Studies of immune response in human tuberculosis

TB (tuberculosis), caused by Mycobacterium tuberculosis (Mtb), continues to be a world-leading killer and a serious global health problem primarily affecting poor people in many developing countries. The difficult situation with TB/HIV co-infection is also a major key challenge to public health. Despite recent advances in TB research, the host- and pathogen-specific factors that lead to protective immunity, particularly in humans, remain unclear. While it is well-established that cell-mediated immunity is required to control TB infection, the role of humoral immunity including Th2 immune responses is debated. This thesis aimed to explore immune responses in human TB and the immunopathogenic mechanisms involved in the progression of clinical TB in both HIV-negative and HIV-positive individuals. To address the aims of this thesis work, well-defined study cohorts of active TB patients were obtained in close collaboration with the Black Lion University Hospital in Addis Ababa, Ethiopia. We used a novel immunodiagnostic test, Antibodies in Lymphocyte Supernatants (ALS), to demonstrate that IgG-secreting plasmablasts were significantly higher in the peripheral circulation of patients with active TB compared to latent TB cases and non-TB controls. Interestingly, BCG-specific IgG titers were particularly high in blood samples from TB/HIV co-infected patients with CD4 T cell counts <200 cells/ml who produced low levels of Mtb-specific IFN-γ in vitro. A technological platform including quantitative assessments of mRNA and protein expression in tissue (lymph nodes), fluids (bronchoalveolar lavage (BAL) and pleura fluid) and peripheral blood, was also used to investigate antimicrobial effector pathways and adverse immune responses in unique clinical samples obtained from the local site of Mtb infection. The results from these studies revealed that TB disease was associated with extensive tissue remodelling including an altered cellular composition, collagen deposition and granuloma formation. Here, the degree of necrotic granuloma formation was particularly prominent in TB/HIV-co-infected patients. Despite granuloma enrichment of activated CD68+ macrophages containing Mtb-antigens, mRNA levels of IFN-γ, TNF-α and IL-17 remained low in Mtb-infected lymph nodes. Accordingly, CD8+ T cells expressing cytolytic and antimicrobial effector molecules perforin and granulysin were low inside the TB lesions, while CD4+FoxP3+ regulatory T cells (Treg) and Th2/Treg cell cytokines IL-13 and TGF-β were up-regulated in the Mtb-infected tissues. The observed shift of the immune response from a Th1/Th17 towards an immunoregulatory phenotype was supported by our finding of a Th2 polarized response in the lung of patients with pulmonary TB. Here, multiplex protein analysis of BAL and plasma samples demonstrated low levels of Th1/Th17 cytokines and the T cell-chemoattractant CCL5, but significantly up-regulated levels of pro-inflammatory cytokines and the Th2 cytokine IL-4. The enhanced Th2 response was associated with increased levels of CCL4, suppressors of cytokine signaling-3 (SOCS3) and mycobacteria-specific IgG in BAL fluid from patients with active pulmonary TB. Contrary, IL-4, CCL4, SOCS3 and IgG-responses remained low in patients with less severe extrapulmonary pleural TB disease, who demonstrated up-regulated levels of both IFN-γ and CCL5. Taken together, our results provide evidence that human TB is associated with impaired Th1 immunity but elevated Th2/immunoregulatory responses and induction of antibody-mediated immunity. Importantly, enhanced Th2 and/or plasmablast responses may be used as relevant biomarkers or immune response signatures of active progressive TB disease that could be explored as potential targets for clinical TB management in the future

Amoebic liver abscess: A 20-year retrospective analysis at Tikur Anbessa Hospital, Ethiopia

No Abstract Available Ethiop.J.Health Dev. Vol.18(3) 2004: 199-20

Prevalence of Helicobacter pylori vacA and cagA genotypes in Ethiopian dyspeptic patients

A total of 300 gastric biopsy samples and 50 Helicobacter pylori isolates were collected from Ethiopian adult dyspeptic patients. The vacA and cagA genes were detected in 90 and 79% of biopsy specimens, respectively, and in 100 and 87% of clinical isolates, respectively. Both genes were detected in 84% of the gastric biopsy samples and in 87% of the clinical isolates. Among vacA genotypes, the s1/m1 genotype was the most common in gastric biopsy samples (48%). The vacA and cagA positive H. pylori strains were detected to a higher degree in patients with chronic active gastritis (71%) than patients with other histopathological findings (29%) (P < 0.05)

Identification and genotyping of the etiological agent of tuberculous lymphadenitis in Ethiopia

In Ethiopia, little has been done to assess how Mycobacterium bovis has contributed to human tuberculosis, though the population routinely consumes unpasteurized milk and raw meat. The aim of this study was to determine the proportion of M. tuberculosis and M. bovis as etiological agents of tuberculous lymphadenitis (TBLN). Patients with lymphadenopathy (n = 171) were included in a cross-sectional study at Butajira Hospital, Southern Ethiopia. Lymph node biopsies were cultured. Patients' HIV status was identified. DNA from positive cultures was tested by PCR to identify M. bovis and M. tuberculosis. Isolates were genotyped by multiplex ligation-dependent probe amplification (MLPA) assay. Among 171 patients, 156 had culture results. Of these, 107 (69%) were positive for M. tuberculosis complex (MTC). Six of the 10 HIV-positive patients were culture positive. M. tuberculosis specific sequences were identified in the DNA of each of 100 samples as assessed by RD10 targeted PCR, and each of the 95 isolates exhibited the M. tuberculosis specific TbD1 deletion by MLPA analysis. No M. bovis was identified. These results indicate that all the isolates were modern M. tuberculosis strains. Furthermore, MLPA studies confirmed that 42% of the isolates showed the Haarlem genotype and 12% displayed sequences compatible with INH resistance. No mutations conferring resistance to ethambutol or rifampicin were detected. Our data showed that M. tuberculosis strains had common characteristics with strains causing pulmonary TB, which appears to be the main etiological agent of TBL

DataSheet_2_Inflammatory immune profiles associated with disease severity in pulmonary tuberculosis patients with moderate to severe clinical TB or anemia.xlsx

BackgroundImmune control of Mycobacterium tuberculosis (Mtb) infection is largely influenced by the extensive disease heterogeneity that is typical for tuberculosis (TB). In this study, the peripheral inflammatory immune profile of different sub-groups of pulmonary TB patients was explored based on clinical disease severity, anemia of chronic disease, or the radiological extent of lung disease.MethodsPlasma samples were obtained from n=107 patients with active pulmonary TB at the time of diagnosis and after start of standard chemotherapy. A composite clinical TB symptoms score, blood hemoglobin status and chest X-ray imaging were used to sub-group TB patients into 1.) mild and moderate-severe clinical TB, 2.) anemic and non-anemic TB, or 3.) limited and extensive lung involvement. Plasma levels of biomarkers associated with inflammation pathways were assessed using a Bio-Plex Magpix 37-multiplex assay. In parallel, Th1/Th2 cytokines were quantified with a 27-multiplex in matched plasma and cell culture supernatants from whole blood stimulated with M. tuberculosis-antigens using the QuantiFERON-TB Gold assay.ResultsClinical TB disease severity correlated with low blood hemoglobin levels and anemia but not with radiological findings in this study cohort. Multiplex protein analyses revealed that distinct clusters of inflammation markers and cytokines separated the different TB disease sub-groups with variable efficacy. Several top-ranked markers overlapped, while other markers were unique with regards to their importance to differentiate the TB disease severity groups. A distinct immune response profile defined by elevated levels of BAFF, LIGHT, sTNF-R1 and 2, IP-10, osteopontin, chitinase-3-like protein 1, and IFNα2 and IL-8, were most effective in separating TB patients with different clinical disease severity and were also promising candidates for treatment monitoring. TB patients with mild disease displayed immune polarization towards mixed Th1/Th2 responses, while pro-inflammatory and B cell stimulating cytokines as well as immunomodulatory mediators predominated in moderate-severe TB disease and anemia of TB.ConclusionsOur data demonstrated that clinical disease severity in TB is associated with anemia and distinct inflammatory immune profiles. These results contribute to the understanding of immunopathology in pulmonary TB and define top-ranked inflammatory mediators as biomarkers of disease severity and treatment prognosis.</p

Vitamin D3 Status and the Association with Human Cathelicidin Expression in Patients with Different Clinical Forms of Active Tuberculosis

Low vitamin D (vitD3) is one of the most common nutritional deficiencies in the world known to be associated with numerous medical conditions including infections such as tuberculosis (TB). In this study, vitD3 status and its association with the antimicrobial peptide, human cathelicidin (LL-37), was investigated in Ethiopian patients with different clinical forms of TB. Patients with active TB (n = 77) and non-TB controls (n = 78) were enrolled in Ethiopia, while another group of non-TB controls (n = 62) was from Sweden. Active TB included pulmonary TB (n = 32), pleural TB (n = 20), and lymph node TB (n = 25). Concentrations of 25-hydroxyvitamin D3 (25(OH)D3) were assessed in plasma, while LL-37 mRNA was measured in peripheral blood and in samples obtained from the site of infection. Median 25(OH)D3 plasma levels in active TB patients were similar to Ethiopian non-TB controls (38.5 versus 35.0 nmol/L) and vitD3 deficiency (&lt;50 nmol/L) was common in both groups (73%). Ethiopians (low latitude) had significantly lower 25(OH)D3 levels compared with Swedish non-TB controls (51.0 nmol/L, high latitude), but vitD3 status was not affected by tuberculin-positivity or HIV infection. Patients with local lymph node TB had significantly higher 25(OH)D3 levels compared with pulmonary TB patients (48.0 versus 29.0 nmol/L). Moreover, plasma 25(OH)D3 levels correlated with local LL-37 expression in granulomatous lesions in TB infected lymph nodes. Instead, systemic LL-37 mRNA expression in blood cells was elevated compared with the site of infection in pulmonary and pleural TB. Low vitD3 status may be associated with an enhanced peripheral expression of LL-37 in patients with intrathoracic TB that could result from chronic inflammation

DataSheet_1_Inflammatory immune profiles associated with disease severity in pulmonary tuberculosis patients with moderate to severe clinical TB or anemia.docx

BackgroundImmune control of Mycobacterium tuberculosis (Mtb) infection is largely influenced by the extensive disease heterogeneity that is typical for tuberculosis (TB). In this study, the peripheral inflammatory immune profile of different sub-groups of pulmonary TB patients was explored based on clinical disease severity, anemia of chronic disease, or the radiological extent of lung disease.MethodsPlasma samples were obtained from n=107 patients with active pulmonary TB at the time of diagnosis and after start of standard chemotherapy. A composite clinical TB symptoms score, blood hemoglobin status and chest X-ray imaging were used to sub-group TB patients into 1.) mild and moderate-severe clinical TB, 2.) anemic and non-anemic TB, or 3.) limited and extensive lung involvement. Plasma levels of biomarkers associated with inflammation pathways were assessed using a Bio-Plex Magpix 37-multiplex assay. In parallel, Th1/Th2 cytokines were quantified with a 27-multiplex in matched plasma and cell culture supernatants from whole blood stimulated with M. tuberculosis-antigens using the QuantiFERON-TB Gold assay.ResultsClinical TB disease severity correlated with low blood hemoglobin levels and anemia but not with radiological findings in this study cohort. Multiplex protein analyses revealed that distinct clusters of inflammation markers and cytokines separated the different TB disease sub-groups with variable efficacy. Several top-ranked markers overlapped, while other markers were unique with regards to their importance to differentiate the TB disease severity groups. A distinct immune response profile defined by elevated levels of BAFF, LIGHT, sTNF-R1 and 2, IP-10, osteopontin, chitinase-3-like protein 1, and IFNα2 and IL-8, were most effective in separating TB patients with different clinical disease severity and were also promising candidates for treatment monitoring. TB patients with mild disease displayed immune polarization towards mixed Th1/Th2 responses, while pro-inflammatory and B cell stimulating cytokines as well as immunomodulatory mediators predominated in moderate-severe TB disease and anemia of TB.ConclusionsOur data demonstrated that clinical disease severity in TB is associated with anemia and distinct inflammatory immune profiles. These results contribute to the understanding of immunopathology in pulmonary TB and define top-ranked inflammatory mediators as biomarkers of disease severity and treatment prognosis.</p

Vitamin D and Phenylbutyrate Supplementation Does Not Modulate Gut Derived Immune Activation in HIV-1

Dysbiosis and a dysregulated gut immune barrier function contributes to chronic immune activation in HIV-1 infection. We investigated if nutritional supplementation with vitamin D and phenylbutyrate could improve gut-derived inflammation, selected microbial metabolites, and composition of the gut microbiota. Treatment-na&iuml;ve HIV-1-infected individuals (n = 167) were included from a double-blind, randomized, and placebo-controlled trial of daily 5000 IU vitamin D and 500 mg phenylbutyrate for 16 weeks (Clinicaltrials.gov NCT01702974). Baseline and per-protocol plasma samples at week 16 were analysed for soluble CD14, the antimicrobial peptide LL-37, kynurenine/tryptophan-ratio, TMAO, choline, and betaine. Assessment of the gut microbiota involved 16S rRNA gene sequencing of colonic biopsies. Vitamin D + phenylbutyrate treatment significantly increased 25-hydroxyvitamin D levels (p &lt; 0.001) but had no effects on sCD14, the kynurenine/tryptophan-ratio, TMAO, or choline levels. Subgroup-analyses of vitamin D insufficient subjects demonstrated a significant increase of LL-37 in the treatment group (p = 0.02), whereas treatment failed to significantly impact LL-37-levels in multiple regression analysis. Further, no effects on the microbiota was found in number of operational taxonomic units (p = 0.71), Shannon microbial diversity index (p = 0.82), or in principal component analyses (p = 0.83). Nutritional supplementation with vitamin D + phenylbutyrate did not modulate gut-derived inflammatory markers or microbial composition in treatment-na&iuml;ve HIV-1 individuals with active viral replication

Daily Nutritional Supplementation with Vitamin D3 and Phenylbutyrate to Treatment-Naïve HIV Patients Tested in a Randomized Placebo-Controlled Trial

Poor nutritional status is common among human immunodeficiency virus (HIV)-infected patients including vitamin D (vitD3) deficiency. We conducted a double-blinded, randomized, and placebo-controlled trial in Addis Ababa, Ethiopia, to investigate if daily nutritional supplementation with vitD3 (5000 IU) and phenylbutyrate (PBA, 2 &times; 500 mg) could mediate beneficial effects in treatment-na&iuml;ve HIV patients. Primary endpoint: the change in plasma HIV-1 comparing week 0 to 16 using modified intention-to-treat (mITT, n = 197) and per-protocol (n = 173) analyses. Secondary endpoints: longitudinal HIV viral load, T cell counts, body mass index (BMI), middle-upper-arm circumference (MUAC), and 25(OH)D3 levels in plasma. Baseline characteristics were detectable viral loads (median 7897 copies/mL), low CD4+ (median 410 cells/&micro;L), and elevated CD8+ (median 930 cells/&micro;L) T cell counts. Most subjects were vitD3 deficient at enrolment, but a gradual and significant improvement of vitD3 status was demonstrated in the vitD3 + PBA group compared with placebo (p &lt; 0.0001) from week 0 to 16 (median 37.5 versus 115.5 nmol/L). No significant changes in HIV viral load, CD4+ or CD8+ T cell counts, BMI or MUAC could be detected. Clinical adverse events were similar in both groups. Daily vitD3 + PBA for 16 weeks was well-tolerated and effectively improved vitD3 status but did not reduce viral load, restore peripheral T cell counts or improve BMI or MUAC in HIV patients with slow progressive disease. Clinicaltrials.gov NCT01702974