Improved sensitivity of an interferon-gamma release assay (T-SPOT.TB™) in combination with tuberculin skin test for the diagnosis of latent tuberculosis in the presence of HIV co-infection

Background Interferon-gamma release assays (IGRA) are more specific than the tuberculin skin test (TST) for the diagnosis of Mycobacterium tuberculosis infection. Data on sensitivity are controversial in HIV infection. Methods IGRA (T-SPOT.TB) was performed using lymphocytes stored within 6 months before culture-confirmed tuberculosis was diagnosed in HIV-infected individuals in the Swiss HIV Cohort Study. Results 64 individuals (69% males, 45% of non-white ethnicity, median age 35 years (interquartile range [IQR] 31-42), 28% with prior AIDS) were analysed. Median CD4 cell count was 223 cells/μl (IQR 103-339), HIV-RNA was 4.7 log10 copies/mL (IQR 4.3-5.2). T-SPOT.TB resulted positive in 25 patients (39%), negative in 18 (28%) and indeterminate in 21 (33%), corresponding to a sensitivity of 39% (95% CI 27-51%) if all test results were considered, and 58% (95% CI 43-74%) if indeterminate results were excluded. Sensitivity of IGRA was independent of CD4 cell count (p = 0.698). Among 44 individuals with available TST, 22 (50%) had a positive TST. Agreement between TST and IGRA was 57% (kappa = 0.14, p = 0.177), and in 34% (10/29) both tests were positive. Combining TST and IGRA (at least one test positive) resulted in an improved sensitivity of 67% (95% CI 52-81%). In multivariate analysis, older age was associated with negative results of TST and T-SPOT.TB (OR 3.07, 95% CI 1,22-7.74, p = 0.017, per 10 years older). Conclusions T-SPOT.TB and TST have similar sensitivity to detect latent TB in HIV-infected individuals. Combining TST and IGRA may help clinicians to better select HIV-infected individuals with latent tuberculosis who qualify for preventive treatment.

Improved antiretroviral treatment outcome in a rural African setting is associated with cART initiation at higher CD4 cell counts and better general health condition

Background Data on combination antiretroviral therapy (cART) in remote rural African regions is increasing. Methods We assessed prospectively initial cART in HIV-infected adults treated from 2005 to 2008 at St. Francis Designated District Hospital, Ifakara, Tanzania. Adherence was assisted by personal adherence supporters. We estimated risk factors of death or loss to follow-up by Cox regression during the first 12 months of cART. Results Overall, 1,463 individuals initiated cART, which was nevirapine-based in 84.6%. The median age was 40 years (IQR 34-47), 35.4% were males, 7.6% had proven tuberculosis. Median CD4 cell count was 131 cells/μl and 24.8% had WHO stage 4. Median CD4 cell count increased by 61 and 130 cells/μl after 6 and 12 months, respectively. 215 (14.7%) patients modified their treatment, mostly due to toxicity (56%), in particular polyneuropathy and anemia. Overall, 129 patients died (8.8%) and 189 (12.9%) were lost to follow-up. In a multivariate analysis, low CD4 cells at starting cART were associated with poorer survival and loss to follow-up (HR 1.77, 95% CI 1.15-2.75, p = 0.009; for CD4 <50 compared to >100 cells/μl). Higher weight was strongly associated with better survival (HR 0.63, 95% CI 0.51-0.76, p < 0.001 per 10 kg increase). Conclusions cART initiation at higher CD4 cell counts and better general health condition reduces HIV related mortality in a rural African setting. Efforts must be made to promote earlier HIV diagnosis to start cART timely. More research is needed to evaluate effective strategies to follow cART at a peripheral level with limited technical possibilities.

Development and validation of decision rules to guide frequency of monitoring CD4 cell count in HIV-1 infection before starting antiretroviral therapy

Background Although CD4 cell count monitoring is used to decide when to start antiretroviral therapy in patients with HIV-1 infection, there are no evidence-based recommendations regarding its optimal frequency. It is common practice to monitor every 3 to 6 months, often coupled with viral load monitoring. We developed rules to guide frequency of CD4 cell count monitoring in HIV infection before starting antiretroviral therapy, which we validated retrospectively in patients from the Swiss HIV Cohort Study. Methodology/Principal Findings We built up two prediction rules (“Snap-shot rule” for a single sample and “Track-shot rule” for multiple determinations) based on a systematic review of published longitudinal analyses of CD4 cell count trajectories. We applied the rules in 2608 untreated patients to classify their 18 061 CD4 counts as either justifiable or superfluous, according to their prior ≥5% or <5% chance of meeting predetermined thresholds for starting treatment. The percentage of measurements that both rules falsely deemed superfluous never exceeded 5%. Superfluous CD4 determinations represented 4%, 11%, and 39% of all actual determinations for treatment thresholds of 500, 350, and 200×106/L, respectively. The Track-shot rule was only marginally superior to the Snap-shot rule. Both rules lose usefulness for CD4 counts coming near to treatment threshold. Conclusions/Significance Frequent CD4 count monitoring of patients with CD4 counts well above the threshold for initiating therapy is unlikely to identify patients who require therapy. It appears sufficient to measure CD4 cell count 1 year after a count >650 for a threshold of 200, >900 for 350, or >1150 for 500×106/L, respectively. When CD4 counts fall below these limits, increased monitoring frequency becomes advisable. These rules offer guidance for efficient CD4 monitoring, particularly in resource-limited settings.

Equine CD4(+) CD25(high) T cells exhibit regulatory activity by close contact and cytokine-dependent mechanisms in vitro

Horses are particularly prone to allergic and autoimmune diseases, but little information about equine regulatory T cells (Treg) is currently available. The aim of this study therefore was to investigate the existence of CD4(+) Treg cells in horses, determine their suppressive function as well as their mechanism of action. Freshly isolated peripheral blood mononuclear cells (PBMC) from healthy horses were examined for CD4, CD25 and forkhead box P3 (FoxP3) expression. We show that equine FoxP3 is expressed constitutively by a population of CD4(+) CD25(+) T cells, mainly in the CD4(+) CD25(high) subpopulation. Proliferation of CD4(+) CD25(-) sorted cells stimulated with irradiated allogenic PBMC was significantly suppressed in co-culture with CD4(+) CD25(high) sorted cells in a dose-dependent manner. The mechanism of suppression by the CD4(+) CD25(high) cell population is mediated by close contact as well as interleukin (IL)-10 and transforming growth factor-beta1 (TGF-beta1) and probably other factors. In addition, we studied the in vitro induction of CD4(+) Treg and their characteristics compared to those of freshly isolated CD4(+) Treg cells. Upon stimulation with a combination of concanavalin A, TGF-beta1 and IL-2, CD4(+) CD25(+) T cells which express FoxP3 and have suppressive capability were induced from CD4(+) CD25(-) cells. The induced CD4(+) CD25(high) express higher levels of IL-10 and TGF-beta1 mRNA compared to the freshly isolated ones. Thus, in horses as in man, the circulating CD4(+) CD25(high) subpopulation contains natural Treg cells and functional Treg can be induced in vitro upon appropriate stimulation. Our study provides the first evidence of the regulatory function of CD4(+) CD25(+) cells in horses and offers insights into ex vivo manipulation of Treg cells.

Intraperitoneal Echinococcus multilocularis infection in mice modulates peritoneal CD4+ and CD8+ regulatory T cell development

Intraperitoneal proliferation of the metacestode stage of Echinococcus multilocularis in experimentally infected mice is followed by an impaired host immune response favoring parasite survival. We here demonstrate that infection in chronically infected mice was associated with a 3-fold increase of the percentages of CD4+ and CD8+ peritoneal T (pT) cells compared to uninfected controls. pT cells of infected mice expressed high levels of IL-4 mRNA, while only low amounts of IFN-gamma mRNA were detected, suggesting that a Th2-biased immune response predominated the late stage of disease. Peritoneal dendritic cells from infected mice (AE-pDCs) expressed high levels of TGF-beta mRNA and very low levels of IL-10 and IL-12 (p40) mRNA, and the expression of surface markers for DC-maturation such as MHC class II (Ia) molecules, CD80, CD86 and CD40 was down-regulated. In contrast to pDCs from non-infected mice, AE-pDCs did not enhance Concanavalin A (ConA)-induced proliferation when added to CD4+ pT and CD8+ pT cells of infected and non-infected mice, respectively. In addition, in the presence of a constant number of pDCs from non-infected mice, the proliferation of CD4+ pT cells obtained from infected animals to stimulation with ConA was lower when compared to the responses of CD4+ pT cells obtained from non-infected mice. This indicated that regulatory T cells (Treg) may interfere in the complex immunological host response to infection. Indeed, a subpopulation of regulatory CD4+ CD25+ pT cells isolated from E. multilocularis-infected mice reduced ConA-driven proliferation of CD4+ pT cells. The high expression levels of Foxp3 mRNA by CD4+ and CD8+ pT cells suggested that subpopulations of regulatory CD4+ Foxp3+ and CD8+ Foxp3+ T cells were involved in modulating the immune responses within the peritoneal cavity of E. multilocularis-infected mice.

CD4(+)CD25(+) T cells expressing FoxP3 in Icelandic horses affected with insect bite hypersensitivity

Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis caused by bites of midges from the genus Culicoides. We have shown previously that peripheral blood mononuclear cells (PBMC) from IBH-affected horses produce higher levels of IL-4 and lower levels of IL-10 and TGF-beta1 than those from healthy horses, suggesting that IBH is associated with a reduced regulatory immune response. FoxP3 is a crucial marker of regulatory T cells (Tregs). Here we have determined the proportion of CD4(+)CD25(+)FoxP3(+) T cells by flow cytometry in PBMC directly after isolation or after stimulation with Culicoides extract or a control antigen (Tetanus Toxoid). There were no differences between healthy and IBH horses either in the proportion of FoxP3(+)CD4(+)CD25(+) cells in freshly isolated PBMC or in the following stimulation with Tetanus Toxoid. However, upon stimulation of PBMC with the allergen, expression of FoxP3 by CD4(+)CD25(+high) and CD4(+)CD25(+dim) cells was significantly higher in healthy than in IBH horses. Addition of recombinant IL-4 to PBMC from healthy horses stimulated with the allergen significantly decreased the proportion of FoxP3 expressing cells within CD4(+)CD25(+high). These results suggest that IBH is associated with a decreased number of allergen-induced Tregs. This could be a consequence of the increased IL-4 production by PBMC of IBH-affected horses.

Comparative analysis of four lipoproteins from Mycoplasma mycoides subsp. mycoides Small Colony identifies LppA as a major T-cell antigen

Control of contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC), remains an important goal in Africa. Subunit vaccines triggering B and T-cell responses could represent a promising approach. To this aim, the T-cell immunogenicity of four MmmSC lipoproteins (LppA, LppB, LppC and LppQ), present in African strains and able to elicit humoral response, was evaluated. In vitro assays revealed that only LppA was recognized by lymph node lymphocytes taken from three cattle, 3 weeks after MmmSC exposure. Maintenance of the LppA-specific response, relying on CD4 T-cells and IFN gamma production, was then demonstrated 1 year after infection. LppA is thus an important target for the CD4 T-cells generated early after MmmSC infection and persisting in the lymph nodes of recovered cattle. Its role as a protective antigen and ability to in vivo trigger both arms of the host immune response remain to be evaluated.

Comparative analysis of canine monocyte- and bone-marrow-derived dendritic cells

Dendritic cells (DC) represent a heterogeneous cell family of major importance for innate immune responses against pathogens and antigen presentation during infection, cancer, allergy and autoimmunity. The aim of the present study was to characterize canine DC generated in vitro with respect to their phenotype, responsiveness to toll-like receptor (TLR) ligands and T-cell stimulatory capacity. DC were derived from monocytes (MoDC) and from bone marrow hematopoietic cells cultured with either Flt3-ligand (FL-BMDC) or with GM-CSF (GM-BMDC). All three methods generated cells with typical DC morphology that expressed CD1c, CD11c and CD14, similar to macrophages. However, CD40 was only found on DC, CD206 on MPhi and BMDC, but not on monocytes and MoDC. CD1c was not found on monocytes but on all in vitro differentiated cells. FL-BMDC and GM-BMDC were partially positive for CD4 and CD8. CD45RA was expressed on a subset of FL-BMDC but not on MoDC and GM-BMDC. MoDC and FL-DC responded well to TLR ligands including poly-IC (TLR2), Pam3Cys (TLR3), LPS (TLR4) and imiquimod (TLR7) by up-regulating MHC II and CD86. The generated DC and MPhi showed a stimulatory capacity for lymphocytes, which increased upon maturation with LPS. Taken together, our results are the basis for further characterization of canine DC subsets with respect to their role in inflammation and immune responses.

CD27 signaling increases the frequency of regulatory T cells and promotes tumor growth

Signaling of the TNF receptor superfamily member CD27 activates costimulatory pathways to elicit T- and B-cell responses. CD27 signaling is regulated by the expression of its ligand CD70 on subsets of dendritic cells and lymphocytes. Here, we analyzed the role of the CD27-CD70 interaction in the immunologic control of solid tumors in Cd27-deficient mice. In tumor-bearing wild-type mice, the CD27-CD70 interaction increased the frequency of regulatory T cells (Tregs), reduced tumor-specific T-cell responses, increased angiogenesis, and promoted tumor growth. CD27 signaling reduced apoptosis of Tregs in vivo and induced CD4(+) effector T cells (Teffs) to produce interleukin-2, a key survival factor for Tregs. Consequently, the frequency of Tregs and growth of solid tumors were reduced in Cd27-deficient mice or in wild-type mice treated with monoclonal antibody to block CD27 signaling. Our findings, therefore, provide a novel mechanism by which the adaptive immune system enhances tumor growth and may offer an attractive strategy to treat solid tumors.

Mean CD4 cell count changes in patients failing a first-line antiretroviral therapy in resource-limited settings

Background Changes in CD4 cell counts are poorly documented in individuals with low or moderate-level viremia while on antiretroviral treatment (ART) in resource-limited settings. We assessed the impact of on-going HIV-RNA replication on CD4 cell count slopes in patients treated with a first-line combination ART. Method Naïve patients on a first-line ART regimen with at least two measures of HIV-RNA available after ART initiation were included in the study. The relationships between mean CD4 cell count change and HIV-RNA at 6 and 12 months after ART initiation (M6 and M12) were assessed by linear mixed models adjusted for gender, age, clinical stage and year of starting ART. Results 3,338 patients were included (14 cohorts, 64% female) and the group had the following characteristics: a median follow-up time of 1.6 years, a median age of 34 years, and a median CD4 cell count at ART initiation of 107 cells/μL. All patients with suppressed HIV-RNA at M12 had a continuous increase in CD4 cell count up to 18 months after treatment initiation. By contrast, any degree of HIV-RNA replication both at M6 and M12 was associated with a flat or a decreasing CD4 cell count slope. Multivariable analysis using HIV-RNA thresholds of 10,000 and 5,000 copies confirmed the significant effect of HIV-RNA on CD4 cell counts both at M6 and M12. Conclusion In routinely monitored patients on an NNRTI-based first-line ART, on-going low-level HIV-RNA replication was associated with a poor immune outcome in patients who had detectable levels of the virus after one year of ART.

CD4 cell count and the risk of AIDS or death in HIV-Infected adults on combination antiretroviral therapy with a suppressed viral load: a longitudinal cohort study from COHERE

Background Most adults infected with HIV achieve viral suppression within a year of starting combination antiretroviral therapy (cART). It is important to understand the risk of AIDS events or death for patients with a suppressed viral load. Methods and Findings Using data from the Collaboration of Observational HIV Epidemiological Research Europe (2010 merger), we assessed the risk of a new AIDS-defining event or death in successfully treated patients. We accumulated episodes of viral suppression for each patient while on cART, each episode beginning with the second of two consecutive plasma viral load measurements <50 copies/µl and ending with either a measurement >500 copies/µl, the first of two consecutive measurements between 50–500 copies/µl, cART interruption or administrative censoring. We used stratified multivariate Cox models to estimate the association between time updated CD4 cell count and a new AIDS event or death or death alone. 75,336 patients contributed 104,265 suppression episodes and were suppressed while on cART for a median 2.7 years. The mortality rate was 4.8 per 1,000 years of viral suppression. A higher CD4 cell count was always associated with a reduced risk of a new AIDS event or death; with a hazard ratio per 100 cells/µl (95% CI) of: 0.35 (0.30–0.40) for counts <200 cells/µl, 0.81 (0.71–0.92) for counts 200 to <350 cells/µl, 0.74 (0.66–0.83) for counts 350 to <500 cells/µl, and 0.96 (0.92–0.99) for counts ≥500 cells/µl. A higher CD4 cell count became even more beneficial over time for patients with CD4 cell counts <200 cells/µl. Conclusions Despite the low mortality rate, the risk of a new AIDS event or death follows a CD4 cell count gradient in patients with viral suppression. A higher CD4 cell count was associated with the greatest benefit for patients with a CD4 cell count <200 cells/µl but still some slight benefit for those with a CD4 cell count ≥500 cells/µl.

Toxicity of thienopyridines on human neutrophil granulocytes and lymphocytes

Thienopyridines can cause neutropenia and agranulocytosis. The aim of the current investigations was to compare cytotoxicity of ticlopidine, clopidogrel, clopidogrel carboxylate and prasugrel for human neutrophil granulocytes with the toxicity for lymphocytes and to investigate underlying mechanisms. For granulocytes, clopidogrel, ticlopidine, clopidogrel carboxylate and prasugrel were concentration-dependently toxic starting at 10μM. Cytotoxicity could be prevented by the myeloperoxidase inhibitor rutin, but not by the cytochrome P450 inhibitor ketoconazole. All compounds were also toxic for lymphocytes, but cytotoxicity started at 100μM and could not be prevented by rutin or ketoconazole. Granulocytes metabolized ticlopidine, clopidogrel, clopidogrel carboxylate and prasugrel, and metabolization was inhibited by rutin, but not by ketoconazole. Metabolism of these compounds by lymphocytes was much slower and could not be inhibited by ketoconazole or rutin. In neutrophils, all compounds investigated decreased the electrical potential across the inner mitochondrial membrane, were associated with cellular accumulation of ROS, mitochondrial loss of cytochrome c and induction of apoptosis starting at 10μM. All of these effects could be inhibited by rutin, but not by ketoconazole. Similar findings were obtained in lymphocytes; but compared to neutrophils, the effects were detectable only at higher concentrations and were not inhibited by rutin. In conclusion, ticlopidine, clopidogrel, clopidogrel carboxylate and prasugrel are toxic for both granulocytes and lymphocytes. In granulocytes, cytotoxicity is more accentuated than in lymphocytes and depends on metabolization by myeloperoxidase. These findings suggest a mitochondrial mechanism for cytotoxicity for both myeloperoxidase-associated metabolites and, at higher concentrations, also for the parent compounds.

P-glycoprotein expression in lamina propria lymphocytes of duodenal biopsy samples in dogs with chronic idiopathic enteropathies

P-glycoprotein (p-gp) is a transmembrane protein functioning as a drug-efflux pump in the intestinal epithelium. Human patients with inflammatory bowel disease (IBD) who fail to respond to treatment with steroids express high levels of p-gp in lamina propria lymphocytes. The purpose of this study was to investigate p-gp expression in duodenal biopsy samples of dogs with chronic enteropathies and to evaluate the expression of p-gp after treatment with a known inducer of p-gp (prednisolone). Duodenal biopsy samples from 48 dogs were evaluated immunohistochemically with the mouse monoclonal antibody C219 for expression of p-gp in lamina propria lymphocytes. Biopsy samples were available from 15 dogs after treatment with prednisolone and 16 dogs after dietary therapy alone ("elimination diet"). Treatment with prednisolone resulted in an increase in p-gp expression (P=0.005). In contrast, dietary treatment alone produced no significant change in p-gp expression (P=0.59). A low p-gp score before initiation of steroid treatment was significantly associated with a positive response to treatment (P=0.01). These results indicate that lamina propria lymphocyte expression of p-gp is upregulated after prednisolone treatment in dogs with IBD, and that mucosal expression of p-gp may be of value in predicting the response to therapy.

Anti-HIV state but not apoptosis depends on IFN signature in CD4+ T cells

To gain insights into the molecular mechanisms underlying early host responses to HIV in the CD4(+) T cell target population, we examined gene expression in CD4(+) T cells isolated 24 h after ex vivo HIV infection of lymphocyte aggregate cultures derived from human tonsils. Gene profiling showed a distinct up-regulation of genes related to immune response and response to virus, notably of IFN-stimulated genes (ISGs), irrespective of the coreceptor tropism of the virus. This mostly IFN-alpha-dependent gene signature suggested the involvement of plasmacytoid dendritic cells, a principal component of the antiviral immune response. Indeed, depletion of plasmacytoid dendritic cells before HIV inoculation abrogated transcriptional up-regulation of several ISGs and resulted in increased levels of HIV replication. Treatment with a blocking anti-IFN-alphaR Ab yielded increased HIV replication; conversely, HIV replication was decreased in pDC-depleted cultures treated with IFN-alpha. Among up-regulated ISGs was also TRAIL, indicating a potential role of the IFN signature in apoptosis. However, a blocking anti-TRAIL Ab did not abrogate apoptosis of CD4(+) T cells in CXCR4-tropic HIV-infected cultures, suggesting the involvement of pathways other than TRAIL mediated. We conclude that acute HIV infection of lymphoid tissue results in up-regulation of ISGs in CD4(+) T cells, which induces an anti-HIV state but not apoptosis.