Efficacy of Mycobacterium indicus pranii immunotherapy as an adjunct to chemotherapy for tuberculosis and underlying immune responses in the lung.

BACKGROUND: The 9-month-long chemotherapy of tuberculosis often results in poor compliance and emergence of drug-resistant strains. So, improved therapeutic strategy is urgently needed. Immunotherapy could be beneficial for the effective management of the disease. Previously we showed the protective efficacy of Mycobacterium indicus pranii (MIP) when given as prophylactic vaccine in animal models of tuberculosis. METHODS: We sought to investigate whether MIP can be used as an adjunct to the chemotherapy in guinea pig models of tuberculosis. Efficacy of MIP was evaluated when given subcutaneously or by aerosol. RESULTS: MIP-therapy as an adjunct to the chemotherapy was found to be effective in accelerating bacterial killing and improving organ pathology. MIP-immunotherapy resulted in higher numbers of activated antigen-presenting cells and lymphocytes in the infected lungs and also modulated the granulomatous response. Early increase in protective Th1 immune response was observed in the immunotherapy group. Following subsequent doses of MIP, decrease in the inflammatory response and increase in the immunosuppressive response was observed, which resulted in the improvement of lung pathology. CONCLUSION: MIP immunotherapy is a valuable adjunct to chemotherapy for tuberculosis. Aerosol route of immunotherapy can play a crucial role for inducing immediate local immune response in the lung

Nocardial Mycetoma: Diverse Clinical Presentations

Nocardia spp are gram-positive, aerobic, acid-fast bacteria which exist as saprophytes in nature. Invasive disseminated infections are particularly common in immunocompromised or debilitated hosts. Superficial infections with Nocardia spp occur as a result of local trauma and contamination of the wound. Clinically, it presents as acute infection (abscesses or cellulitis), mycetoma, or sporotrichoid infection. Differential diagnosis includes eumycetoma, chromomycosis, blastomycosis, coccidioidomycosis, sporotrichosis, tuberculosis, botryomycosis, syphilis, yaws, and neoplasia. Its diagnosis is confirmed by demonstrating the causative organism in exudates (as granules), tissue specimens, or cultures. Early diagnosis will obviate need for drastic surgical measures as early institution of chemotherapy is effective in most patients. However, its diagnosis is often delayed due to diverse clinical presentations and for want of clinical suspicion, particularly in non-endemic areas. This paper presents 4 clinical forms of this not so uncommon disease, emphasizing the importance of high index of clinical suspicion, especially in non-endemic regions; and the significance of repeated examination of exudates for Nocardia granules for an early diagnosis

Evaluation of lung pathology.

<p>Lung pathology was monitored both microscopically and macroscopically. Bar diagram shows gross pathological score of each experimental group after 60 days or 30 days of therapy. Data represents the mean value of four animals in each group. * <i>p</i><0.05, ** <i>p</i><0.01. Gross pathological score was calculated based on the scoring system which represents number of nodules, haemorrhagic spots, cavities, degree of necrosis, caseation etc as mentioned in the material and methods. Histopathology of lungs of different experimental groups was evaluated after 60 days or 30 days of therapy and the representative photomicrographs are shown below the respective graphs., The microscopic sections from control group, group treated with only drug and drug + MIP aerosol group have been shown in the upper row (10×) and in the lower row (40×). Photomicrographs of lower magnification show the overall distribution of granuloma with plenty of healthy alveolar spaces in drug+MIP group; while the 40× images show the organization of granuloma.</p

Effect of therapy on mRNA expression level of various cytokines in lung.

<p>The expression level of mRNAs for different cytokines were measured by relative quantification method. Expression level of each gene was normalized against the house keeping gene GAPDH and calibrated against the expression of the same gene versus GAPDH in uninfected (naive) animal. Data represents the mean value of four animals in each group.</p

Monitoring of activated T cells in the infected lungs during therapy.

<p>Percentage of activated CD4+ T cells (<b>A</b>) and CD8+ T cells (<b>B</b>) in drug or drug plus MIP group were evaluated at different time points during the course of therapy. The percentage of respective cells in infected control group has been shown in the inset of bar diagram. Activated T cells were analyzed based on the expression of homing receptors as shown in the representative contour plots and compared at 15 days and 30 days of therapy. In each plot, region R6 represents T cells with high homing receptor expression and the corresponding percentage value is mentioned inside the region. Data represents the mean value of four animals in each group. * <i>p</i><0.05. Uninfected animal (naive) was taken as uninfected negative control and untreated animal at 30 days post infection was taken as infected positive control.</p

Bacterial load in infected animals following chemotherapy or chemotherapy + immunotherapy.

<p>Bacterial load in lung and spleen of guinea pigs following different schedules of therapy as compared to control untreated animals. The chemotherapy was given either for 60 days (1^st row) or for 30 days (2^nd row). The schematic diagram in each panel shows the detailed schedule of the therapy in the drug-treated and drug + immunotherapy treated groups. Bacterial counts are expressed in log value of colony forming units. Data represents the mean CFU of four animals in each group. * <i>p</i><0.05, ** <i>p</i><0.01.</p

Evaluation of cellular immune response inside the granuloma of infected lungs.

<p>Scattergrams show the numbers of CD4+ T cells, CD8+ T cells, granulocytes and MHC II+ cells per unit area of granuloma from different experimental groups after 30 days of therapy. The number of different immune cells inside the granuloma in the lung sections from different experimental groups were counted microscopically and expressed as number of cells per unit area of granuloma. Minimum of 1000 cells were counted from randomly selected, at least 10 fields. Sections were taken from lung samples of 4 animals per group. The representative photomicrographs show the positively stained cells with red arrow in control group, drug or drug + MIP group in each panel. DAB was used as substrate in staining granulocytes while VIP red was substrate for staining other types of cells. * <i>p</i><0.05, ** <i>p</i><0.01.</p

Evaluation of neutrophil infiltration and MHC expressions in the infected lung.

<p>The expression of MHC I, MHC II and percentage of neutrophils were monitored at different time points during the course of therapy. The bar diagram shows the mean percentage of MHC expression on gated population or neutrophil content in the lungs of drug or drug plus MIP group. Data represents the mean value of four animals in each group. * <i>p</i><0.05. Entire lymphocyte negative region was gated based on forward versus side scatter and MHC expression or neutrophil infiltration was analyzed on the gated region. The representative plots show the MHC expression or neutrophil infiltration in different experimental groups along with the suitable negative and positive controls. Percentage value has been mentioned in the assigned region in each plot.</p