HTLV-1 infection is associated with a history of active tuberculosis among family members of HTLV-1-infected patients in Peru.

The purpose of this study was to assess the association between human T-lymphotropic virus 1 (HTLV-1) and a lifetime history of active tuberculosis (TB) among relatives of HTLV-1-infected patients. We reviewed clinical charts of all relatives of HTLV-1-infected index cases who attended our institute in Lima from 1990-2004. The data of 1233 relatives was analysed; 394 (32.0%) were HTLV-1 positive. Eighty-one subjects (6.6%) had a history of active TB, including 45/394 (11.4%) HTLV-1-positive and 36/839 (4.3%) HTLV-1-negative relatives (P<0.001). On multivariate analysis, three factors were associated with TB history: HTLV-1 infection (adjusted OR 2.5, 95% CI 1.6-3.9), age (adjusted OR 1.3, 95% CI 1.1-1.5 per 10-year age increase) and relation to the index case (adjusted OR 2.6, 95% CI 1.3-5.1, for siblings vs. spouses of index cases). In conclusion, HTLV-1 infection may increase the susceptibility to active TB. In populations where both infections are frequent, such an association could affect the dynamics of TB.

HTLV-1 infection is frequent among out-patients with pulmonary tuberculosis in northern Lima, Peru.

SETTING: Tuberculosis (TB) and human T-lymphotropic virus 1 (HTLV-1) are frequent in Peru. The prevalence of HTLV-1 among Peruvian TB patients is unknown. OBJECTIVE: To determine the prevalence of HTLV-1, HTLV-2 and the human immunodeficiency virus (HIV) in out-patients with TB and to compare HTLV-1-infected patients with seronegative patients. DESIGN: Cross-sectional study including subjects aged 18-65 years diagnosed with smear-positive pulmonary TB at health centres in northern Lima from November 2004 to August 2005. HTLV and HIV screening was performed using enzyme-linked immunosorbent assay; HTLV-1 and HTLV-2 were confirmed using line immunoassay. RESULTS: There were 311 participants with a median age of 29 years; 173 (56%) were men. HTLV-1 prevalence was 5.8% (18/311, 95%CI 3.2-8.4) and HIV prevalence was 1.3% (4/304, 95%CI 0.4-3.3). HTLV-2 was not diagnosed. In comparison with HIV- and HTLV-seronegative patients, HTLV-1-infected subjects were older (median age 44 vs. 28, P < 0.001) and were more likely to have been born in the southern Andes (OR 4.4, 95%CI 1.6-11.9). They were also more likely to report a history of TB deaths in the family (OR 5.4, 95%CI 1.7-16.8) and had more sputum smear results graded as 3+ (OR 4.1, 95%CI 1.5-11.2). CONCLUSION: HTLV-1 screening among Peruvian TB patients is important. Because 3+ sputum smears are frequent and mortality is high among relatives, families of HTLV-1/TB-positive cases merit special attention.

Proviral load and immune markers associated with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in Peru.

Human T-lymphotropic virus type 1 (HTLV-1) is the aetiological agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The objective of this study is to identify which ex vivo and in vivo markers are associated independently with HAM/TSP in a Peruvian population. Eighty-one subjects (33 men/48 women) were enrolled: 35 presented with HAM/TSP, 33 were asymptomatic HTLV-1 carriers (ACs) and 13 were HTLV-1-seronegative controls (SCs). Ex vivo markers included T cell proliferation and Th1 [interferon (IFN)-gamma], Th2 [interleukin (IL)-4, IL-5], proinflammatory [tumour necrosis factor (TNF)-alpha] and anti-inflammatory (IL-10) cytokine production in non-stimulated peripheral blood mononuclear cell (PBMC) cultures. In vivo CD4(+) T cell count, markers of Th1 [interferon-inducible protein (IP)-10] and Th2 (sCD30) activity in plasma and HTLV-1 proviral load in PBMCs were also evaluated. In univariate analysis, several markers, including T cell proliferation, IFN-gamma, IP-10, sCD30 and proviral load were associated with HAM/TSP, but in a multiple logistic regression analysis only the proviral load remained associated significantly with disease manifestation [adjusted OR 9.10 (1.24-66.91)]. Our findings suggest that HAM/TSP is associated primarily with proviral load, whereas the observed association with some immune markers seems secondary.

Ex vivo induction of viral antigen-specific CD8 T cell responses using mRNA-electroporated CD40-activated B cells.

Cell-based immunotherapy, in which antigen-loaded antigen-presenting cells (APC) are used to elicit T cell responses, has become part of the search for alternative cancer and infectious disease treatments. Here, we report on the feasibility of using mRNA-electroporated CD40-activated B cells (CD40-B cells) as alternative APC for the ex vivo induction of antigen-specific CD8(+) T cell responses. The potential of CD40-B cells as APC is reflected in their phenotypic analysis, showing a polyclonal, strongly activated B cell population with high expression of MHC and co-stimulatory molecules. Flow cytometric analysis of EGFP expression 24 h after EGFP mRNA-electroporation showed that CD40-B cells can be RNA transfected with high gene transfer efficiency. No difference in transfection efficiency or postelectroporation viability was observed between CD40-B cells and monocyte-derived dendritic cells (DC). Our first series of experiments show clearly that peptide-pulsed CD40-B cells are able to (re)activate both CD8+ and CD4(+) T cells against influenza and cytomegalovirus (CMV) antigens. To demonstrate the ability of viral antigen mRNA-electroporated CD40-B cells to induce virus-specific CD8+ T cell responses, these antigen-loaded cells were co-cultured in vitro with autologous peripheral blood mononuclear cells (PBMC) for 7 days followed by analysis of T cell antigen-specificity. These experiments show that CD40-B cells electroporated with influenza M1 mRNA or with CMV pp65 mRNA are able to activate antigen-specific interferon (IFN)-gamma-producing CD8(+) T cells. These findings demonstrate that mRNA-electroporated CD40-B cells can be used as alternative APC for the induction of antigen-specific (memory) CD8(+) T cell responses, which might overcome some of the drawbacks inherent to DC immunotherapy protocols.

Involvement of interleukin 18 in Crohn's disease: evidence from in vitro analysis of human gut inflammatory cells and from experimental colitis models.

An imbalance of immunoregulatory factors and/or cells contributes to uncontrolled mucosal T cell activation and inflammation in Crohn's disease (CD). Bioactive interleukin (IL)-18 has been shown to be produced by macrophages in CD lesions. We report here that T cells freshly isolated from inflamed tissue of CD patients (and not T cells from control intestinal tissue) were responsive to IL-18. In the presence of IL-18, these T cells produced more interferon (IFN)-gamma and less IL-10. To analyse further the role of IL-18 in this disease, an acute and a chronic model of murine colitis were used. IL-18 mRNA was significantly enhanced in trinitrobenzene sulphonic acid (TNBS) induced colitis, and treatment with IL-18 binding protein (IL-18BPa), which neutralizes IL-18 bioactivity, significantly reduced the severity of colitis. However, IL-18BPa did not affect the course of chronic colitis in CD45RBhighCD4+ T cell reconstituted SCID mice. Production of IFN-gamma in lamina propria mononuclear cell cultures from IL-18BPa-treated SCID mice was decreased, but at the same time fewer lamina propria CD4+ T cells harvested from IL-18BPa-treated mice compared to non-treated mice were in apoptosis. We conclude that IL-18 clearly has a modulatory role in the inflammatory cascade of CD and experimental colitis by affecting IFN-gamma and IL-10 production, and apoptosis. In view of the divergent effects of IL-18 neutralization in the two different murine colitis models, it is unlikely that IL-18 is at the top of this cascade.

Activation of beta-chemokines and CCR5 in persons infected with human immunodeficiency virus type 1 and tuberculosis.

Tuberculosis (TB) in human immunodeficiency virus type 1 (HIV-1)-infected persons is associated with progression of HIV-1 disease. The expression of macrophage inflammatory protein (MIP)-1alpha and CCR5 was assessed in HIV-1-infected patients with pulmonary TB (HIV-1/PTB) and without PTB (HIV-1/C), PTB patients not infected with HIV-1 (PTB), and control subjects. Mycobacterium tuberculosis (MTB)-induced MIP-1alpha production was lower in peripheral blood mononuclear cells (PBMC) of HIV-1/PTB patients than in those of PTB patients (P< .05) and was lower in PBMC of HIV-1/C patients than in those of control subjects (P< .005). However, MIP-1alpha production was higher in PBMC of HIV/PTB patients than in those of HIV-1/C patients (P< .01). The pattern of MTB-induced RANTES production was similar to that of MIP-1alpha. However, MTB induced greater expression of mRNA for CCR5 in PBMC of HIV-1/PTB patients than in those of HIV-1/C patients (P< .04). Furthermore, the MTB-induced HIV p24 antigen level in PBMC of HIV-1/PTB patients with a CD4 cell count <500 cells/microL was higher (P< .05) than that in HIV-1/C patients. Thus, perturbations in chemokine pathways in HIV-1/PTB patients may accelerate HIV-1 disease.

T cell activation, apoptosis and cytokine dysregulation in the (co)pathogenesis of HIV and pulmonary tuberculosis (TB).

Immune parameters were compared in four groups of Ugandan subjects: HIV-and HIV+ adult patients with active pulmonary TB (HIV- PTB n = 38; HIV+ PTB n = 28), patients with HIV infection only (n = 26) and PPD+ healthy controls (n = 25). Compared with healthy controls, CD4 and CD8 T cells from patients with HIV and/or PTB expressed more activation markers (HLA-DR, CD38); their CD8 T cells expressed more CD95 (pre-apoptosis) and less CD28 (co-stimulatory receptor). Peripheral blood mononuclear cells (PBMC) of patients with either HIV or PTB were impaired in interferon-gamma (IFN-gamma) production upon antigenic stimulation. PTB (with or without HIV) was characterized by monocytosis, granulocytosis, increased transforming growth factor-beta 1 production and PPD-induced apoptosis. In vivo CD4 T cell depletion, in vitro increased spontaneous CD4 T cell apoptosis and defects in IFN-gamma responses upon mitogenic stimulation were restricted to HIV+ subjects (with or without PTB). Overlapping and distinctive immune alterations, associated with PTB and HIV, might explain mutual unfavourable influences of both diseases.

Modeling HIV transfer between dendritic cells and T cells: importance of HIV phenotype, dendritic cell-T cell contact and T-cell activation.

OBJECTIVE: To study the requirements for HIV transfer between dendritic cells (DC) and CD4 T cells, using an in vitro model, combined with flow cytometry. METHODS: Immature DC and macrophages (MA) were generated from monocytes. After infection, DC or MA were cultured alone or with purified CD4 T cells. Intracellular HIV was measured, using (1) the monocyte (MO)-tropic AD8 HIV, endowed with enhanced green fluorescent protein (EGFP); and (2) intracellular staining of laboratory HIV strains and clones from primary isolates. RESULTS: (1) Clone AD8-EGFP infected DC and MA with equal efficiency, but the virus was preferentially transferred from DC to autologous T cells. (2) DC were more productively infected with R5/NSI, as compared to X4/SI, HIV, but both HIV phenotypes were easily transmitted to autologous T4 cells. (3) HIV-infected DC transferred the virus to T cells across a semi-permeable membrane, if the T cells were in contact with non-infected DC. (4) Co-culture of T cells with autologous non-infected DC induced T-cell activation. HIV-infected DC selectively increased HLA-DR on T cells and HLA-DR (+) T cells were preferential targets for HIV transfer. (5) Resting Ba-L-infected CD4 T cells were able to transmit the virus 'inversely' to co-cultured DC. CONCLUSION: HIV transfer between monocyte-derived dendritic cells and autologous CD4 T cells was directly demonstrated using flow cytometry. The transfer proceeded in both directions, depended on cellular contact and was associated with partial T-cell activation. This model, representing relevant in vivo targets of HIV, is useful to further investigate interactions between HIV, DC and T cells, without the need for primary ex vivo DC.

Hyperreactive malaria in expatriates returning from sub-Saharan Africa.

The extreme presentation of hyperreactive malaria is hyperreactive malarial splenomegaly syndrome (HMS). Some patients present with a less pronounced syndrome. To investigate whether the degree of splenomegaly correlates with the degree of immune stimulation, whether prophylaxis or recent treatment play a role, and whether short therapy alone is effective, we examined retrospectively the medical records of expatriates with exposure to P. falciparum who attended our outpatient department from 1986 to 1997, particularly subacute symptoms or signs, strongly elevated malarial antibodies and elevated total serum IgM. We analysed duration of stay, prophlyaxis intake, spleen size, serum IgM levels and response to antimalarial treatment. Serum IgM levels were significantly higher in patients with larger splenomegaly. The use of chloroquine alone as treatment for presumptive or proved malaria attacks was correlated with larger spleen size. Short adequate antimalarial therapy resulted in marked improvement or complete recovery. In nine patients the hyperreactive response reappeared after re-exposure, in four of them twice. We conclude that patients with subacute symptoms but without gross splenomegaly may have very high levels of IgM and malarial antibodies, and relapse on re-exposure, suggesting the existence of a variant of the hyperreactive malarial splenomegaly syndrome without gross splenomegaly.

A dual infection/competition assay shows a correlation between ex vivo human immunodeficiency virus type 1 fitness and disease progression.

This study was designed to examine the impact of human immunodeficiency virus type 1 (HIV-1) fitness on disease progression through the use of a dual competition/heteroduplex tracking assay (HTA). Despite numerous studies on the impact of HIV-1 diversity and HIV-specific immune response on disease progression, we still do not have a firm understanding of the long-term pathogenesis of this virus. Strong and early CD8-positive cytotoxic T-cell and CD4-positive T-helper cell responses directed toward HIV-infected cells appear to curb HIV pathogenesis. However, the rate at which the virus infects the CD4(+) T-cell population and possibly destroys the HIV-specific immune response may also alter the rate of disease progression. For HIV-1 fitness studies, we established conditions for dual HIV-1 infections of peripheral blood mononuclear cells (PBMC) and a sensitive HTA to measure relative virus production. A pairwise comparison was then performed to estimate the relative fitness of various non-syncytium-inducing/CCR5-tropic (NSI/R5) and syncytium-inducing/CXCR4-tropic (SI/X4) HIV-1 isolates. Four HIV-1 strains (two NSI/R5 and two SI/X4) with moderate ex vivo fitness were then selected as controls and competed against primary HIV-1 isolates from an HIV-infected Belgian cohort. HIV-1 isolates from long-term survivors (LTS) were outcompeted by control strains and were significantly less fit than HIV-1 isolates from patients with accelerated progression to AIDS (PRO). In addition, NSI/R5 HIV-1 isolates from PRO overgrew control SI/X4 strains, suggesting that not all SI/X4 HIV-1 isolates replicate more efficiently than all NSI/R5 isolates. Finally, there were strong, independent correlations between viral load and the total relative fitness values of HIV-1 isolates from PRO (r = 0.84, P = 0.033) and LTS (r = 0.86, P = 0.028). Separation of the PRO and LTS plots suggest that HIV-1 fitness together with viral load may be a strong predictor for the rate of disease progression.

The HIV-2 genotype and the HIV-1 syncytium-inducing phenotype are associated with a lower virus replication in dendritic cells.

During sexual transmission, HIV infects the mucosal dendritic cells and is transferred to CD4 T cells. Whether HIV variants of a particular genetic (sub)type or phenotype selectively infect dendritic cells (DC) or are preferentially transferred to T cells remains highly controversial. To avoid the cumbersome use of primary dendritic cells, in vitro dendritic cell models were generated from precursors, either hematopoietic progenitor cells (HPC) or monocytes (MO). Productive infection in the dendritic cells and transfer of the virus to T cells was assessed for a range of HIV variants. HPC-derived dendritic cells (HPC-DC) were more susceptible to HIV-1 than to HIV-2 isolates. The HIV-1 group O strains were more productive in HPC-DC than group M, but amongst the latter, no subtype-related difference was observed. Both non-syncytium-inducing (NSI) and SI HIV isolates and lab strains could productively infect HPC-DC, albeit with a different efficiency. Adding blocking antibodies confirmed that both CCR-5 and CXCR-4 co-receptors were functional. Biological HIV-1 clones of the NSI/R5 phenotype infected more readily HPC-DC than SI/X4 clones. MO-derived dendritic cells were, however, more exclusive in their preference for NSI/R5 clones. Some HIV variants, that did not grow readily in HPC-DC alone, could be rescued by adding resting or pre-activated T cells. The present data show that HIV-2 isolates and SI clones replicate less in model-DC, but no preference for a particular HIV-1 subtype was evident. Co-culture with T cells could "correct" a limited growth in dendritic cells. Clearly, both intrinsic dendritic cell susceptibility and enhancement by T cells are explained only partly by HIV genotype and phenotype. The in vitro dendritic cell models seem useful tools to further unravel interactions between HIV, DC, and T cells.

GP130 and c-kit signalling, initiated by the sIL-6R/IL-6 complex, is insufficient to expand the primitive adult bone marrow CD34+CD38- pre-CFU cell.

It has previously been shown that gp130 and c-kit signalling synergize for the ex vivo expansion of human cord blood (CB) CD34+ haematopoietic progenitor cells. We were interested in evaluating this synergy within an ontogenetically different haematopoietic tissue [i.e. adult bone marrow (BM)] and on a more primitive progenitor subset (i.e. CD34+ CD38-cells), which are highly enriched for pre-colony forming unit (CFU) cells. These cells were plated out in a primary liquid culture supplemented with either interleukin (IL)-6+stem cell factor (SCF), IL-6+ SCF+soluble IL-6 receptor (sIL-6R), IL-6+SCF+sIL-6R+IL3+IL-1 or SCF+IL-3+IL-6+IL-1. Cell counting after liquid culture revealed an absolute expansion of 2.2-, 4.1-, 89.5- and 65.7-fold compared with initial cell input for the four-cytokine combinations, respectively. The secondary read-out assay revealed that this cell expansion in the liquid culture also resulted in CFU generation, with absolute cloning efficiencies of 0.002, 0.024, 12.13 and 7.73 (per cell initially present) for the respective cytokine combinations. These results indicate that gp130 and c-kit signalling alone (i.e. using IL6+SCF+sIL-6R), in terms of both cell number and CFU generation, insufficiently stimulate primitive adult BM CD34+CD38- haematopoietic cells in order to reach a CFU generation comparable with that obtained after multifactor stimulation. Adding sIL-6R to the multifactor stimulation and compared with this multifactor stimulation, a 1.7-fold synergy in terms of cell expansion and a 3.0-fold synergy in terms of CFU generation are obtained. The sIL-6R/IL-6 complex thus has a narrower spectrum of action on primitive adult BM CD34+CD38- cells than on CB CD34+ cells.

Decreased CD40 ligand induction in CD4 T cells and dysregulated IL-12 production during HIV infection.

During HIV infection various cytokines are overproduced in early stages, whereas in advanced disease cytokines of the T helper 1 type (e.g. interferon-gamma (IFN-gamma)) are selectively deficient. During antigenic stimulation, the production of type-1 cytokines is enhanced by IL-12, secreted by antigen-presenting cells (APC) after their interaction with activated CD4 T cells. Two factors are essential in this process: priming APC with IFN-gamma and triggering the CD40 receptor on APC by CD40 ligand (CD40L). In view of the importance of this pathway, we compared its regulation in HIV-infected and control subjects. After cross-linking of the T cell receptor (TCR)/CD3 complex, the proportional expression of CD40L was similar on CD4+ T cells from controls and from patients with high circulating CD4 T counts (> 500/microl), but CD40L up-regulation was significantly reduced in patients with more advanced disease. Simultaneous triggering of the costimulatory receptor CD28 on T cells through its natural ligand CD80 partly corrected the CD40L defect in patients with intermediate CD4 T counts (200-500), but not in AIDS patients. Early production of IFN-gamma was preserved in lymphocytes from HIV+ patients. The expression of CD40 on peripheral monocytes from HIV+ subjects was increased in a disease stage-related fashion. Stimulation of mononuclear cells through cell-bound CD40L and soluble IFN-gamma induced significantly higher IL-12 in cultures from patients with > 200 circulating CD4 T cells, whereas IL-12 production was marginally decreased in cultures from patients with < 200 CD4 T cells, compared with healthy control cultures. In conclusion, our data suggest that impaired CD40L induction on CD4 T cells contributes to deficient type-1 responses through decreased IL-12 production in AIDS infection, whereas enhanced CD40-mediated IL-12 production in less advanced stages might contribute to increased levels of various cytokines in early disease

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.

Nonviral transfection of distinct types of human dendritic cells: high-efficiency gene transfer by electroporation into hematopoietic progenitor- but not monocyte-derived dendritic cells.

Human dendritic cells (DC) are highly professional antigen presenting cells for the priming of naive cytotoxic T cells. Gene transfer in DC would be a useful strategy to load DC with relevant de novo synthesized antigens for immunotherapeutical purposes. As a first step towards a DC-based gene therapy, we examined the efficiency of nonviral transfection in different types of cultured human dendritic cells with a humanized red-shifted green fluorescent protein reporter gene. Plasmid DNA transfection by electroporation or lipofection was used to transfect CD34+ progenitor cell-derived DC (PC-DC) and Langerhans' cells (PC-LC), as well as monocyte-derived DC (Mo-DC). While lipofection was unsuccessful in all types of DC, we obtained high-efficiency gene transfer by electroporation in PC-LC (16%) and PC-DC (12%). In contrast, electroporation was strikingly less efficient in Mo-DC (< or = 2%). The potent allostimulatory capacity of DC was still retained in electroporated PC-DC and PC-LC. In conclusion, electroporation of antigen expressing plasmid DNA is an efficient tool for nonviral gene transfer in PC-DC and PC-LC, but not in Mo-DC and could be useful for the development of DC-based tumor immunotherapy.