Zahra Toossi

Influence of vitamin D deficiency and vitamin D receptor polymorphisms on tuberculosis among Gujarati Asians in west London: a case-control study

BACKGROUND Susceptibility to disease after infection by Mycobacterium tuberculosis is influenced by environmental and host genetic factors. Vitamin D metabolism leads to activation of macrophages and restricts the intracellular growth of M. tuberculosis. This effect may be influenced by polymorphisms at three sites in the vitamin D receptor (VDR) gene. We investigated the interaction between serum vitamin D (25-hydroxycholecalciferol) concentrations and VDR genotype on susceptibility to tuberculosis. METHODS This study was a hospital-based case-control analysis of Asians of Gujarati origin, a mainly vegetarian immigrant population with a high rate of tuberculosis. We typed three VDR polymorphisms (defined by the presence of restriction endonuclease sites for Taq1, Bsm1, and Fok1) in 91 of 126 untreated patients with tuberculosis and 116 healthy contacts who had been sensitised to tuberculosis. Serum 25-hydroxycholecalciferol was recorded in 42 contacts and 103 patients. FINDINGS 25-hydroxycholecalciferol deficiency was associated with active tuberculosis (odds ratio 2.9 [95% CI 1.3-6.5], p=0.008), and undetectable serum 25-hydroxycholecalciferol (<7 nmol/L) carried a higher risk of tuberculosis (9.9 [1.3-76.2], p=0.009). Although there was no significant independent association between VDR genotype and tuberculosis, the combination of genotype TT/Tt and 25-hydroxycholecalciferol deficiency was associated with disease (2.8 [1.2-6.5]) and the presence of genotype ff or undetectable serum 25-hydroxycholecalciferol was strongly associated with disease (5.1 [1.4-18.4]). INTERPRETATION 25-hydroxycholecalciferol deficiency may contribute to the high occurrence of tuberculosis in this population. Polymorphisms in the VDR gene also contribute to susceptibility when considered in combination with 25-hydroxycholecalciferol deficiency.

A role for CD4+CD25+ T cells in regulation of the immune response during human tuberculosis

Active tuberculosis (TB) is associated with prolonged suppression of Mycobacterium tuberculosis (MTB)‐specific immune responses, but mechanisms involved are understood incompletely. We investigated a potential role for CD4+CD25+ regulatory T cells in depressed anti‐MTB immunity by evaluating serially CD4 cell phenotype and interferon (IFN)‐γ production by mononuclear cells from patients with TB. At diagnosis, frequencies of CD4+CD25+ T cells were increased in blood from TB patients compared to healthy purified protein derivative (PPD)‐positive controls (with a history of prior TB exposure), and remained elevated at completion of therapy (6 months). By contrast, expression of another activation marker, CD69, by CD4 T cells was increased at diagnosis, but declined rapidly to control levels with treatment. Among CD4+CD25+ T cells from TB patients at diagnosis those expressing high levels of CD25, probably representing regulatory T cells, were increased 2·9‐fold when compared to control subjects, while MTB‐stimulated IFN‐γ levels in whole blood supernatants were depressed. A role for CD4+CD25+ T cells in depressed IFN‐γ production during TB was substantiated in depletion experiments, where CD25+‐depleted CD4 T cells produced increased amounts of IFN‐γ upon MTB stimulation compared to unseparated T cells. At follow‐up, IFN‐γ production improved most significantly in blood from TB patients with high baseline frequencies of CD4+CD25+ T cells (more than threefold higher than controls for both total and CD25hi+ CD4 T cells), who also had a significant drop in frequencies of both total and ‘regulatory’ CD4+CD25+ T cells in response to treatment. Expansion of CD4+CD25+ regulatory T cells during active TB may play a role in depressed T cell IFN‐γ production.

Changes in Human Immunodeficiency Virus Type 1 Fitness and Genetic Diversity during Disease Progression

ABSTRACT This study examined the relationship between ex vivo human immunodeficiency virus type 1 (HIV-1) fitness and viral genetic diversity during the course of HIV-1 disease. Primary HIV-1 isolates from 10 patients at different time points were competed against control HIV-1 strains in peripheral blood mononuclear cell (PBMC) cultures to determine relative fitness values. Patient HIV-1 isolates sequentially gained fitness during disease at a significant rate that directly correlated with viral load and HIV-1 env C2V3 diversity. A loss in both fitness and viral diversity was observed upon the initiation of antiretroviral therapy. A possible relationship between genotype and phenotype (virus replication efficiency) is supported by the parallel increases in ex vivo fitness and viral diversity during disease, of which the correlation is largely based on specific V3 sequences. Syncytium-inducing, CXCR4-tropic HIV-1 isolates did have higher relative fitness values than non-syncytium-inducing, CCR5-tropic HIV-1 isolates, as determined by dual virus competitions in PBMC, but increases in fitness during disease were not solely powered by a gradual switch in coreceptor usage. These data provide in vivo evidence that increasing HIV-1 replication efficiency may be related to a concomitant increase in HIV-1 diversity, which in turn may be a determining factor in disease progression.

GENERALIZED IMMUNE ACTIVATION IN PULMONARY TUBERCULOSIS: CO-ACTIVATION WITH HIV INFECTION

Parameters of immune activation/differentiation were studied in a group of newly diagnosed HIV− and HIV+ pulmonary tuberculosis (TB) patients. Compared with controls, HLA‐DR expression on both CD4 and CD8 T cells from the HIV− TB patients was approximately doubled; HLA‐DR on T cells from the HIV+ group was tripled. The monocytes from both groups of patients expressed abnormally high levels of the Fcγ receptors I and III. Serum levels of tumour necrosis factor‐alpha (TNF‐α), neopterin and β2‐microglobulin were increased in HIV− and even more so in HIV+ TB patients. The expression of HLA‐DR on T cell subsets and of FcγR on monocytes correlated with each other, but not with serum activation markers. This pattern of non‐specific activation during TB infection may be associated with enhanced susceptibility to HIV infection.

Virological and Immunological Impact of Tuberculosis on Human Immunodeficiency Virus Type 1 Disease

Unlike other opportunistic infections associated with human immunodeficiency virus (HIV) type 1, tuberculosis (TB) occurs throughout the course of HIV-1 infection, and, as a chronic infection, its impact on viral activity is sustained. In dually infected subjects, HIV-1 load and heterogeneity are increased both locally and systemically during active TB. Studies over the past decade have indicated that Mycobacterium tuberculosis (MTB) infection supports HIV-1 replication and dissemination through the dysregulation of host cytokines, chemokines, and their receptors. Furthermore, concentrations of HIV-1 inhibitory chemokines are limited during TB and at sites of MTB infection. Cumulatively, these data indicate that TB provides a milieu of continuous cellular activation and irregularities in cytokine and chemokine circuits that are permissive of viral replication and expansion in situ. I address new research that has identified the basis for the augmentation of HIV-1 replication during TB and discuss potential immunotherapies to contain viral expansion during TB.

Apoptosis and T Cell Hyporesponsiveness in Pulmonary Tuberculosis

Mycobacterium tuberculosis (MTB)-induced T cell responses are depressed in peripheral blood mononuclear cells of persons with newly diagnosed pulmonary tuberculosis (TB), and levels of interferon (IFN)-gamma remain low even after completion of antituberculous therapy. Loss of MTB-reactive T cells through apoptotic mechanisms could account for this prolonged T cell hyporesponsiveness. T cell apoptosis was studied in TB patients and healthy control subjects. Both spontaneous and MTB-induced apoptosis (in CD4 and non-CD4 T cells) from TB patients was increased when compared with healthy control subjects, whereas coculture with control antigen (candida) had no effect on T cell apoptosis in either group of study subjects. An inverse correlation existed between increased MTB-induced T cell apoptosis and IFN-gamma and interleukin (IL)-2 immunoreactivities. Successful antituberculous chemotherapy resulted in a 50% reduction in both spontaneous and MTB-induced apoptosis, which coincided with 3- and 8-fold increases in levels of MTB-stimulated IL-2 and IFN-gamma, respectively. These data indicate that apoptotic pathways are operant during active MTB infection and may contribute to deletion of MTB-reactive T cells and the immunopathogenesis of this disease.

Mycobacterium tuberculosis (MTB)-stimulated production of nitric oxide by human alveolar macrophages and relationship of nitric oxide production to growth inhibition of MTB.

SETTING Although nitric oxide (NO) is a major proximate mediator of microbicidal activity in murine macrophages against intracellular pathogens including mycobacteria, its production by and effector role in human macrophages is not clear. OBJECTIVE To determine the capacity of Mycobacterium tuberculosis (MTB) to stimulate NO in human monocytes (MN) and alveolar macrophages (AM) and to assess the relationship between NO production and intracellular growth of MTB. DESIGN NO production (measured as nitrite) by MTB (H37Ra)-infected macrophages and intracellular growth of MTB were measured in cells from 17 healthy subjects. RESULTS MTB (5:1, MTB:cells) stimulated little to no NO by MN, but induced NO in AM at days 4 and 7 after infection. There was, however, variability in the response by AM to MTB: among seven subjects MTB-induced NO was low (4 +/- 2 microM, mean +/- SE); six subjects were moderate (56 +/- 11); four subjects were high (502 +/- 167). NO synthase inhibitors inhibited the production of NO by AM but did not significantly affect the intracellular growth of MTB, although a trend towards increased intracellular growth was seen on day 4 of culture. Intracellular growth of MTB in AM from low NO producers was significantly higher than that in AM from moderate NO producers, P < or = 0.05. Inducible NO synthase (iNOS) mRNA by RT-PCR was constitutively expressed by both MN and AM, but was further stimulated by MTB in AM > MN; MTB-induced iNOS protein was present in both MN and AM by Western blot analysis. CONCLUSION Thus, MTB-infected human AM are capable of producing NO and NO production correlates with intracellular growth inhibition of MTB in AM suggesting that NO may serve either directly or indirectly as a mycobactericidal mediator in human tissue macrophages.

Intestinal helminth co-infection has a negative impact on both anti-Mycobacterium tuberculosis immunity and clinical response to tuberculosis therapy

The impact of intestinal helminth infection on Mycobacterium tuberculosis (MTB)‐specific immune responses during active tuberculosis (TB) is not known. We investigated the role of intestinal helminth infection in anti‐MTB immunity by evaluating both cellular phenotype and cytokine profiles in patients with TB and patients with concomitant TB and intestinal helminth infection (TB + Helm) during TB therapy. Twenty‐seven per cent of TB patients enrolled for the study were co‐infected with at least one intestinal helminth. At baseline, absolute frequencies of leucocytes, monocytes and eosinophils from TB and TB + Helm patients differed from healthy subjects. Concomitant intestinal helminth infection in TB + Helm patients had a negative impact (P < 0·05) on absolute frequencies of CD3+, CD4+, CD8+, natural killer (NK) T and CD4+ CD25high T cell subsets when compared to either TB patients or healthy controls. Differences in CD4+ T cell frequencies were accompanied by lower interferon (IFN)‐γ and elevated and sustained interleukin (IL)‐10 levels in whole blood (WB) cultures from TB + Helm compared to TB patients. In addition to a depressed anti‐MTB immunity, TB + Helm patients also presented with more severe radiological pulmonary disease, with a significant difference (P = 0·013) in the number of involved lung zones at the end of TB treatment. The above data may indicate that concomitant intestinal helminth infection in patients with newly diagnosed TB skews their cytokine profile toward a T helper 2 response, which could favour persistent MTB infection and a more protracted clinical course of the disease.

Sputum Cytokine Levels in Patients with Pulmonary Tuberculosis as Early Markers of Mycobacterial Clearance

ABSTRACT Sputum and serum from patients with active pulmonary tuberculosis (TB), healthy purified protein derivative-positive adults, and patients with bacterial pneumonia were collected to simultaneously assess local immunity in the lungs and peripheral blood. To determine whether cytokine profiles in sputum from TB patients and control subjects were a reflection of its cellular composition, cytospin slides were prepared in parallel and assessed for the presence of relative proportions of epithelial cells, neutrophils, macrophages, and T cells. Gamma interferon (IFN-γ) in sputum from TB patients was markedly elevated over levels for both control groups. With anti-TB therapy, IFN-γ levels in sputum from TB patients decreased rapidly and by week 4 of treatment were comparable to those in sputum from controls. Further, IFN-γ levels in sputum closely followed mycobacterial clearance. Although detected at fourfold-lower levels, IFN-γ immunoreactivities in serum followed kinetics in sputum. TNF-α, interleukin 8 (IL-8) and IL-6 also were readily detected in sputum from TB patients at baseline and responded to anti-TB therapy. In contrast to IFN-γ, however, TNF-α and IL-8 levels also were elevated in sputum from pneumonia controls. These data indicate that sputum cytokines correlate with disease activity during active TB of the lung and may serve as potential early markers for sputum conversion and response to anti-TB therapy.

Regulation of Interleukin-12 by Interleukin-10, Transforming Growth Factor-β, Tumor Necrosis Factor-α, and Interferon-γ in Human Monocytes Infected with Mycobacterium tuberculosis H37Ra

Regulation of interleukin (IL)-12 production by coexpression of tumor necrosis factor (TNF)-alpha, IL-10, and transforming growth factor (TGF)-beta in human monocytes infected with Mycobacterium tuberculosis H37Ra was analyzed. Also, since IL-12 induces interferon (IFN)-gamma, the effect of IFN-gamma on IL-12 expression was examined. IL-12 mRNA was measured by reverse transcriptase-polymerase chain reaction and IL-12 protein by ELISA. IL-12 p35 mRNA was constitutive and inducible. IL-12 p70 protein paralleled IL-12 p40 protein expression. TNF-alpha protein expression occurred earlier than IL-12 p40 protein but was not required for IL-12 induction. Addition or neutralization of TGF-beta did not significantly alter IL-12 induction. In contrast, recombinant IL-10 reduced IL-12 and neutralization of IL-10 minimally enhanced IL-12. A pronounced increase in IL-12 followed IFN-gamma pretreatment, which selectively up-regulated IL-12 p35 mRNA. Further understanding of operative cytokine networks during M. tuberculosis infection may improve strategies for vaccine development and immunotherapy.