Consolidation of first remission using radioimmunotherapy with yttrium-90-ibritumomab-tiuxetan in adult patients with Burkitt lymphoma

The addition of anti-CD20 antibodies to high intensity polychemotherapy regimens has improved response and survival rates in newly diagnosed patients with Burkitt lymphoma (BL). However, the role of additional anti-CD20 directed radioimmunotherapy for consolidation of first remission (CR1) has not been reported so far in BL patients receiving rituximab during first-line treatment. We compared five BL patients receiving Y-90-IT radioimmunotherapy consolidation in CR1 to 22 consecutive BL patients without consolidation. We observed that Y-90-IT treatment was associated with clinically relevant myelosuppression. After a median follow-up of 50 months, none of the patients with Y-90-IT treatment relapsed, and no patient died. In contrast, one patient (4.5%) in the non-Y-90-IT group relapsed (50 months-PFS 95.5%; p = 0.6336), and one patient died (50 months-OS 95.5%; p = 0.6171). In conclusion, our data suggest that survival rates are excellent and equal in rituximab pretreated BL patients with or without Y-90-IT consolidation in first remission.

Q fever outbreak in the terraced vineyards of Lavaux, Switzerland

Coxiella burnetii infection (Q fever) is a widespread zoonosis with low endemicity in Switzerland, therefore no mandatory public report was required. A cluster of initially ten human cases of acute Q fever infections characterized by prolonged fever, asthenia and mild hepatitis occurred in 2012 in the terraced vineyard of Lavaux. Epidemiological investigations based on patients' interviews and veterinary investigations included environmental sampling as well as Coxiella-specific serological assay and molecular examinations (real-time PCR in vaginal secretions) of suspected sheep. These investigations demonstrated that 43% of sheep carried the bacteria whereas 30% exhibited anti-Coxiella antibodies. Mitigation measures, including limiting human contacts with the flock, hygiene measures, flock vaccination and a public official alert, have permitted the detection of four additional human cases and the avoidance of a much larger outbreak. Since November 2012, mandatory reporting of Q fever to Swiss public health authorities has been reintroduced. A close follow up of human cases will be necessary to identify chronic Q fever.

Molecular epidemiology of hepatitis B virus infection in Switzerland: a retrospective cohort study.

BACKGROUND Chronic hepatitis B virus (HBV) infection affects up to 7 % of the European population. Specific HBV genotypes are associated with rapid progression to end-stage liver disease and sub-optimal interferon treatment responses. Although the geographic distribution of HBV genotypes differs between regions, it has not been studied in Switzerland, which lies at the crossroads of Europe. METHODS In a retrospective analysis of 465 HBV samples collected between 2002 and 2013, we evaluated the HBV genotype distribution and phylogenetic determinants, as well as the prevalence of serological evidence of hepatitis delta, hepatitis C and HIV infections in Switzerland. Baseline characteristics of patients were compared across their region of origin using Fisher's exact test and ANOVA, and risk factors for HBeAg positivity were assessed using logistic regression. RESULTS The Swiss native population represented 15.7 % of HBV-infected patients living in Switzerland. In the overall population, genotype D was most prevalent (58.3 %), whereas genotype A (58.9 %) was the predominant genotype among the Swiss native population. The prevalence of patients with anti-HDV antibodies was 4.4 %. Patients of Swiss origin were most likely to be HBeAg-positive (38.1 %). HBV genotypes of patients living in Switzerland but sharing the same original region of origin were consistent with their place of birth. CONCLUSIONS The molecular epidemiology of HBV infection in Switzerland is driven by migration patterns and not by the genotype distribution of the native population. The prevalence of positive anti-HDV antibodies in our cohort was very low.

Classical swine fever virus replicon particles lacking the Erns gene: a potential marker vaccine for intradermal application.

Classical swine fever virus replicon particles (CSF-VRP) deficient for E(rns) were evaluated as a non-transmissible marker vaccine. A cDNA clone of CSFV strain Alfort/187 was used to obtain a replication-competent mutant genome (replicon) lacking the sequence encoding the 227 amino acids of the glycoprotein E(rns) (A187delE(rns)). For packaging of A187delE(rns) into virus particles, porcine kidney cell lines constitutively expressing E(rns) of CSFV were established. The rescued VRP were infectious in cell culture but did not yield infectious progeny virus. Single intradermal vaccination of two pigs with 10(7) TCID(50) of VRP A187delE(rns) elicited neutralizing antibodies, anti-E2 antibodies, and cellular immune responses determined by an increase of IFN-gamma producing cells. No anti-E(rns) antibodies were detected in the vaccinees confirming that this vaccine represents a negative marker vaccine allowing differentiation between infected and vaccinated animals. The two pigs were protected against lethal challenge with the highly virulent CSFV strain Eystrup. In contrast, oral immunization resulted in only partial protection, and neither CSFV-specific antibodies nor stimulated T-cells were found before challenge. These data represent a good basis for more extended vaccination/challenge trials including larger numbers of animals as well as more thorough analysis of virus shedding using sentinel animals to monitor horizontal spread of the challenge virus.

Immune response in pemphigus and beyond: progresses and emerging concepts

Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are two severe autoimmune bullous diseases of the mucosae and/or skin associated with autoantibodies directed against desmoglein (Dsg) 3 and/or Dsg1. These two desmosomal cadherins, typifying stratified epithelia, are components of cell adhesion complexes called desmosomes and represent extra-desmosomal adhesion receptors. We herein review the advances in our understanding of the immune response underlying pemphigus, including human leucocyte antigen (HLA) class II-associated genetic susceptibility, characteristics of pathogenic anti-Dsg antibodies, antigenic mapping studies as well as findings about Dsg-specific B and T cells. The pathogenicity of anti-Dsg autoantibodies has been convincingly demonstrated. Disease activity and clinical phenotype correlate with anti-Dsg antibody titers and profile while passive transfer of anti-Dsg IgG from pemphigus patients' results in pemphigus-like lesions in neonatal and adult mice. Finally, adoptive transfer of splenocytes from Dsg3-knockout mice immunized with murine Dsg3 into immunodeficient mice phenotypically recapitulates PV. Although the exact pathogenic mechanisms leading to blister formation have not been fully elucidated, intracellular signaling following antibody binding has been found to be necessary for inducing cell-cell dissociation, at least for PV. These new insights not only highlight the key role of Dsgs in maintenance of tissue homeostasis but are expected to progressively change pemphigus management, paving the way for novel targeted immunologic and pharmacologic therapies.

Transmission of border disease virus from a persistently infected calf to seronegative heifers in early pregnancy

BACKGROUND: This study describes the transmission of border disease virus (BDV) from a persistently infected calf to seronegative heifers in early pregnancy, resulting in persistently infected fetuses. On day 50 of pregnancy (= day 0 of the infection phase), six heifers were co-housed in a free stall with a bull calf persistently infected with BDV (pi BVD) for 60 days. The heifers underwent daily clinical examination, and blood samples were collected regularly for detection of pestiviral RNA and anti-pestivirus antibodies. After day 60 (= day 110 of pregnancy), the heifers were slaughtered, and the fetuses and placentae underwent post-mortem and immunohistochemical examination and RT-PCR for viral RNA detection. RESULTS: Three heifers had mild viraemia from day 8 to day 14, and by day 40 all heifers had pestivirus antibodies identified as anti-BDV antibodies in the serum neutralisation test. The placenta of the three viraemic heifers had histological evidence of inflammation, and fetal organs from these heifers were positive for pestivirus antigen by immunohistochemical examination and for BD viral RNA by RT-PCR and sequencing. Thus, co-housing of heifers in early pregnancy with a pi-BDV calf led to seroconversion in all heifers and persistent fetal infection in three. CONCLUSIONS: Considering that pi-BDV cattle can infect other cattle and lead to persistent infection of the fetus in pregnant cows, BDV should not be ignored in the context of the mandatory BVDV eradication and monitoring program. This strongly suggests that BDV should be taken into account in BVD eradication and control programs.

A Lentiviral Vector Expressing Japanese Encephalitis Virus-like Particles Elicits Broad Neutralizing Antibody Response in Pigs.

BACKGROUND Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in Southeast Asia. Vaccination of domestic pigs has been suggested as a "one health" strategy to reduce viral disease transmission to humans. The efficiency of two lentiviral TRIP/JEV vectors expressing the JEV envelope prM and E glycoproteins at eliciting protective humoral response was assessed in a mouse model and piglets. METHODOLOGY/PRINCIPAL FINDINGS A gene encoding the envelope proteins prM and E from a genotype 3 JEV strain was inserted into a lentiviral TRIP vector. Two lentiviral vectors TRIP/JEV were generated, each expressing the prM signal peptide followed by the prM protein and the E glycoprotein, the latter being expressed either in its native form or lacking its two C-terminal transmembrane domains. In vitro transduction of cells with the TRIP/JEV vector expressing the native prM and E resulted in the efficient secretion of virus-like particles of Japanese encephalitis virus. Immunization of BALB/c mice with TRIP/JEV vectors resulted in the production of IgGs against Japanese encephalitis virus, and the injection of a second dose one month after the prime injection greatly boosted antibody titers. The TRIP/JEV vectors elicited neutralizing antibodies against JEV strains belonging to genotypes 1, 3, and 5. Immunization of piglets with two doses of the lentiviral vector expressing JEV virus-like particles led to high titers of anti-JEV antibodies, that had efficient neutralizing activity regardless of the JEV genotype tested. CONCLUSIONS/SIGNIFICANCE Immunization of pigs with the lentiviral vector expressing JEV virus-like particles is particularly efficient to prime antigen-specific humoral immunity and trigger neutralizing antibody responses against JEV genotypes 1, 3, and 5. The titers of neutralizing antibodies elicited by the TRIP/JEV vector are sufficient to confer protection in domestic pigs against different genotypes of JEV and this could be of a great utility in endemic regions where more than one genotype is circulating.

The low abundance of U7 snRNA is partly determined by its Sm binding site.

In transient expression studies after DNA transfection of HeLa cells, the mouse U7 gene produces only approximately 30% of the RNA produced by a mouse U1b gene. This difference persists even when the transfected genes have all their 5' and 3' flanking sequences exchanged suggesting a post-transcriptional effect. When the special U7 Sm binding site is mutated to a consensus derived from the major snRNAs (Sm-opt), the U7 RNA level increases 4- to 5-fold, whereas no RNA is detected from a U7 gene with a non-functional Sm binding site (Sm-mut). Moreover, U1b genes with the U7 Sm binding site yield reduced RNA levels. The Sm-opt site also alters the cellular behaviour of the corresponding U7 snRNA. It accumulates to a higher level in the nucleus than wild type U7 RNA, and is better immunoprecipitable with anti-Sm antibodies. Injection experiments in Xenopus oocytes indicate that the U7 genes with either Sm-opt or Sm-mut sites produce similar amounts of RNA as wild type U7, but that they differ in opposing ways in the processing of precursors to mature size U7 snRNA and in nuclear accumulation. However, in reconstitution experiments using Xenopus oocytes, we show that U7 Sm-opt RNA, despite its efficient nuclear accumulation, is not active in 3' processing of histone pre-mRNA, whereas wild type U7 RNA is assembled into functional snRNPs, which correctly process histone pre-mRNA substrate. This suggests a functional importance of the special U7 Sm sequence.

The Topoisomerase I Inhibitor Irinotecan and the Tyrosyl-DNA Phosphodiesterase 1 Inhibitor Furamidine Synergistically Suppress Murine Lupus Nephritis

OBJECTIVE The treatment of lupus nephritis is still an unmet medical need requiring new therapeutic approaches. Our group found recently that irinotecan, an inhibitor of topoisomerase I (topo I), reversed proteinuria and prolonged survival in mice with advanced lupus nephritis. While irinotecan is known to stabilize the complex of topo I and DNA, the enzyme tyrosyl-DNA phosphodiesterase 1 (TDP-1) functions in an opposing manner by releasing topo I from DNA. Therefore, we undertook this study to test whether the TDP-1 inhibitor furamidine has an additional effect on lupus nephritis when used in combination with irinotecan. METHODS NZB/NZW mice were treated with low-dose irinotecan and furamidine either alone or in combination beginning at age 26 weeks. DNA relaxation was visualized using gel electrophoresis. Binding of anti-double-stranded DNA (anti-dsDNA) antibodies to DNA modified by topo I, TDP-1, and the topo I inhibitor camptothecin was determined by enzyme-linked immunosorbent assay. RESULTS Compared to treatment with either agent alone, simultaneous treatment with low-dose irinotecan and furamidine significantly improved survival of NZB/NZW mice. Similar to what has been previously shown for irinotecan alone, the combination treatment did not change the levels of anti-dsDNA antibodies. In vitro, recombinant TDP-1 increased topo I-mediated DNA relaxation, resulting in enhanced binding of anti-dsDNA antibodies. In combination with topo I and camptothecin, TDP-1 reversed the inhibitory effects of camptothecin on DNA relaxation and anti-dsDNA binding. CONCLUSION Affecting DNA relaxation by the enzymes topo I and TDP-1 and their inhibitors may be a promising approach for the development of new targeted therapies for systemic lupus erythematosus.

Absence of Hepatitis Delta Infection in a Large Rural HIV Cohort in Tanzania

OBJECTIVES The epidemiological and clinical determinants of hepatitis delta virus (HDV) infection in sub-Saharan Africa are ill-defined. We determined the prevalence of HDV infection in HIV/hepatitis B virus (HBV)-co-infected individuals in rural Tanzania. DESIGN AND METHODS We screened all hepatitis B virus (HBV)-infected adults under active follow-up in the Kilombero and Ulanga Antiretroviral Cohort (KIULARCO) for anti-HDV antibodies. In positive samples, we performed a second serological test and nucleic acid amplification. Demographic and clinical characteristics at initiation of antiretroviral therapy (ART) were compared between anti-HDV-negative and positive patients. RESULTS Among 222 HIV/HBV-coinfected patients on ART, 219 (98.6%) had a stored serum sample available and were included. Median age was 37 years, 55% were female, 46% had WHO stage III/IV HIV disease and median CD4 count was 179 cells/μL. The prevalence of anti-HDV positivity was 5.0% (95% confidence interval 2.8%-8.9%). There was no significant predictor of anti-HDV positivity. HDV could not be amplified in any of the anti-HDV-positive patients and the second serological test was negative in all of them. CONCLUSIONS We found no confirmed case of HDV infection among over 200 HIV/HBV-co-infected patients in Tanzania. As false-positive serology results are common, screening results should be confirmed with a second test.

High affinity targeting of CD23 inhibits IgE synthesis in human B cells.

The low-affinity IgE receptor FcϵRII (CD23) is part of the regulatory system controlling IgE synthesis in human B cells and exists in membrane and soluble forms. Binding of IgE to CD23 has been described to have stabilizing effects and to prevent cleavage of CD23. Previous experiments using anti-CD23 antibodies reduced IgE synthesis but were difficult to interpret as the antibody Fc part might also mediate feedback mechanisms. The purpose of this study was to investigate the regulatory role of CD23, by using designed ankyrin repeat proteins (DARPins) that specifically recognize CD23. Anti-CD23 DARPins were isolated by ribosome display and were produced as monovalent and bivalent constructs. Affinities to CD23 were measured by surface plasmon resonance. IgE synthesis and up-regulation of CD23 in human peripheral B cells were induced by IL-4 and anti-CD40 antibody. We assessed CD23 expression and its stabilization by FACS and used an ELISA for detecting soluble CD23. IgE synthesis was measured by ELISA and real-time PCR. Surface plasmon resonance revealed affinities of the DARPins to CD23 in the pico-molar range. Anti-CD23 DARPins strongly inhibited binding of IgE to CD23 and share thus a similar binding epitope as IgE. The DARPins stabilized membrane CD23 and reduced IgE synthesis in an isotype specific manner. Furthermore, the anti-CD23 DARPins decreased IgE transcript through inhibition of mature Cϵ RNA synthesis suggesting a posttranscriptional control mechanism. This study demonstrates that targeting CD23 alone is sufficient to inhibit IgE synthesis and suggests that a negative signaling occurs directly through the CD23 molecule.

Absence of Active Hepatitis C Virus Infection in Human Immunodeficiency Virus Clinics in Zambia and Mozambique.

Few studies have evaluated the prevalence of replicating hepatitis C virus (HCV) infection in sub-Saharan Africa. Among 1812 individuals infected with human immunodeficiency virus, no patient in rural Mozambique and 4 patients in urban Zambia were positive for anti-HCV antibodies. Of these, none had confirmed HCV replication.

Validation of an enzyme-linked immunosorbent assay (ELISA) for the measurement of canine S100A12.

BACKGROUND Canine S100 calcium-binding protein A12 (cS100A12) shows promise as biomarker of inflammation in dogs. A previously developed cS100A12-radioimmunoassay (RIA) requires radioactive tracers and is not sensitive enough for fecal cS100A12 concentrations in 79% of tested healthy dogs. An ELISA assay may be more sensitive than RIA and does not require radioactive tracers. OBJECTIVE The purpose of the study was to establish a sandwich ELISA for serum and fecal cS100A12, and to establish reference intervals (RI) for normal healthy canine serum and feces. METHODS Polyclonal rabbit anti-cS100A12 antibodies were generated and tested by Western blotting and immunohistochemistry. A sandwich ELISA was developed and validated, including accuracy and precision, and agreement with cS100A12-RIA. The RI, stability, and biologic variation in fecal cS100A12, and the effect of corticosteroids on serum cS100A12 were evaluated. RESULTS Lower detection limits were 5 μg/L (serum) and 1 ng/g (fecal), respectively. Intra- and inter-assay coefficients of variation were ≤ 4.4% and ≤ 10.9%, respectively. Observed-to-expected ratios for linearity and spiking recovery were 98.2 ± 9.8% (mean ± SD) and 93.0 ± 6.1%, respectively. There was a significant bias between the ELISA and the RIA. The RI was 49-320 μg/L for serum and 2-484 ng/g for fecal cS100A12. Fecal cS100A12 was stable for 7 days at 23, 4, -20, and -80°C; biologic variation was negligible but variation within one fecal sample was significant. Corticosteroid treatment had no clinically significant effect on serum cS100A12 concentrations. CONCLUSIONS The cS100A12-ELISA is a precise and accurate assay for serum and fecal cS100A12 in dogs.

Hepatitis C in HIV-infected individuals: a systematic review and meta-analysis of estimated prevalence in Africa.

INTRODUCTION Although hepatitis C virus (HCV) screening is recommended for all HIV-infected patients initiating antiretroviral therapy, data on epidemiologic characteristics of HCV infection in resource-limited settings are scarce. METHODS We searched PubMed and EMBASE for studies assessing the prevalence of HCV infection among HIV-infected individuals in Africa and extracted data on laboratory methods used. Prevalence estimates from individual studies were combined for each country using random-effects meta-analysis. The importance of study design, population and setting as well as type of test (anti-HCV antibody tests and polymerase chain reactions) was examined with meta-regression. RESULTS Three randomized controlled trials, 28 cohort studies and 121 cross-sectional analyses with 108,180 HIV-infected individuals from 35 countries were included. The majority of data came from outpatient populations (55%), followed by blood donors (15%) and pregnant women (14%). Based on estimates from 159 study populations, anti-HCV positivity prevalence ranged between 3.3% (95% confidence interval (CI) 1.8-4.7) in Southern Africa and 42.3% (95% CI 4.1-80.5) in North Africa. Study design, type of setting and age distribution did not influence this prevalence significantly. The prevalence of replicating HCV infection, estimated from data of 29 cohorts, was 2.0% (95% CI 1.5-2.6). Ten studies from nine countries reported the HCV genotype of 74 samples, 53% were genotype 1, 24% genotype 2, 14% genotype 4 and 9% genotypes 3, 5 or 6. CONCLUSIONS The prevalence of anti-HCV antibodies is high in HIV-infected patients in Africa, but replicating HCV infection is rare and varies widely across countries.

Novelties in Diabetic Retinopathy

Although diabetic retinopathy (DR) remains a leading cause of vision loss, the last decade has brought significant advances in the diagnosis and treatment of this common complication of diabetes mellitus. First, optical coherence tomography allows for noninvasive imaging of the retina, in particular, the macula, with very high resolution, thus facilitating the management of diabetic macular edema. In addition, recent advances in the understanding of the pathophysiology of DR, in particular, the key role of cytokines, such as vascular endothelial growth factor (VEGF), have led to the development of anti-VEGF antibodies for intraocular use. Anti-VEGF therapies have largely replaced laser photocoagulation for the treatment of diabetic macular edema. The benefit of intravitreal anti-VEGF in diabetic macular edema has been proven in numerous large randomized controlled trials. Moreover, a role of inflammation in DR has been recognized, and several mainly steroid-based, anti-inflammatory agents for intravitreal treatment have been shown to be effective. Despite these recent advances, strict systemic control of glycemia remains the cornerstone of the management of DR, significantly reducing ocular complications. This chapter will provide an overview of current and novel concepts of DR and will allude to promising novel therapeutic options for this sight-threatening disease.