Epstein-Barr Virus load and immune activation in Human Immunodeficiency Virus type 1-infected patients

Background

Patients infected with HIV-1 are at high risk of developing Epstein-Barr Virus (EBV)-related diseases. Chronic immune activation is a hallmark of HIV-1 pathogenesis and may play a role in B-cell stimulation and expansion of EBV-infected cells.

Objectives

The aim of the study was to define the relationship between parameters of immune activation and EBV load in HIV-1-infected subjects.

Study design

A total of 156 HIV-1-infected patients were studied. EBV types 1 and 2 were quantified on peripheral blood mononuclear cells by multiplex real-time PCR. Plasma levels of cytokines and lipopolysaccharide (LPS) were determined by immunoenzymatic assays. B-cell activation was analyzed by flow cytometry.

Results

EBV-DNA was detected in 114 patients, and in all but 3 was EBV type 1. The median [interquartile] EBV-DNA load was 43[1-151] copies/10⁵ PBMC. EBV-DNA load was higher in patients with detectable HIV-1 plasma viremia, despite good immunological status (CD4 > 500 cells/μl), than in patients with undetectable HIV-1 plasma viremia regardless of immunological status (46[5-136] copies/10⁵ cells vs 17[1-56] copies/10⁵ cells, p = 0.008). Patients with high EBV-DNA load (>median value) had higher levels of LPS and proinflammatory cytokines (IL-6, IL-10 and TNF-α) than patients with low EBV load. Furthermore, percentages of activated B-cells correlated with EBV-DNA load (r_s = 0.754; p < 0.001).

Conclusions

Overall, these findings indicate a strong association between HIV-1 viremia, markers of immune activation and EBV load and suggest that persistence of HIV-1 viremia and immune activation, regardless of peripheral CD4 cell depletion/repopulation, may favor expansion of EBV-infected cells and onset of EBV-related malignancies.     

Dynamic changes in HCV RNA levels and viral quasispecies in a patient with chronic hepatitis C after telaprevir-based treatment

Background

Telaprevir is a selective inhibitor of the hepatitis C virus NS3·4A serine protease. Treatment with telaprevir resulted in a rapid HCV-RNA decline in chronic hepatitis C genotype 1 patients.

Objectives

To report the clinical and viral course of a patient treated with telaprevir in combination with pegylated interferon-alpha-2a and ribavirin in a Phase 2 clinical trial (PROVE3).

Study design

This previous non-responder to interferon based therapy was treated for 40 weeks with a telaprevir, pegylated interferon alpha-2a, and ribavirin regimen. Viral sequencing and phylogenetic analysis were performed before, during and after therapy.

Results

The patient, a 54 years old male patient, experienced a viral relapse 4 weeks post-treatment and HCV-RNA levels continued to increase 14 weeks post-treatment (150,000 IU/mL). The viral population, which was wild type at baseline, consisted of only V36A variants at both of these post-treatment timepoints. Subsequently, this patient had a transient disappearance of HCV-RNA for more than 1 year in the absence of antiviral therapy. Thereafter, HCV-RNA reappeared again with a viral population consisting of only wild type virus. Phylogenetic analysis of NS3·4A corresponded with a viral population bottleneck resulting in changes in viral quasispecies.

Conclusion

In this case report, significant viral load reductions resulted in a genetic bottleneck leading to a reduction of variability in the hepatitis C viral population. We hypothesize that the reduction in viral heterogeneity potentially led to a reduced viral capacity to adapt to a host immune response leading to a transient loss of detectable HCV-RNA.     

Immune cell profile in invasive cholesteatomas: Preliminary findings

Background

Cholesteatoma consists of keratinizing squamous epithelium, granulation tissue and keratin plugs. The pathogenesis of cholesteatoma may be related to alterations in the stromal immune cell infiltrate.

Objective

To examine the immunophenotypic characteristics of the immune cell infiltrate in invasive cholesteatomas.

Materials and methods

This study included 12 patients with invasive cholesteatomas causing wide bone erosion of the mastoid, middle ear structures, and the bony plates of middle ear cleft. Diagnosis of invasiveness was based on the clinical, radiological and intraoperative findings. Canal wall-down surgical approach was done in all cases to control the disease process. We used the cholesteatomatous tissue specimens to perform immunohistochemical stains for B cells (CD20), T cells (CD3), histiocytes (CD68) and Langerhans' cells (CD1a). Mouse monoclonal antibodies and immunoperoxidase staining methods were used. The results of immunohistology were scored as mean values of positively stained immune cells. The data were compared with findings in 10 specimens of external ear skin (control group).

Results

Immunohistochemistry showed highly significant (p < 0.00) counts of immune cells in invasive cholesteatomas (CD3: 4.7 ± 0.4, CD68:4.6 ± 0.5, CD20: 0.8 ± 0.1 and CD1a: 0.8  ± 0.1) compared to those in external canal skin (control group: CD3:0.8 ± 0.3, CD68: 1.0 ± 0.4, CD20: 0.2 ± 0.1 and CD1a: 0.1 ± 0.1). In cholesteatomas, the predominant of CD3⁺ T lymphocytes and CD68⁺ cells (histiocytes). Rare CD20⁺ cells and CD1a⁺ cells (Langerhans' cells) were also observed.

Conclusions

This preliminary study describes the profile of the immune cell infiltrate in invasive cholesteatomas. The numeric dominance of CD3⁺ cells and CD68⁺ cells suggests that cell-mediated immunity has important role in the development of cholesteatoma and in its autodestructive properties. Further studies are recommended to categorize the T cell subsets in different stages of cholesteatomas.     

Phenotypic characterization of the infiltrating immune cells in normal prostate, benign nodular prostatic hyperplasia and prostatic adenocarcinoma

Background

Immune cell infiltrate is a constant feature in normal prostate, benign nodular prostatic hyperplasia and prostatic adenocarcinoma. This study elaborates on the cells of the immune system present in normal prostate, benign nodular prostatic hyperplasia and prostatic adenocarcinoma.

Hypothesis

Here, we hypothesized that “the development of benign nodular prostatic hyperplasia and prostatic adenocarcinoma is associated with numeric alterations of the immune cell infiltrate”.

Materials and methods

A total of 50 transurethral prostatic resection specimens, each entailing normal prostate, benign nodular prostatic hyperplasia and high grade prostatic adenocarcinoma were evaluated for the density and phenotype of the immune cells using immunohistological methods and mouse monoclonal antibodies decorating T cells (CD3), histiocytes (CD68) and B lymphocytes (CD20).

Results

Immune cell infiltrate was composed of T cells, histiocytes and B-lymphocytes. CD⁺3 T lymphocytes and CD68⁺ cells were the predominant cell populations. We observed variations in the density of the immune cells among the normal prostate, benign nodular prostatic hyperplasia and high grade prostatic adenocarcinoma. Compared with normal prostate, benign nodular prostatic hyperplasia had a statistically significant high density of immune cells (3.4 ± 0.4versus 13.5 ± 1.0, P < 0.00). In contrast, a significant decrease in the counts of these cells was observed in high-grade prostatic adenocarcinoma compared to benign nodular prostatic hyperplasia (13.5 ± 1.0 versus 5.2 ± 0.3, P < 0.01).

Conclusions

The increased density of immune cells (predominantly CD⁺3 T cells) in benign nodular prostatic hyperplasia suggests that the initial response to cellular damage is mediated by cell-mediated immunity. The decreased density of immune cells in high-grade prostatic adenocarcinoma may reflect immunosuppression. The underlying mechanisms of these numeric variations are open for further investigations.     

CD4 T cell nadir independently predicts the magnitude of the HIV reservoir after prolonged suppressive antiretroviral therapy

Background

The level of HIV-1 integrated DNA in CD4 T cells was reported to predict the evolution of untreated HIV-1 infection independently of CD4 cell counts or plasma HIV-1 RNA levels. However, the relevance of reservoir level while on efficient antiretroviral therapy (ART) is still unknown.

Objectives

To evaluate factors that may contribute to the establishment and maintenance of HIV-1 reservoir size in ART-treated HIV-1-infected adults with complete suppression of viremia.

Study design

35 subjects receiving ART with plasma HIV-1 RNA below the limit of detection for an average duration of 3.2 years were studied. A highly sensitive PCR was used to assess HIV-1 integrated DNA levels in sorted CD4 T cells.

Results

The mean HIV-1 integrated DNA was 300 ± 7 copies/10⁶ CD4 cells (range 10-1408). In univariate analysis, the levels of HIV-1 proviral DNA appeared to be independent of duration of HIV-1-infection, duration on ART, time since HIV-1 viral load was undetectable, delay between HIV-1 infection and starting ART, or viral load before starting ART. Conversely, CD4 T cell nadir, CD4/CD8 ratio and, to lesser degree, CD4 T cell counts were inversely associated with HIV-1 proviral DNA levels. In multivariate analysis, only CD4 T cell nadir significantly predicted levels of HIV-1 proviral DNA (P = 0.025).

Conclusions

CD4 T cell nadir strongly predicted reservoir size independently of other factors in HIV-1-infected adults with complete suppression of viremia. Collectively, these results indicate that the extent of CD4 T cell depletion before ART drives the size of the viral reservoir after prolonged therapy.     

Human hepatoma cells transmit surface bound HIV-1 to CD4 T cells through an ICAM-1/LFA-1-dependent mechanism

Background

During the viremic phase of human immunodeficiency virus type-1 (HIV-1) infection, hepatocytes are likely to be constantly exposed to circulating virions. Knowing that a contact between hepatocytes and CD4⁺ T lymphocytes is favoured by the local slow blood flow present within the liver, we hypothesize that hepatic cells can act as a viral reservoir and thus contribute to HIV-1 propagation.

Results

We report that human hepatoma cells bind and internalize HIV-1 particles. Infection of CD4⁺ T cells was found to be much more efficient following a contact with virus-loaded hepatocytes than with cell-free virus. Additional studies suggest that infection of CD4⁺ T cells in trans with hepatocytes carrying virus is primarily due to surface bound HIV-1 particles and relies on LFA-1/ICAM-1 interactions.

Conclusion

This work represents the first demonstration by which circulating CD4⁺ T cells can be potentially infected with HIV-1 through a contact with hepatocytes in the liver.     

Comparison of Abbott RealTime High Risk HPV and Hybrid Capture 2 for the detection of high-risk HPV DNA in a referral population setting

Background

The Abbott RealTime High Risk HPV assay (ART) is an automated multiplex real-time PCR test for detection of DNA from 14 high risk (HR) HPV types in cervical specimens and simultaneous distinction of HPV16 and HPV18 from other HR-HPV.

Objectives

To evaluate the performance of the ART assay in specimens referred for HPV testing to our laboratory (referral population) by comparison with historical data from HC2 and INNO-LiPA as well as histological status, if available.

Study design

412 cervical specimens were collected from women between 18 and 70 years of age: 301 previously tested by HC2 without clinical data and 111 previously tested by HC2 and INNO-LiPA with histological diagnosis of CIN3+.

Results

Our study demonstrated good overall agreement between ART, HC2 and INNO-LiPA. In the group of the CIN3+ specimens HR-HPV was detected by ART in 93.07% (95% CI: 88.12-98.02), while HR-HPV detection rates with HC2 and INNO-LiPA were 91.09% (95% CI: 85.53-96.65) and 95.05% (95% CI: 90.82-99.28), respectively. The typing capability of ART for HPV16, HPV18 and a pool of twelve other HR-HPV types was investigated by comparison with INNO-LiPA demonstrating high overall assay concordance (89.81%; k 0.87).

Conclusions

The Abbott RealTime assay showed similar clinical performance for detection of CIN3+ compared with HC2. The high level of automation and ability to identify HPV16, HPV18 and other HR-HPV make this assay a very attractive option for HR-HPV testing, potentially improving patient management by risk stratification of cytological abnormal populations.     

Use of real-time polymerase chain reaction (rtPCR) as a diagnostic tool for influenza infection in a vaccine efficacy trial

Background

Conventional techniques for diagnosing influenza based on viral cell culture or disease serology have limitations, and molecular assays, such as real-time polymerase chain reaction (rtPCR) are increasingly used.

Objectives

To evaluate the use of rtPCR as a diagnostic tool for the determination of influenza virus infection.

Study design

This prospective, double-blind, placebo-controlled, randomised efficacy study was conducted in persons aged 18-64 years. Cases of influenza-like-illness (ILI), defined as at least one systemic symptom [fever ≥37.8 °C and/or myalgia] and at least one respiratory symptom [cough and/or sore throat] were identified by active and passive surveillance. For each case of suspected ILI, nasal and throat swabs were collected and analysed by viral culture and rtPCR.

Results

227 ILI cases were positive by rtPCR while 64% (145/227) were positive by both rtPCR and culture. For both assays, the maximum percentage of swabs that tested positive was on Day 0, thereafter positive samples by rtPCR remained constant until Day 5 but decreased progressively by culture. All rtPCR positive cases with a viral load of below 4.5 log₁₀ copies/sample were negative by culture. There were however culture negative cases with high viral loads. Vaccine efficacy for influenza was estimated as 54.7% by rtPCR (culture positive or negative) and 61.6% by culture irrespective of match to vaccine strain. Clinical severity was not significantly different between culture positive cases and culture negative but rtPCR positive cases.

Conclusions

rtPCR is a sensitive and specific diagnostic tool for influenza vaccine efficacy studies.     

Comparative assessment of the recognition of domain-specific CD163 monoclonal antibodies in human monocytes explains wide discrepancy in reported levels of cellular surface CD163 expression

Background

CD163 is expressed exclusively on cells of the monocyte/macrophage lineage and is widely used as a marker of human macrophages. Further, it has been suggested as a diagnostic marker of monocyte/macrophage activity in inflammatory conditions and as a therapeutic target. However, studies continue to exhibit great discrepancy in the measured percentage of CD163-expressing blood monocytes in healthy individuals. In this study we sought to clarify this inconsistency in reported levels of CD163 surface expression by a detailed analysis of a panel of CD163 antibodies used in previous studies.

Materials and methods

The cellular distribution of CD163 on human peripheral blood monocytes in freshly drawn blood and peripheral blood mononuclear cells isolated from buffy-coats was investigated by flow cytometry using CD163 monoclonal antibodies recognizing scavenger receptor cysteine-rich (SRCR) domain 1 (MAC2-158), domain 4 (R-20), domain 7 (GHI/61), and domain 9 (RM3/1). The CD163 monoclonal antibodies were characterized in binding and endocytosis experiments in human macrophages and CD163-transfected Flp-In CHO cells. Calcium-dependent ligand binding was assessed using surface plasmon resonance, and the specificity of the CD163 monoclonal antibodies was analyzed by western blotting.

Results and discussion

Flow cytometric analysis revealed that the estimated proportion of CD163-expressing human peripheral blood monocytes increased when using CD163 monoclonal antibodies recognizing epitopes in the N-terminal part of CD163, remote from the membrane surface. Moreover, the proportion of CD163 positive monocytes observed was highly dependent on free calcium. GHI/61 did not exhibit CD163 binding in the presence of calcium as measured by surface plasmon resonance, which was in agreement with the concordant loss of binding in heparin-stabilized whole blood observed by flow cytometry. In contrast, RM3/1 exhibited weak binding to CD163 in the absence of calcium but high affinity binding to CD163 in the presence of calcium. R-20 and MAC2-158 were unaffected by extracellular calcium levels. The latter SRCR domain 1 mAb consistently recognized more than 80% CD163-positive monocytes in human peripheral blood.

Conclusion

Epitope accessibility and extracellular calcium dependence elucidate discrepancies in reported levels of monocytic CD163 expression. Utilizing monoclonal antibodies to the N-terminal part of CD163 more than 80% monocytes in human peripheral blood could be identified as CD163 positive, indicating that most, and conceivably all, human peripheral blood monocytes do express CD163.     

Molecular epidemiology of a large community-based outbreak of hepatitis B in Bristol, UK

Background

A large outbreak of hepatitis B virus (HBV) infection in the UK occurred between 2001 and 2005 in Bristol, UK.

Objectives

To identify HBV strains circulating amongst risk groups in the HBV outbreak cohort.

Study design

Cross-sectional study of acute HBV outbreak cases in Bristol.

Results

HBV sequences from sera of 95 of the 237 cases (40%) were characterised. The majority of cases (77%) were found to carry an HBV variant belonging to genotype D, designated HBV^BV. Eighty-eight percent (36/41) of sequences from injection drug users were HBV^BV as were 70% (19/27) from those with heterosexual intercourse as the primary identified risk factor. Of 15 sequences characterised from cases of pre-outbreak acute or chronic hepatitis B residing in Bristol, 40% also carried HBV^BV; the earliest was from a case identified in 1994.

Conclusion

The findings from this study link the spread of HBV^BV from injecting drug users to the general population through heterosexual intercourse during the outbreak. The molecular sequencing of specimens from this outbreak reports the emergence of HBV^BV, a HBV strain circulating in Bristol and South West England, as the cause of one of the largest outbreaks of acute hepatitis B in the UK.     

Trichodysplasia spinulosa is characterized by active polyomavirus infection

Background

Recently a new polyomavirus was identified in a patient with trichodysplasia spinulosa (TS), a rare follicular skin disease of immunocompromised patients characterized by facial spines and overgrowth of inner root sheath cells. Seroepidemiological studies indicate that TSPyV is ubiquitous and latently infects 70% of the healthy individuals.

Objective

To corroborate the relationship between active TSPyV infection and TS disease by analyzing the presence, load, and precise localization of TSPyV infection in TS patients and in controls.

Study design

TS lesional and non-lesional skin samples were retrieved from TS patients through a PubMed search. Samples were analyzed for the presence and load of TSPyV DNA with quantitative PCR, and for expression and localization of viral protein with immunofluorescence. Findings obtained in TS patients (n = 11) were compared to those obtained in healthy controls (n = 249).

Results

TSPyV DNA detection was significantly associated with disease (P < 0.001), with 100% positivity of the lesional and 2% of the control samples. Quantification revealed high TSPyV DNA loads in the lesional samples (∼10⁶ copies/cell), and low viral loads in the occasionally TSPyV-positive non-lesional and control samples (<10² copies/cell). TSPyV VP1 protein expression was detected only in lesional TS samples, restricted to the nuclei of inner root sheath cells over-expressing trichohyalin.

Conclusions

The high prevalence and load of TSPyV DNA only in TS lesions, and the abundant expression of TSPyV protein in the affected hair follicle cells demonstrate a tight relation between TSPyV infection and TS disease, and indicate involvement of active TSPyV infection in TS pathogenesis.     

Targeting gene delivery to activated vascular endothelium using anti E/P-Selectin antibody linked to PAMAM dendrimers

Introduction

The adhesion molecules P- and E-selectin are expressed on activated endothelial cells, and are good targets for gene therapy aimed at inflammatory disease. We have therefore investigated the potential of targeting PAMAM dendrimers, a non viral vector system, to cells expressing P/E-selectin using a monoclonal antibody that recognises these molecules.

Materials and Methods

We used biotin and avidin to cross-link anti E/P-Selectin monoclonal antibody to pre-formed Superfect-DNA complexes that were then used to transfect reporter genes to CHO cells expressing E-Selectin, cytokine-activated primary Human Saphenous Vein Endothelial Cells (HSVEC) and whole vein segments.

Results

The use of the anti E/P-Selectin antibody increased the transfection efficiency in CHO-E cells, activated HSVEC and saphenous vein segments ex vivo. We also showed that the antibody improved the binding of the complexes onto cells as well as the internalisation kinetics.

Discussion

We demonstrate here that by attaching antibodies onto PAMAM dendrimers the efficiency of transfection can be significantly improved in cells or tissues expressing the receptor. This technology has potential in the treatment of cardiovascular disease by gene therapy but can also be used with different antibodies to target other diseased cells or tissues.     

Lipopolysaccharide levels are elevated in dengue virus infected patients and correlate with disease severity

Background

Although in the majority of cases dengue virus (DENV) infection results in a self-limiting febrile disease, it can cause severe plasma leakage in a minority of patients. The appearance of plasma leakage indicates an increased permeability of the vascular wall. In this study we investigated if DENV infection can lead to leakage of lipopolysaccharide (LPS) from the intestine into the blood of the patient, indicative of an increased permeability of the intestinal mucosal barrier.

Objectives

The aim of this study was to investigate if LPS levels were elevated in DENV infected patients and if these levels correlated with disease severity.

Study design

LPS levels in the blood of DENV infected children were determined using the Limulus Amebocyte Lysate assay. To determine disease severity we used the 1997-WHO criteria, the expert physician's judgement and a score that focused on plasma leakage in particular. Furthermore, the modulatory factors LPS binding protein (LBP) and sCD14, as well as the immune activation marker neopterin were determined.

Results

We showed significantly elevated LPS levels in plasma of DENV infected children compared to healthy controls. The plasma leakage severity score had the strongest correlation with levels of LPS. LBP, sCD14 and neopterin were elevated compared to healthy controls.

Conclusion

In this study we show evidence of elevated LPS levels during DENV infection. Moreover, a correlation between LPS levels and disease severity was found, especially when disease severity was determined in terms of plasma leakage.     

Molecular characterization of a novel entecavir mutation pattern isolated from a multi-drug refractory patient with chronic hepatitis B infection

Background

Prolonged antiviral treatment results in selection and accumulation of resistant strains in quasispecies pool in hepatitis B virus (HBV) infection.

Objectives

The aim of this study was to characterise a novel HBV pattern which shows resistance to lamivudine, adefovir dipivoxil and entecavir using in vitro phenoyping assay.

Study design

A male 36 years old patient diagnosed with anti HBe-positive chronic hepatitis B (CHB) had received lamivudine treatment for 7 years following an initial unsuccessfull interferon treatment. The therapy had been switched to adefovir and then to entecavir when breakthrough occcured during each treatment. This led only to a temporary HBV DNA decline which soon was followed by viral breakthrough despite the lack of known entecavir resistance mutations. Patient died after 9 months of entecavir treatment from liver failure. A total of 434 clones from 6 different serum samples were analysed retrospectively. HBV genomes bearing mutation patterns suggestive of antiviral resistance were analysed by in vitro phenotyping assay.

Results

Dominance of a clone carrying L80LV, L91I, M204I, S219A, N238D, Y245H changes was detected in the last serum sample of the patient just before his death. This pattern displayed 30.4 fold resistance to entecavir when compared with the wild type HBV by in vitro phenotyping assay.

Conclusion

A novel mutation pattern showing a high degree of resistance to entecavir was documented. In this pattern, the S219A and Y245H mutations mainly seem to contribute to the emergence of ETV resistance.     

Quantitation of human herpesvirus-6A, -6B and -7 DNAs in whole blood, mononuclear and polymorphonuclear cell fractions from healthy blood donors

Background

Diagnosis of human herpesvirus-6A (HHV-6A), -6B (HHV-6B) or -7 (HHV-7) infections is often based on the measure of viral load in blood.

Objectives

The aim of this study was to define usual values of HHV-6A, HHV-6B and HHV-7 loads in blood fractions (whole blood [WB], mononuclear cells [PBMCs], polymorphonuclear leukocytes [PMNLs]) of blood donors.

Study design

HHV-6A, HHV-6B and -7 DNAs were quantitated using real-time PCR assays in WB, PBMCs and PMNLs separated on Ficoll or dextran gradients, respectively, for 200 blood donors. Viral loads were expressed as the number of viral genomic copies per million cells (Cop/M) for all fractions, and also per milliliter for WB.

Results

HHV-6B DNA was rarely detected in WB (8%), PBMCs (16.5%), and PMNLs (10.5%), HHV-6A was never detected, whereas HHV-7 DNA was often present in WB (51.5%), PBMCs (62%) and PMNLs (51.5%). Median loads were low with 81 Cop/M in WB, 62 Cop/M in PBMCs and 34.5 Cop/M in PMNLs for HHV-6B, and 129 Cop/M in WB, 225 Cop/M in PBMCs and 62 Cop/M in PMNLs for HHV-7. Viral load expression per million cells and per mL were equivalent. One subject had chromosomally integrated HHV-6 with high viral loads ranging from 2.23 × 10⁶ to 3.21 × 10⁶ Cop/M in all compartments and plasma.

Conclusions

These results allow to propose viral load in WB as a sensitive and suitable marker, with values for healthy subjects at approximately 100 Cop/M for both viruses. The prevalence of chromosomally integrated HHV-6 was 0.5%.     

HCV core antigen testing in HIV- and HBV-coinfected patients, and in HCV-infected patients on hemodialysis

Background

A quantitative HCV core antigen (HCVcoreAg) immunoassay has been developed for the confirmation of viremia in patients with hepatitis C.

Objectives

We evaluated the correlation of HCV RNA and HCVcoreAg in different patient populations without HCV-specific treatment: HIV/HCV-coinfection, HBV/HCV-coinfection, and patients with end-stage renal disease.

Study design

HCVcoreAg was quantified by a fully-automated immunoassay. Correlation of HCVcoreAg with HCV RNA was studied cross-sectionally in HIV/HCV- and HBV/HCV-coinfected patients, as well as before and after hemodialysis in patients with end-stage renal disease.

Results

A concordant positive or negative test result for both HCV RNA and HCVcoreAg was observed in 68 of 71 (96%), 55 of 57 (96%), and in 109 of 109 (100%) samples of patients with HIV- or HBV/HCV-coinfection, and patients undergoing hemodialysis, respectively. HCVcoreAg showed high correlation with HCV RNA in samples from HIV/HCV-coinfected patients and HCV-infected patients undergoing hemodialysis (r = 0.97 and r = 0.94, p < 0.001). There was no overall correlation between HCVcoreAg and HCV RNA in HBV/HCV-coinfected individuals (r = 0.04, p = 0.822). Excluding patients with HCV RNA to HCVcoreAg ratios below 100 and above 10,000 kIU/fmol led to improved correlation (r = 0.53; p = 0.02), but remained worse than for the other cohorts. Overall, HCV RNA to HCVcoreAg ratios did not differ significantly between the different patient populations, though variation tended to be higher in HBV/HCV-coinfected patients. Patients with lower HCV RNA levels tend to have lower HCV RNA/HCVcoreAg ratios.

Conclusions

HCVcoreAg represents a reliable marker of viral replication showing a good correlation with HCV RNA in various patient populations, with some limitations in HBV/HCV-coinfection.     

CD8α⁺ dendritic cells improve collagen-induced arthritis in CC chemokine receptor (CCR)-2 deficient mice

Objective

Dendritic cells (DCs) have long been recognized as potential therapeutic targets of rheumatoid arthritis (RA). Increasing evidence has showed that DCs are capable of suppressing autoimmunity by expanding FoxP3⁺ regulatory T cells (T_reg), which in turn exert immunosuppression by increasing TGFβ-1. In the SKG mice, activated DC prime autoreactive T cells causing autoantibody production and an inflammatory arthritic response. Recently, we reported that CC-chemokine receptor-2 deficient (Ccr2^−/−) mice had impaired DCs migration and reduced CD8α⁺ DCs in the C57Bl/6J mice strain and that these mice were more susceptible to collagen antibody-induced arthritis (CAIA), compared to wild type mice. To examine the mechanism by which DCs contribute to the increased susceptibility of arthritis in Ccr2^−/− mice, we tested the hypothesis that CD8α⁺ DCs are protective (tolerogenic) against autoimmune arthritis by examining the role of CD8α⁺ DCs in Ccr2^−/− and SKG mice.

Methods

To examine the mechanism by which DCs defects lead to the development of arthritis, we used two murine models of experimental arthritis: collagen-induced arthritis (CIA) in DBA1/J mice and zymosan-induced arthritis in SKG mice. DBA1/J mice received recombinant fms-like tyrosine kinase 3 ligand (Flt3L) injections to expand endogenous DCs populations or adoptive transfers of CD8α⁺ DCs.

Results

Flt3L-mediated expansion of endogenous CD8α⁺ DCs resulted in heightened susceptibility of CIA. In contrast, supplementation with exogenous CD8α⁺ DCs ameliorated arthritis in Ccr2^−/− mice and enhanced TGFβ1 production by T cells. Furthermore, SKG mice with genetic inactivation of CCR2 did not affect the numbers of DCs nor improve the arthritis phenotype.

Conclusion

CD8α⁺ DCs were tolerogenic to the development of arthritis. CD8α⁺ DCs deficiency heightened the sensitivity to arthritis in Ccr2^−/− mice. Ccr2 deficiency did not alter the arthritic phenotype in SKG mice suggesting the arthritis in Ccr2^−/− mice was T cell-independent.     

Co-infection par le VHB de génotype G : développement, validation et application d’une technique de quantification spécifique du VHB de génotype G

Goal of the study

A detection and quantification method specific of genotype G hepatitis B virus (G-HBV) was developed and used to study the epidemiology and evolution of G-HBV co-infection during antiviral therapy.

Patients and methods

A multiplex real time PCR was developed and validated on A-HBV and G-HBV plasmid mixes. G-HBV infection was sought for on chronic hepatitis B carriers followed in our institution.

Results

Two primers, flanking the specific G-HBV 36 nucleotide insertion and two probes, including one specific for the insertion, were validated for a multiplex real time PCR. This new tool was well correlated to commercially available quantification systems within a 2 to 7 log₁₀ IU/mL range. On A- and G-HBV clonal mixes, G-HBV was quantified with a precision between 5 to 10%. On HBV-HIV infected patients, a 25% G-HBV prevalence was found, while it was below 1% in HBV mono-infected patients. Longitudinal G-HBV quantifications on five treated patients indicate that G-HBV is not selected by antiviral treatment.

Conclusion

A specific method for G-HBV quantification within multi-genotype mixes was set up. The important G-HBV prevalence in HIV patients was unexpected. Our data are not in favour of an intrinsic resistance of G-HBV to current antivirals.     

Sphingosine-kinase 1 and 2 contribute to oral sensitization and effector phase in a mouse model of food allergy

Background

Sphingosine-1-phosphate (S1P) influences activation, migration and death of immune cells. Further, S1P was proposed to play a major role in the induction and promotion of allergic diseases. However, to date only limited information is available on the role of S1P in food allergy.

Objective

We aimed to investigate the role of sphingosine-kinase (SphK) 1 and 2, the enzymes responsible for endogenous S1P production, on the induction of food allergy.

Methods and results

Human epithelial colorectal CaCo2 cells stimulated in vitro with S1P revealed a decrease of transepithelial resistance and enhanced transport of FITC labeled OVA. We studied the effect of genetic deletion of the enzymes involved in S1P production on food allergy induction using a mouse model of food allergy based on intragastrically (i.g.) administered ovalbumin (OVA) with concomitant acid-suppression. Wild-type (WT), SphK1^−/− and SphK2^−/− mice immunized with OVA alone i.g. or intraperitoneally (i.p.) were used as negative or positive controls, respectively. SphK1- and SphK2-deficient mice fed with OVA under acid-suppression showed reduced induction of OVA specific IgE and IgG compared to WT mice, but had normal responses when immunized by the intraperitoneal route. Flow cytometric analysis of spleen cells revealed a significantly reduced proportion of CD4⁺ effector T-cells in both SphK deficient animals after oral sensitization. This was accompanied by a reduced accumulation of mast cells in the gastric mucosa in SphK-deficient animals compared to WT mice. Furthermore, mouse mast cell protease-1 (mMCP-1) levels, an IgE-mediated anaphylaxis marker, were reliably elevated in allergic WT animals.

Conclusion

Modulation of the S1P homeostasis by deletion of either SphK1 or SphK2 alters the sensitization and effector phase of food allergy.     

Epidemiological and genetic analyses of a diffuse outbreak of hepatitis A in Japan, 2010

Background

Hepatitis A virus (HAV) is still one of the most common causative agents of acute hepatitis in Japan. Although a relatively small number of annual acute hepatitis A cases (approximately 100-150, 0.78-1.17 per million) were recently reported, a larger number of cases (346, 2.71 per million) were reported in 2010.

Objectives

To investigate the causes of the 2010 HAV resurgence in Japan by using molecular epidemiological and genetic analyses.

Study design

HAV specimens were obtained from 61 cases from 22 different prefectures. These viral specimens were genotyped by PCR amplification and sequencing of the VP1/2A region of HAV genome.

Results

Phylogenetic analysis revealed that 61 HAV strains could be divided into three genotypes: IA (44 cases), IB (1 case) and IIIA (16 cases). The IA genotype consisted of two genomic sub-lineages. The sequences of one of the two IA sub-lineages (corresponding to 31 cases) were very similar, 26 of these 31 isolates had 100% identity. The other IA sub-lineage corresponded to strains endemic to Japan. The sequences of Japanese IIIA strains were similar to those of strains that caused a large epidemic in the Republic of Korea from 2007 to 2009.

Conclusions

The resurgence of HAV in 2010 can be attributed to importation of two newly emerged HAV genotypes.