4). Largely, the type of cytokine response depends on the fact that whether the host earlier had been exposed to Mycobacterium Tuberculosis or no, and if has been exposed to Mycobacterium Tuberculosis, what had been the outcome. The clinical presentation of the disease depended upon the interaction of these cytokines. If the host had high levels of proinflammatory (IL-1, Tumor Necrosis factor) and immune inhibitory cytokines (IL-10, IL-4), then patients had an acute exudative presentation or large abscess as observed in this study. High levels of Tumor Necrosis factor caused widespread destruction and depending upon the type of cellular profile of exudation, the host exhibited the type and consistency of pus [Figure - 3]. Many of these patients with acute exudative presentation exhibited scar marks of old healed tubercular lesion, untreated or partially treated, but healed by natural immunity [Figure - 4].
Immunomodulation: Clinical unresponsiveness to first line antitubercular drugs is an increasing worldwide problem. , In developing countries, modern diagnostic techniques like Bactec and PCR methods of organism identification and sensitivity are available at very few centers. Moreover, the positive yield of culture by routine methods (as on LJ media) is very very low as skeletal tuberculosis is considered a paucibacillary disease. The problem is further compounded by ill-advised use of antibiotics like quinolones before a definitive diagnosis of tuberculosis is made leading to further reduction in positive culture yields. In such a situation clinical parameters in assessing the response assumed huge importance, which was done in the present study. We relied on clinical parameters rather than culture reports to label non-responsiveness. In poor countries where much can not be elaborated on the seed (Mycobacterium/ pathogen), immunomodulation of the soil (host), aimed at combating immunosuppresion and enhancing the host's own immune response is an attractive approach to supplement conventional chemotherapy.
Robert Koch's attempt at immunomodulation fell into disrepute as repeated injections of old tuberculin could cure as well as kill the host. Sphalinger reported remarkable success with immunotherapy in 1930s after modifying Kochs therapy  . The concept that immune reactivity in mycobacterial disease is a double edged sword (able to confer protection, but also to cause tissue destruction as well) has been a topic of a lengthy controversy. It has only recently resolved by Mosman's demonstration of two functional subsets of T helper cells Th1 and Th2. Thus the modern approach is not simply to boost immunity but to replace an inappropriate reaction by an appropriate one  . Immunotherapy with Mycobacterium vaccae, a scotochromogenic, rapidly growing organism has been suggested by many workers prominent among them being Stanford and Grange  . M vaccae has been found to prevent further tissue destruction in tuberculosis and restore protective immunity against the causative organism by causing a Th2 to Th1 shift  . Encouraging results in leprosy and tuberculosis have been reported following immunotherapy with Mycobacterium vaccae, which restored the protective immunity responses , . Onyebujoh et al reported beneficial influence on clinical recovery and survival by immunotherapy with M. vaccae , .
In the present study, a combination of various immuno-modulatory agents was used. Bacille Calmette-Guerin (BCG) has been shown to produce an immunostimulant effect  . BCG has been shown to even inhibit tumour growth. ,,,,,, Miller et al observed that BCG infected mice showed increased immune response to an additional antigenic stimulus  . Ishibashi et al reported non-specific augmentation of immune response with BCG due to generalized systemic activation of lymphoid system  . Moreover they observed long lasting effect of this immunepotentiation. BCG is capable of inducing beneficial TH1 type immunological response in standard or high doses as is Mycobacterium vaccae vaccine ,,,,, . In a double blind, placebo-controlled study, Hoft et al demonstrated BCG induced significant increase in Mycobacterial specific T-cell proliferative and TH1 type cytokine responses  . The clinical adverse effects of BCG injection include hypersensitivity and shock, chills, fever, malaise, and immune complex disease  . In the present series no reaction related to BCG vaccination was observed expect in one patient who showed abscess formation at site of injection.
Levamisole initially was synthesized as an antihelminthic agent. Subsequent studies in human beings demonstrated that levamisole can increase delayed hypersensitivity and/ or T-cell mediated immunity  . Levamisole has been used in Hodgkin's disease, rheumatoid arthritis, and most recently in adjuvant chemotherapy of colorectal cancer, although it is not completely clear whether or not the effect in the latter is immunologic ,,, . In the present study no side effect directly related to Levamisole was observed. DPT vaccine also has a generalized immunostimulatory effect  . Tuli is credited for inducing the concept of immunomodulation in osteoarticular tuberculosis  . He reported on 35 cases of osteoarticular tuberculosis considered clinically resistant to multidrug therapy. He supplemented standard chemotherapy in these patients with immunomodulation by oral Levamisole, BCG and DPT vaccination. Of the 35 patients, 31 showed favorable clinicoradiological response within 6 to 8 weeks of execution of immunomodulatory therapy. However, he did not present supportive laboratory data indicating immunomodulation. In the present study 23 patients out of 32 responded to a combination of adjuvant immunotherapy and ATT. Immunomodulation will not work in a situation where the cause of persistence of symptoms is because of a sequestrum or a cavity, though the tubercular process has been largely controlled by antitubercular drugs [Figure - 5].
The therapeutic attack on the extracellular bacilli M. tuberculoses poses few problems when they are drugsusceptible. Being actively replicating these bacilli are amenable to killing by most of the antitubercular drugs, which readily diffuse into the body fluids  . As the patients with active disease feel ill, they can usually be relied upon to take their drugs. The problems in therapy arise when only intracellular persistors remain. As these metabolize very slowly and, probably, intermittently, the number of drugs that are effective is greatly reduced and these must be given for several months  . As the patients often feel well, noncompliance is frequent problem and supervision is required, at considerable additional cost to health services. "Drug Defaulters" are one of the major sources of spreading multidrug resistant Mycobacteria in the community , . It is against this population of residual bacilli, probably mostly intracellular, that immunotherapy coupled with chemotherapy is directed. Another important advantage of immunomodulation could be reduction in total duration of chemotherapy required to combat the disease. In third world countries, where the disease is rampant, and resources minimal, this approach would definitely reduce the number of drug defaulter patients.
The foreseeable advantage of combined therapy (chemotherapy and immunomodulation) lies in the hope of development and deployment of therapeutic measures that will correct suboptimal immune effects, in both tuberculosis and HIV/AIDS. The day is not far when we will have cytokines (Interleukin) therapy available to treat Osteoarticular Tuberculosis, as an adjunct to antitubercular treatment. Let us work, not only on the BUG (Mycobacterium tuberculosis) but also on the bearer (the host, the infected person). The future of Immunepotentiation looks bright.
Acknowledgement : I am profoundly thankful to Prof. SM Tuli for constant scientific guidance and our research team - Prof. Sudhir Kumar, Prof. V Ramachandran, Prof. Geeta Dev, Prof. AK Jain, Dr Biren Nadkarni, Dr Anil Agarwal, Dr Anand Vadehra and Dr Rishi for untiring scientific and technical support.
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[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5]
[Table - 1], [Table - 2], [Table - 3], [Table - 4], [Table - 5], [Table - 6]