Arming of MAIT Cell Cytolytic Antimicrobial Activity Is Induced by IL-7 and Defective in HIV-1 Infection.

Mucosa-associated invariant T (MAIT) cells represent a large innate-like evolutionarily conserved antimicrobial T-cell subset in humans. MAIT cells recognize microbial riboflavin metabolites from a range of microbes presented by MR1 molecules. MAIT cells are impaired in several chronic diseases including HIV-1 infection, where they show signs of exhaustion and decline numerically. Here, we examined the broader effector functions of MAIT cells in this context and strategies to rescue their functions. Residual MAIT cells from HIV-infected patients displayed aberrant baseline levels of cytolytic proteins, and failed to mobilize cytolytic molecules in response to bacterial antigen. In particular, the induction of granzyme B (GrzB) expression was profoundly defective. The functionally impaired MAIT cell population exhibited abnormal T-bet and Eomes expression patterns that correlated with the deficiency in cytotoxic capacity and cytokine production. Effective antiretroviral therapy (ART) did not fully restore these aberrations. Interestingly, IL-7 was capable of arming resting MAIT cells from healthy donors into cytotoxic GrzB+ effector T cells capable of killing bacteria-infected cells and producing high levels of pro-inflammatory cytokines in an MR1-dependent fashion. Furthermore, IL-7 treatment enhanced the sensitivity of MAIT cells to detect low levels of bacteria. In HIV-infected patients, plasma IL-7 levels were positively correlated with MAIT cell numbers and function, and IL-7 treatment in vitro significantly restored MAIT cell effector functions even in the absence of ART. These results indicate that the cytolytic capacity in MAIT cells is severely defective in HIV-1 infected patients, and that the broad-based functional defect in these cells is associated with deficiency in critical transcription factors. Furthermore, IL-7 induces the arming of effector functions and enhances the sensitivity of MAIT cells, and may be considered in immunotherapeutic approaches to restore MAIT cells.

Lack of Association Between Interleukin 28B Polymorphism and Vertical Transmission of Hepatitis C.

OBJECTIVES: Single genetic nucleotide polymorphism (rs12979860) near the gene for interleukin 28B (IL28B) is known to be of importance for frequency of spontaneous clearance and treatment outcome in interferon-based therapies in patients with hepatitis C virus (HCV) infection. The aim of the present study was to investigate whether IL28B polymorphism in children and/or their mothers plays a role in vertical transmission of HCV (HCV-VT). METHODS: Plasma samples from 59 infected women, 76 uninfected children born to infected mothers, and 47 children with known vertically transmitted HCV infection, were analysed for IL28B polymorphism and classified by the IL28B genotype (C/C, C/T, and T/T) and by viral genotype. RESULTS: The proportion of children with genotype C/C was the same in the vertically infected (36%, 17/47) and the exposed uninfected children (38%, 29/76). No difference was seen when stratifying for viral genotype. There was no association between mothers' IL28B genotype and the risk of vertical transmission. CONCLUSIONS: Regardless of viral genotype we found no association between IL28B genotype and the risk of HCV-VT. The IL28B genotype CC, which has been shown to be favourable in other settings, was not protective of HCV-VT. Thus, other factors possibly associated with the risk of HCV-VT need to be explored.

Occasional spontaneous clearance of chronic hepatitis C virus in HIV-infected individuals.

The IL28B genotype has been found to have a strong influence on spontaneous clearance of acute HCV both in HCV mono- and HIV/ the HCV co-infected patients. Spontaneous clearance of chronic HCV without HCV treatment is rare. Here, we report on three chronic HCV cases co-infected with HIV with spontaneous clearance of their HCV infection, all with the IL28B CC genotype. These cases were derived from a surveillance of the total HIV/HCV co-infected cohort in Sweden (n =4 66). The estimated frequency of spontaneous clearance of chronic HCV infection in our cohort was calculated to be 0.6-4.7%. Our cases lend some support to the initiation of ART prior to HCV treatment in HIV/HCV co-infected patients. Furthermore, HCV-RNA testing should be recommended immediately before initiation of HCV treatment, to find the subset of HIV/HCV co-infected patients with IL28B CC that may have cleared their chronic infection spontaneously.

Improving on the ability of endogenous hepatitis B core antigen to prime cytotoxic T lymphocytes.

Hepatitis B virus core antigen (HBcAg) is thought to be a major target for specific cytotoxic T cells (CTLs) in hepatitis B virus infections. A single dose of hepatitis C virus nonstructural 3/4A DNA (<5 microg) effectively primes functional specific CTLs, independently of CD4(+) T helper cells and by different routes of immunization. In contrast, HBcAg-specific CTL priming was T helper cell dependent and highly sensitive to the dose and route of delivery. Although CTL priming was improved 10-fold by codon optimization and in vivo electroporation, low levels of DNA still failed to prime CTLs effectively. Only high doses (5 microg) of codon-optimized HBcAg delivered by in vivo electroporation primed in vivo lytic and polyfunctional CTLs. The ability of endogenous HBcAg to prime CTLs is surprisingly inefficient and differs from that of nonstructural 3/4A. This has important implications for the design of HBcAg-based therapeutic vaccines in humans.

Do bats use guano and urine stains to find new roosts? Tests with three group-living bats.

Many animals use social cues to find refuges. Bats can find roosts using the echolocation and social calls of conspecifics, but they might also use scent cues, a possibility which is less studied. The entrances of bat roosts are often marked by guano and urine, providing possible scent cues. We conducted eight experiments to test whether bats use the scent of guano and urine to find potential roosts. In field experiments, we tested if Molossus molossus (velvety free-tailed bats) in Panama and Eptesicus fuscus (big brown bats) in Ohio would investigate artificial roost boxes that were scented with guano and urine more often than a paired unscented control. We did not detect any difference in flights near the scented versus unscented roosts, and we detected only one entrance into any artificial roost (scented). In six captive experiments, we tested for the attraction of Desmodus rotundus (common vampire bats) and Molossus molossus to areas scented with guano and urine, under several conditions. Results were mixed, but overall suggested that the scent of guano and urine does not act as a strong lure for the tested bat species. We suggest that further tests of olfaction-based roost choice in bats should manipulate existing scent cues on familiar roosts.

Virus-specific T cell immune response in children and adolescents with chronic hepatitis B virus infection.

OBJECTIVES: To study the hepatitis B-specific T cell-mediated immune response in chronically infected children and adolescents. PATIENTS AND METHODS: In all, 36 HBsAg-positive patients, 2 to 19 years old, were included. There were 9 HBeAg-positive patients with normal levels of alanine aminotransferase (ALT) (group 1), 18 HBeAg-positive patients with elevated ALT (group 2), and 9 HBeAg-negative, anti-HBe-positive patients (group 3). Four patients in group 2 were treated with interferon during the study. In all patients, HBcAg-specific T cell proliferation and ALT levels were prospectively studied in repeated samples for a mean follow-up time of 1.6 years. The baseline HBV-DNA and plasma cytokine levels were determined, and genotypes were analyzed. RESULTS: The percent of patients with at least 1 sample indicating T cell proliferation was 55% in group 1 and 89% in groups 2 and 3, respectively (P = 0.07 group 1 vs group 2, P = 0.013 group 1 vs the combined groups 2 and 3). Tendencies for positive correlations between the degree of T cell proliferation and ALT levels were noted in groups 1 and 3 and for negative correlations in HBeAg seroconverting patients of group 2. In patients with successful interferon treatment, a pattern of more vigorous T cell proliferation than in patients with spontaneous seroconversion was noted. CONCLUSIONS: A majority of patients showed signs of ongoing T cell proliferation. The continuation of the T cell-mediated immune response seems to be of importance in maintaining the HBeAg seroconversion over time.

Chlorinated paraffins leaking from hand blenders can lead to significant human exposures.

BACKGROUND: Chlorinated paraffins (CPs, polychlorinated n-alkanes) are versatile, high-production-volume chemicals. A previous study indicated that hand blenders leak CPs into prepared food. OBJECTIVES: (1) to estimate exposure to CPs from hand blender use compared to background CP exposure from diet; (2) to assess the risk from human dietary exposure to CPs from hand blender use; (3) to investigate how hand blenders leak out CPs. METHODS: CPs were analyzed in food market baskets, in cooking oil/water samples (1g oil/100mL water) mixed using 16 different hand blenders, and in dismantled components of the hand blenders. RESULTS: Dietary intake of CPs from food market baskets was calculated to be 4.6µg/day per capita for Swedish adults. Total CP amounts in oil/water leakage samples ranged from <0.09 to 120µg using the hand blenders once. CP leakage showed no decreasing levels after 20 times of hand blender usage. CP profiles in the leakage samples matched those of self-lubricating bearings and/or polymer components disassembled from the hand blenders. CONCLUSIONS: Usage of 75% of the hand blenders tested will lead to increased human exposure to CPs. The intake of CPs for Swedish adults by using hand blenders once a day can raise their daily dietary intake by a factor of up to 26. The 95th percentile intake of CPs via using the hand blenders once a day exceeded the TDI for Swedish infants with a body weight <7.2kg. CP leakage came from blender components which contain CPs. The leakage may last several hundred times of hand blender use.

Low levels of microbial translocation marker LBP are associated with sustained viral response after anti-HCV treatment in HIV-1/HCV co-infected patients.

BACKGROUND: Microbial translocation (MT) contributes to immune activation during HIV and HCV infections. We investigated the kinetics of MT markers during anti-HCV and anti-HIV treatments, and if baseline plasma levels of lipopolysaccharide (LPS), lipopolysaccharide binding protein (LBP) and soluble CD14 (sCD14) could predict anti-HCV treatment outcome. METHODS: Plasma from 78 HIV-infected patients was evaluated for LPS, LBP and sCD14. The patients starting anti-HCV treatment (with ongoing antiretroviral (ART) treatment) were categorized into sustained viral responders (SVR; n = 21) or non-responders (NR; n = 15) based on treatment outcome. ART starting subjects--were categorized into chronically HCV-infected (CH; n = 24) and mono-infected (HIV; n = 18), based on the HCV infection status. Samples were collected before start (at baseline) of pegylated-interferon-alpha/ribavirin (peg-IFN/RBV) or antiretroviral-therapy and two years after treatment start (at follow up). χ2-test, non-parametric statistics and logistic regression were applied to determine the associations with treatment response and changes of the soluble markers. RESULTS: Plasma levels of LPS and sCD14 were elevated in all subjects before antiviral-treatment but remained unchanged at follow-up. Elevated levels of LBP were present in patients with HIV and HIV/HCV co-infection and were reduced by ART. Additionally, higher levels of LBP were present at baseline in NR vs. SVR. Higher levels of LBP at baseline were associated with non-response to peg-IFN/RBV treatment in both bivariate (OR: 0.19 95% CI: 0.06-0.31, p = 0.004) and multivariate analysis (OR: 1.43, 95% CI: 1.1-1.86, p = 0.07). CONCLUSION: In HIV/HCV co-infected patients high baseline LBP levels are associated with non-response to peg-IFN/RBV therapy. Plasma LBP (decreased by ART) may be a more relevant MT marker than LPS and sCD14.

Kinetics of microbial translocation markers in patients on efavirenz or lopinavir/r based antiretroviral therapy.

OBJECTIVES: We investigated whether there are differences in the effects on microbial translocation (MT) and enterocyte damage by different antiretroviral therapy (ART) regimens after 1.5 years and whether antibiotic use has impact on MT. In a randomized clinical trial (NCT01445223) on first line ART, patients started either lopinavir/r (LPV/r) (n = 34) or efavirenz (EFV) containing ART (n = 37). Lipopolysaccharide (LPS), sCD14, anti-flagellin antibodies and intestinal fatty acid binding protein (I-FABP) levels were determined in plasma at baseline (BL) and week 72 (w72). RESULTS: The levels of LPS and sCD14 were reduced from BL to w72 (157.5 pg/ml vs. 140.0 pg/ml, p = 0.0003; 3.13 ug/ml vs. 2.85 ug/ml, p = 0.005, respectively). The levels of anti-flagellin antibodies had decreased at w72 (0.35 vs 0.31 [OD]; p<0.0004), although significantly only in the LPV/r arm. I-FABP levels increased at w72 (2.26 ng/ml vs 3.13 ng/ml; p<0.0001), although significantly in EFV treated patients only. Patients given antibiotics at BL had lower sCD14 levels at w72 as revealed by ANCOVA compared to those who did not receive (Δ = -0.47 µg/ml; p = 0.015). CONCLUSIONS: Markers of MT and enterocyte damage are elevated in untreated HIV-1 infected patients. Long-term ART reduces the levels, except for I-FABP which role as a marker of MT is questionable in ART-experienced patients. Why the enterocyte damage seems to persist remains to be established. Also antibiotic usage may influence the kinetics of the markers of MT. TRIAL REGISTRATION: ClinicalTrials.gov NCT01445223.

High levels of HMGB1 in plasma may be due to ex vivo cell necrosis.

BACKGROUND: High mobility group box 1 protein (HMGB1) is considered an important biological marker during inflammation and malignancies. Here, we evaluated sample handling and effects of ex vivo necrosis on HMGB1 levels. MATERIALS AND METHODS: Plasma samples were obtained from healthy volunteers (n=14) simulating the standard laboratory conditions, overnight incubations and harsh treatment. HMGB1 levels were evaluated by ELISA or western blot. Additionally, levels of hemoglobin, hemolysis index and lactate dehydrogenase were measured. RESULTS: Plasma levels of HMGB1 were 9-fold increased in samples stored overnight at room temperature, as compared to those processed directly. The rapid centrifugation prevented the increase of HMGB1 in stored samples. Hemoglobin, hemolysis index and lactate dehydrogenase concentrations showed significant correlations with HMGB1 levels. CONCLUSION: Handling of blood samples is important for the accurate estimation of systemic HMGB1. We propose that all samples with high HMGB1 concentrations should be evaluated for markers of ex vivo necrosis.

Elevated plasma levels of lipopolysaccharide and high mobility group box-1 protein are associated with high viral load in HIV-1 infection: reduction by 2-year antiretroviral therapy.

OBJECTIVE: To investigate plasma levels of high mobility group box-1 protein (HMGB1), a marker of tissue necrosis and immune activation, as well as lipopolysaccharide (LPS), a marker of bacterial translocation, in HIV-1-infected patients. DESIGN: We studied 32 HIV-1-positive patients who had responded to antiretroviral therapy with undetectable viremia after 2 years, 10 nonresponders and 19 healthy controls. METHODS: HMGB1 was analyzed by ELISA, and LPS by Lamilus colometric assay. Nonparametric statistics were applied. RESULTS: In naive HIV-1 patients, HMGB1 and LPS were elevated as compared with controls (P < 0.001). LPS levels were higher in African and Oriental patients compared with whites (P = 0.007). Notably, viral load was two-fold higher in patients with LPS, and HMGB1 was above median as compared with other patients (P = 0.005). This association was largely driven by African patients, who had a five-fold increased viral load in the presence of elevated LPS and HMGB1. After 2 years of effective antiretroviral therapy, LPS was reduced to the same median level as in the control group (P < 0.001), and HMGB1 was also reduced (P = 0.001), whereas no reductions were seen in nonresponders. CONCLUSION: The new findings are the association of elevated plasma levels of LPS and HMGB1 with high viral load, as well as the normalized levels of LPS, and the reduction of HMGB1 after 2 years of effective antiretroviral therapy. As LPS and HMGB1 tend to form immunologically active complexes in vitro, we propose that such complexes may be involved in the immune activation and pathogenesis of HIV-1 infection.

Improved cell mediated immune responses after successful re-vaccination of non-responders to the hepatitis B virus surface antigen (HBsAg) vaccine using the combined hepatitis A and B vaccine.

We successfully re-vaccinated hepatitis B virus (HBV) vaccine non-responders using a double dose of the combined hepatitis A virus (HAV) and HBV vaccine. The hope was to improve priming of hepatitis B surface antigen (HBsAg)-specific cell mediated immune response (CMI) by an increased antigen dose and a theoretical adjuvant-effect from the local presence of a HAV-specific CMI. A few non-responders had a detectable HBsAg-specific CMI before re-vaccination. An in vitro detectable HBsAg-specific CMI was primed equally effective in non-responders (58%) as in first time vaccine recipients (68%). After the third dose a weak, albeit significant, association was observed between the magnitude of HBsAg-specific proliferation and anti-HBs levels. This regimen improves the priming of HBsAg-specific CMIs and antibodies.

In vivo clearance of hepatitis C virus nonstructural 3/4A-expressing hepatocytes by DNA vaccine-primed cytotoxic T lymphocytes.

A key question in the development of a therapeutic vaccine against hepatitis C virus (HCV) is whether vaccine-primed T cells enter the liver and eliminate HCV-expressing hepatocytes. In the absence of an infectious small-animal model, we evaluated liver homing of vaccine-primed T cells in mice with transient hepatic transgene expression of the HCV nonstructural 3/4A (NS3/4A) protein. We found that T cells primed by transdermal DNA-based vaccination entered the liver and cleared NS3/4A-expressing hepatocytes in transiently transgenic CD8(+/+) mice but not in CD8(-/-) mice. Hence, peripherally primed NS3/4A-specific CD8(+) T cells home to the liver and clear HCV protein-expressing hepatocytes.

Priming of cytotoxic T cell responses to exogenous hepatitis B virus core antigen is B cell dependent.

The hepatitis B virus (HBV) core antigen (HBcAg) has a unique ability to bind a high frequency of naive human and murine B cells. The role of HBcAg-binding naive B cells in the immunogenicity of HBcAg is not clear. The HBcAg-binding properties of naive B cells were characterized using HBcAg particles with mutated spike region (residues 76-85) sequences. Deletion of residues 76-85 (HBcDelta76-85) destroyed naive B cell binding, whereas deletion of residues 79-85 did not. HBcAg particles with an Ile instead of the natural Ala at position 80 did not bind naive B cells, whereas reversion of Ile80-->Ala restored B cell binding. Destroying the B cell-binding ability of HBcAg had a marginal effect on the overall B cell immunogenicity of the different particles, suggesting that they were equally efficient in priming T helper cells. Therefore, the importance of HBcAg-binding B cells is studied with relation to the priming of HBcAg-specific cytotoxic T cells (CTLs). The role of HBcAg-binding B cells in the priming of HBcAg-specific CTLs was evaluated by immunization with endogenous HBcAg (DNA immunization) and exogenous recombinant HBcAg particles. Endogenous HBcAg primed HBcAg-specific CTLs in wild-type and B cell-deficient mice, whereas exogenous HBcAg primed HBcAg-specific CTLs only in wild-type mice. Importantly, HBcDelta76-85 did not prime CTLs despite the presence of B cells. Thus, the ability of exogenous HBcAg particles to prime specific CTLs is B cell dependent, suggesting a possible role for HBcAg-binding B cells in HBV infections.