Urea-Mediated Cross-Presentation of Soluble Epstein-Barr Virus BZLF1 Protein

Soluble extracellular proteins usually do not enter the endogenous human leukocyte antigen (HLA) I–dependent presentation pathway of antigen-presenting cells, strictly impeding their applicability for the re-stimulation of protein-specific CD8+ cytotoxic T lymphocytes (CTL). Here we present for the Epstein-Barr virus (EBV) BZLF1 a novel strategy that facilitates protein translocation into antigen-presenting cells by its solubilisation in high molar urea and subsequent pulsing of cells in presence of low molar urea. Stimulation of PBMC from HLA-matched EBV-seropositive individuals with urea-treated BZLF1 but not untreated BZLF1 induces an efficient reactivation of BZLF1-specific CTL. Urea-treated BZLF1 (uBZLF1) enters antigen-presenting cells in a temperature-dependent manner by clathrin-mediated endocytosis and is processed by the proteasome into peptides that are bound to nascent HLA I molecules. Dendritic cells and monocytes but also B cells can cross-present uBZLF1 in vitro. The strategy described here has potential for use in the development of improved technologies for the monitoring of protein-specific CTL

Immunologische und epidemiologische Aspekte antigenspezifischer T- und B-Zell-Antworten gegen humanpathogene Parvoviren

Die adaptive Immunantwort spielt bei der Kontrolle und Eliminierung von Infektionserregern eine zentrale Rolle und verleiht dem Organismus einen meist lebenslangen Schutz gegen Reinfektionen. Der Nachweis von Immunreaktionen gegen Pathogene ist unverzichtbar für die Forschung und klinische Diagnostik, da hierdurch erregerspezifische Therapien ermöglicht werden. Dies gilt auch für Infektionen mit Parvoviren, welchen zunehmend klinische Relevanz zugesprochen wird. In der Familie der Parvoviridae findet man zwei humanpathogene Vertreter: Das Parvovirus B19 (B19V) und das Humane Bocavirus (HBoV). Ersteres wurde vor drei Jahrzehnten als Verursacher der Ringelröteln in Kindern beschrieben. HBoV hingegen wurde erst kürzlich entdeckt und konnte bis heute nicht eindeutig mit Erkrankungen in Verbindung gebracht werden. In dieser Arbeit sollten durch die Analyse der HBoV- und B19V-spezifischen Immunantwort (i) Informationen zur Epidemiologie und Krankheitsassoziation von HBoV-Infektionen in gesunden Probanden und Patienten mit Infektionserkrankungen gewonnen werden und (ii) untersucht werden, ob nachweisbare Mengen an B19V-Genomen in Herzbiopsien von Patienten mit akuter Myokarditis oder dilatativer Kardiomyopathie kausal mit der Entstehung von Herzerkrankungen in Verbindung gebracht werden können

No detection of human bocavirus in amniotic fluid samples from fetuses with hydrops or isolated effusions.

BACKGROUND: Human bocavirus (HBoV) is a recently identified parvovirus associated with respiratory disease in infants. Animal bocaviruses have been shown to cause intrauterine infection, fetal anasarca and abortion in late gestation. OBJECTIVES: To investigate whether HBoV infection is associated with fetal hydrops, fetal anemia or isolated fetal effusions. STUDY DESIGN: We determined the prevalence of HBoV and parvovirus B19 (B19) DNA in amniotic fluid samples from fetuses with hydrops, anemia or isolated effusions using different real-time PCR protocols, and the HBoV IgG and IgM positivity rate in pregnant women with fetal hydrops or normal ultrasound findings by a non-commercial virus-like particle-based enzyme immunoassay. RESULTS: None of 87 amniotic fluid samples tested was HBoV DNA positive. Twelve of 60 fetuses with hydrops or anemia were found B19 DNA positive. Anti-HBoV IgG antibodies were detected in 100% (19/19) and 94% (47/50) of serum samples from pregnant women with fetal hydrops and normal ultrasound findings, respectively. All serum samples were found negative for anti-HBoV IgM. CONCLUSION: We suggest that HBoV is not a common cause of fetal hydrops, anemia or isolated effusions. This has to be confirmed by further studies of proven gestational HBoV infection

Adaptive immune responses against parvovirus B19 in patients with myocardial disease.

BACKGROUND: Parvovirus B19 (B19V)-DNA is frequently detected in endomyocardial biopsies (EMBs) from patients with acute myocarditis (AMC) and dilated cardiomyopathy (DCM), but also in various healthy tissues. The clinical relevance of this DNA-persistence is unclear. OBJECTIVES: To investigate potential pathogenic influences of B19V-DNA in EMBs, we analyzed B19V-specific adaptive immune responses in AMC/DCM patients and healthy controls. STUDY DESIGN: 15 AMC/DCM patients with detectable B19V-DNA in EMBs and 51 controls were analyzed for signs of acute B19V-infections and virus-specific immune responses by PCR, ELISA, Western line, and ELISpot-assays. RESULTS: Productive B19V-infection was determined in three patients. Slightly lower levels of B19V-specific T-cells were observed in patients as compared to the controls, no differences were observed in virus-specific serology. Viral DNA-load in EMBs could not be correlated to the number of B19V-specific T-cells. No differences in T-cell response, viremia and/or serological markers indicative for viral pathogenesis were observed in patients with inflammatory cardiomyopathy. CONCLUSIONS: Discrepancies in B19V-specific adaptive immunity were not observed in AMC/DCM patients as compared to controls. The data indicate that the exclusive detection of B19V-DNA in EMBs is not sufficient to associate B19V with AMC/DCM but should be complemented with additional virological and immunological parameters in further studies

Prevalence of parvovirus B19 and human bocavirus DNA in the heart of patients with no evidence of dilated cardiomyopathy or myocarditis.

BACKGROUND: Although the DNA of parvovirus B19 (B19V) is frequently detected in patients with dilated cardiomyopathy or myocarditis, whether the parvovirus causes disease is questionable, since even in healthy individuals the virus persists in various tissues. The same question applies to human bocavirus (HBoV). We have determined the prevalence and quantity of B19V and HBoV DNA in heart tissue of patients who were not experiencing virus-related heart diseases and analyzed whether the seroprevalence corresponded to DNA prevalence in the heart. METHODS: Samples of left-atrium heart tissue and serum were obtained from 100 patients who underwent open-heart surgery. Serum immunoglobulin (Ig) G and IgM against proteins encoded by B19V and HBoV were detected by enzyme-linked immunoabsorption assay and immunoblotting. B19V and HBoV DNA concentrations were determined by quantitative real-time polymerase chain reaction (PCR) in heart tissue and serum samples. Nested PCRs for VP1, K71, and GT3 identified the B19V genotypes. RESULTS: The prevalences of serum IgG specific for B19V and HBoV were 85% and 96%, respectively. Of all the patients, 85% had B19V DNA detected in heart tissues, and 4% displayed low-level B19V viremia, whereas only 5% of heart tissue samples and none of the serum samples demonstrated HBoV DNA. The sensitivity of B19V serological testing for B19V DNA in heart samples was 0.96 (95% confidence interval, 0.92-1.0). Specificity was 0.8 (95% confidence interval, 0.6-1.0), and the positive predictive value was 0.96 (95% confidence interval, 0.92-1.0). B19V genotypes 1 and 2 were present in 11% and 89% of heart tissues samples, respectively. B19V genotype 3 was not detected in any of the samples. CONCLUSIONS: Our data suggest that B19V but not HBoV demonstrates a lifelong persistence in the heart. The detection of B19V DNA in heart tissue showed no correlation with clinical symptoms. We strongly recommend that serological testing become a standardized procedure for future studies, to obtain representative data concerning the prevalence of B19V in the heart

Vascular Endothelial Growth Factor Receptors VEGFR-2 and VEGFR-3 are localised primarily to vasculature in human primary solid cancers

Purpose: Vascular endothelial growth factor (VEGF) signaling is key to tumor angiogenesis and is an important target in the development of anticancer drugs. However, VEGF receptor (VEGFR) expression in human cancers, particularly the relative expression of VEGFR-2 and VEGFR-3 in tumor vasculature versus tumor cells, is poorly defined. Experimental Design: VEGFR-2– and VEGFR-3–specific antibodies were identified and used in the immunohistochemical analysis of human primary cancers and normal tissue. The relative vascular localization of both receptors in colorectal and breast cancers was determined by coimmunofluorescence with vascular markers. Results: VEGFR-2 and VEGFR-3 were expressed on vascular endothelium but not on malignant cells in 13 common human solid tumor types (n > 400, bladder, breast, colorectal, head and neck, liver, lung, skin, ovarian, pancreatic, prostate, renal, stomach, and thyroid). The signal intensity of both receptors was significantly greater in vessels associated with malignant colorectal, lung, and breast than adjacent nontumor tissue. In colorectal cancers, VEGFR-2 was expressed on both intratumoral blood and lymphatic vessels, whereas VEGFR-3 was found predominantly on lymphatic vessels. In breast cancers, both receptors were localized to and upregulated on blood vessels. Conclusions: VEGFR-2 and VEGFR-3 are primarily localized to, and significantly upregulated on, tumor vasculature (blood and/or lymphatic) supporting the majority of solid cancers. The primary clinical mechanism of action of VEGF signaling inhibitors is likely to be through the targeting of tumor vessels rather than tumor cells. The upregulation of VEGFR-3 on tumor blood vessels indicates a potential additional antiangiogenic effect for dual VEGFR-2/VEGFR-3–targeted therapy

Structural Differences Explain Diverse Functions of Plasmodium Actins

Actins are highly conserved proteins and key players in central processes in all eukaryotic cells. The two actins of the malaria parasite are among the most divergent eukaryotic actins and also differ from each other more than isoforms in any other species. Microfilaments have not been directly observed in Plasmodium and are presumed to be short and highly dynamic. We show that actin I cannot complement actin II in male gametogenesis, suggesting critical structural differences. Cryo-EM reveals that Plasmodium actin I has a unique filament structure, whereas actin II filaments resemble canonical F-actin. Both Plasmodium actins hydrolyze ATP more efficiently than α-actin, and unlike any other actin, both parasite actins rapidly form short oligomers induced by ADP. Crystal structures of both isoforms pinpoint several structural changes in the monomers causing the unique polymerization properties. Inserting the canonical D-loop to Plasmodium actin I leads to the formation of long filaments in vitro. In vivo, this chimera restores gametogenesis in parasites lacking actin II, suggesting that stable filaments are required for exflagellation. Together, these data underline the divergence of eukaryotic actins and demonstrate how structural differences in the monomers translate into filaments with different properties, implying that even eukaryotic actins have faced different evolutionary pressures and followed different paths for developing their polymerization properties