Research Questions
1. Provide a detailed description of the histopathology of the spinal TB abscess in HIV-infected and –uninfected specimens
2. Immunolocalise macrophages and T-cells infiltrating the spinal TB abscess
3. Compare, quantitatively, the T-cell infiltration in HIV-infected with HIV-uninfected specimens
Fit with Africa Centre portfolio/Background
The incidence of spinal TB (Potts disease, TB spondylitis), a devastating disease, characterized by bone destruction and deformity in advanced disease, has increased dramatically in recent years at a primary referral hospital in KwaZulu-Natal, SA. The anti-TB immune response is dominated by macrophages and lymphocytes however, their specific phenotypes, functionality and relative distribution within the spinal TB lesion remain unclear. Understanding the immunopathology of spinal TB would greatly impact approaches to diagnosis, therapeutic interventions and ultimately, patient outcomes. This is of particular relevance in HIV/TB endemic regions such as KZN where the spinal TB disease profile has paralleled that of both the pulmonary TB and HIV epidemics. A detailed microscopic investigation of spinal TB biopsies was conducted at the PHRI, New Jersey, (NY) to provide the first histopathology description of granuloma formation in mono- and –dual spinal TB infection; and the first immunolocalisation study of the impact of co-infection on immune cell homing to the site of disease.
Methods/study design
Patients were recruited at the Spinal Unit of the King George V Referral Hospital, Durban, SA as part of a molecular investigation of spinal TB and HIV co-infection (for degree purposes). Specimens, collected during surgery from HIV-infected and –uninfected patients were processed for light microscopy, ultra-thin sections were cut and one of these sections were stained with Haematoxylin and Eosin (H&E). The remaining sections remained unstained for immunolocalisation experiments were CD3+, CD4+, CD8+ and CD68+ (macrophage) cells were localized using an indirect immunoperoxidase method. Positive cells, denoted by a positive chromogenic reaction, were manually counted. CD4+ and CD8+ cells were expressed as a percentage of the total T-cell count as indicated by the CD3 stain.
Preliminary Results
1. Histopathology
Typical granuloma formation was noted in both HIV+ and HIV- biopsies with macrophages, polymorphonuclear and mononuclear cells, T-cells and multinucleated giant cells (Langerharns cells). There was evidence of vasularisation, thrombosis, granuloma formation, extensive immune cell infiltration, and lymph drainage, as well as nerve bundles in both HIV+ and HIV- extra-dural specimens. Biopsies collected adjacent to collapsed vertebrae displayed evidence of active osteogenesis and bone resorption.
2. Immunolocalisation
Image analysis following immunolocalisation of CD68, CD4, CD8 and CD3 antibodies demonstrated that CD68+ macrophage and total T-cell (CD3+ cells) infiltration to the site of infection was similar in HIV- and HIV+ specimens. CD4+ T-cells were consistently observed within the granuloma and to a limited degree on the periphery of the granuloma while CD8+ T-cells were observed largely on the periphery of the granuloma. This pattern of CD4+ and CD8+ T-cell distribution was independent of HIV status. There was no statistical difference in total T-cell (CD3+) infiltration when comparing the two study groups however, CD4+ T-cell counts were significantly greater (p<0.01) in HIV- compared with HIV+ specimens. In contrast percentage CD8+ T-cells (p<0.05) was significantly greater in the HIV+ group. A significant negative correlation (R2=0.60) was observed between circulatory and tissue specific CD4+ T-cells in the HIV- group while a positive relationship (R2=0.63) was noted in the HIV+ group.
Further Work and timelines
The completion of a more detailed statistical analysis of the data and the subsequent completion of the related manuscript is pending. It is anticipated both will be complete within the first quarter of 2010.
In addition, the findings of this study have prompted further research questions such as:
1. Are the T-cells localized to the isolated anatomical site under chronic immune stimulation, the site of spinal TB and HIV-1 co-infection, poly-functional? Partial funding for this work has been granted by the SAOA and the study will commence as soon as ethical approval has been received from the UKZN IRB. The anticipated date of completion for this work is the end of 2010.
2. Does chronic immune stimulation at this isolated site of co-infection create anatomical and cellular HIV-1 viral compartments?
Policy implications
The findings of this investigation are novel and provide the first histopathological and immunohistological description of the immunopathogenesis of spinal TB. The findings of this study would suggest that CD4+ T-cells are preferentially homed to the site of infection in immune competent individuals but switch to a primarily CD8+ T-cell response when CD4+ cells become depleted. The study also illustrated that the pathologies and deformity observed in advanced disease may be a result of chronic immune stimulation and the continued influx of immune-stimulatory cells promoted by bacterial fragments or antibodies rather than live bacteria. This has implications to the duration of anti-TB therapy.