Sensitization to wasp venom does not induce autoantibodies leading to infertility

Antigenic cross-reactivity has been described between the venom allergen (antigen 5) and mammalian testis proteins. Based on an allergen database we have previously shown that allergens can be represented by allergen motifs. A motif group was found containing venom antigen 5 sequences from different vespids. Using an optimized amino acid profile based on antigen 5 sequences for searching cross-reactive proteins, three human semen proteins belonging to the family of cysteine-rich secretory proteins (hCRISP) were found in the Swiss Protein database. To analyze antigenic cross-reactivity between antigen 5 and hCRISPs, antigen 5 from yellow jacket venom (Ves v 5) and two hCRISPs (CRISP-2 and -3) were chosen and produced as recombinant proteins in E. coli. A correlation was found between antibodies reacting with rVes v 5 and rhCRISP-2, -3 in a small human sera population indicating the presence of cross-reactive antibodies in human serum. Using intravenous immunoglobulin (IVIg), a therapeutic multidonor IgG preparation, cross-reactive antibodies were isolated that recognize rVes v 5, hCRISP-2 and -3 suggesting the presence of common epitopes between Ves v 5 and hCRISPs. However this cross-reactivity seems not to be linked to allergy to wasp venom as we could show no correlation between increasing CAP-class IgE level to wasp venom and IgG to sperm extract and hCRISPs. These data suggest that higher sensitization to wasp venom does not induce more antibodies against autoantigens and might not represent a higher risk to develop autoantibodies leading to infertility.

Drug targeting using OX7-immunoliposomes: correlation between Thy1.1 antigen expression and tissue distribution in the rat

OX7 monoclonal antibody F((ab')2) fragments directed against Thy1.1 antigen can be used for drug targeting by coupling to the surface of drug-loaded liposomes. Such OX7-conjugated immunoliposomes (OX7-IL) were used recently for drug delivery to rat glomerular mesangial cells, which are characterized by a high level of Thy1.1 antigen expression. In the present study, the relationship between OX7-IL tissue distribution and target Thy1.1 antigen localization in different organs in rat was investigated. Western blot and immunohistofluorescence analysis revealed a very high Thy1.1 expression in brain cortex and striatum, thymus and renal glomeruli. Moderate Thy1.1 levels were observed in the collecting ducts of kidney, lung tissue and spleen. Thy1.1 was not detected in liver and heart. There was a poor correlation between Thy1.1 expression levels and organ distribution of fluorescence- or (14)C-labeled OX7-IL. The highest overall organ density of OX7-IL was observed in the spleen, followed by lung, liver and kidney. Heart and brain remained negative. With respect to intra-organ distribution, a localized and distinct signal was observed in renal glomerular mesangial cells only. As a consequence, acute pharmacological (i.e. toxic) effects of doxorubicin-loaded OX7-IL were limited to renal glomeruli. The competition with unbound OX7 monoclonal antibody F((ab')2) fragments demonstrated that the observed tissue distribution and acute pharmacological effects of OX7-IL were mediated specifically by the conjugated OX7 antibody. It is concluded that both the high target antigen density and the absence of endothelial barriers are needed to allow for tissue-specific accumulation and pharmacological effects of OX7-IL. The liposomal drug delivery strategy used is therefore specific toward renal glomeruli and can be expected to reduce the risk of unwanted side effects in other tissues.

Antiaggregatory and proangiogenic effects of a novel recombinant human dual specificity anti-integrin antibody

BACKGROUND: beta(3)-Integrins are involved in platelet aggregation via alpha(IIb)beta(3) [glycoprotein (GP)IIb-GPIIIa], and in angiogenesis via endothelial alpha(V)beta(3). Cross-reactive ligands with antiaggregatory and proangiogenic effects, both desirable in peripheral vasculopathies, have not yet been described. OBJECTIVES: In vitro and in vivo characterization of antiaggregatory and proangiogenic effects of two recombinant human Fab fragments, with emphasis on beta(3)-integrins. METHODS: Recombinant Fab fragments were obtained by phage display technology. Specificity, affinity and IC(50) were determined by immunodot assays, enzyme-linked immunosorbent assay (ELISA), and Scatchard plot analysis, and by means of human umbilical vein endothelial cells (HUVECs). Functional analyses included ELISA for interaction with fibrinogen binding to GPIIb-GPIIIa, flow cytometry for measurement of activation parameters and competitive inhibition experiments, human platelet aggregometry, and proliferation, tube formation and the chorioallantoic membrane (CAM) assay for measurement of angiogenic effects. RESULTS: We observed specific and high-affinity binding to an intact GPIIb-GPIIIa receptor complex of two human Fab autoantibody fragments, with no platelet activation. Dose-dependent fibrinogen binding to GPIIb-GPIIIa and platelet aggregation were completely inhibited. One Fab fragment was competitively inhibited by abciximab and its murine analog monoclonal antibody (mAb) 7E3, whereas the other Fab fragment bound to cultured HUVECs, suggesting cross-reactivity with alpha(V)beta(3), and also demonstrated proangiogenic effects in tube formation and CAM assays. CONCLUSIONS: These Fab fragments are the first entirely human anti-GPIIb-GPIIIa Fab fragments with full antiaggregatory properties; furthermore, they do not activate platelets. The unique dual-specificity anti-beta(3)-integrin Fab fragment may represent a new tool for the study and management of peripheral arterial vasculopathies.

The moderating effect of personal mastery and the relations between stress and Plasminogen Activator Inhibitor-1 (PAI-1) antigen

OBJECTIVE: This study tested whether feelings of personal control over one's life circumstances (i.e., personal mastery) would attenuate the relations between stress (i.e., negative life events and caregiving distress) and Plasminogen Activator Inhibitor (PAI)-1 antigen, an inhibitor of fibrinolysis implicated in the development of cardiovascular disease. DESIGN: Seventy-one spousal dementia caregivers were assessed for plasma levels of PAI-1 antigen, negative life events, caregiver distress, and feelings of personal mastery. Regression analysis was used to determine if personal mastery moderated the relations between stress (i.e., life stress and caregiving distress) and PAI-1 antigen levels. MAIN OUTCOME MEASURE: Plasminogen activator inhibitor (PAI)-1 antigen in plasma. RESULTS: After controlling for other factors associated with PAI-1 antigen levels, negative life events were positively associated with plasma PAI-1 antigen concentrations in participants low in personal mastery (beta = .31; p = .050) but not in individuals high in personal mastery (beta = .22; p = .184). The moderating effect of mastery on the relations between caregiving distress and PAI-1 antigen did not reach statistical significance (p = .091). CONCLUSIONS: These data suggest that mastery may protect individuals from some of the alterations in hemostatic factors that have been linked to cardiovascular risk.

Recombinant human erythropoiesis-stimulating agents and mortality in patients with cancer: a meta-analysis of randomised trials

BACKGROUND: Erythropoiesis-stimulating agents reduce anaemia in patients with cancer and could improve their quality of life, but these drugs might increase mortality. We therefore did a meta-analysis of randomised controlled trials in which these drugs plus red blood cell transfusions were compared with transfusion alone for prophylaxis or treatment of anaemia in patients with cancer. METHODS: Data for patients treated with epoetin alfa, epoetin beta, or darbepoetin alfa were obtained and analysed by independent statisticians using fixed-effects and random-effects meta-analysis. Analyses were by intention to treat. Primary endpoints were mortality during the active study period and overall survival during the longest available follow-up, irrespective of anticancer treatment, and in patients given chemotherapy. Tests for interactions were used to identify differences in effects of erythropoiesis-stimulating agents on mortality across prespecified subgroups. FINDINGS: Data from a total of 13 933 patients with cancer in 53 trials were analysed. 1530 patients died during the active study period and 4993 overall. Erythropoiesis-stimulating agents increased mortality during the active study period (combined hazard ratio [cHR] 1.17, 95% CI 1.06-1.30) and worsened overall survival (1.06, 1.00-1.12), with little heterogeneity between trials (I(2) 0%, p=0.87 for mortality during the active study period, and I(2) 7.1%, p=0.33 for overall survival). 10 441 patients on chemotherapy were enrolled in 38 trials. The cHR for mortality during the active study period was 1.10 (0.98-1.24), and 1.04 (0.97-1.11) for overall survival. There was little evidence for a difference between trials of patients given different anticancer treatments (p for interaction=0.42). INTERPRETATION: Treatment with erythropoiesis-stimulating agents in patients with cancer increased mortality during active study periods and worsened overall survival. The increased risk of death associated with treatment with these drugs should be balanced against their benefits. FUNDING: German Federal Ministry of Education and Research, Medical Faculty of University of Cologne, and Oncosuisse (Switzerland).

In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications

We describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications.

Cathepsin G is differentially expressed in primary human antigen-presenting cells

Cathepsins are required for the processing of antigens in order to make them suitable for loading on major histocompatibility complex (MHC) class II molecules, for subsequent presentation to CD4(+) T cells. It was shown that antigen processing in monocyte-derived dendritic cells (DC), a commonly used DC model, is different from that of primary human DC. Here, we report that the two subsets of human myeloid DC (mDC) and plasmacytoid DC (pDC) differ in their cathepsin distribution. The serine protease cathepsin G (CatG) was detected in mDC1, mDC2, pDC, cortical thymic epithelial cells (cTEC) and high levels of CatG were determined in pDC. To address the role of CatG in the processing and presentation of a Multiple Sclerosis-associated autoantigen myelin basic protein (MBP), we used a non-CatG expressing fibroblast cell line and fibroblasts, which were preloaded with purified CatG. We find that preloading fibroblasts with CatG results in a decrease of MBP84-98-specific T cell proliferation, when compared to control cells. Our data suggest a different processing signature in primary human antigen-presenting cells and CatG may be of functional importance.

Antigen Processing and Presentation in Multiple Sclerosis

CD4(+) T cells play a central role in the pathogenesis of multiple sclerosis (MS). Generation, activation and effector function of these cells crucially depends on their interaction with MHC II-peptide complexes displayed by antigen presenting cells (APC). Processing and presentation of self antigens by different APC therefore influences the disease course at all stages. Selection by thymic APC leads to the generation of autoreactive T cells, which can be activated by peripheral APC. Reactivation by central nervous system APC leads to the initiation of the inflammatory response resulting in demyelination. In this review we will focus on how MHC class II antigenic epitopes are created by different APC from the thymus, the periphery and from the brain, and will discuss the relevance of the balance between creation and destruction of such epitopes in the context of MS. A solid understanding of these processes offers the possibility for designing future therapeutic strategies.

New respiratory enterovirus and recombinant rhinoviruses among circulating picornaviruses

Rhinoviruses and enteroviruses are leading causes of respiratory infections. To evaluate genotypic diversity and identify forces shaping picornavirus evolution, we screened persons with respiratory illnesses by using rhinovirus-specific or generic real-time PCR assays. We then sequenced the 5 untranslated region, capsid protein VP1, and protease precursor 3CD regions of virus-positive samples. Subsequent phylogenetic analysis identified the large genotypic diversity of rhinoviruses circulating in humans. We identified and completed the genome sequence of a new enterovirus genotype associated with respiratory symptoms and acute otitis media, confirming the close relationship between rhinoviruses and enteroviruses and the need to detect both viruses in respiratory specimens. Finally, we identified recombinants among circulating rhinoviruses and mapped their recombination sites, thereby demonstrating that rhinoviruses can recombine in their natural host. This study clarifies the diversity and explains the reasons for evolution of these viruses.

Divergent adaptation of hepatitis C virus genotypes 1 and 3 to human leukocyte antigen-restricted immune pressure

Many hepatitis C virus (HCV) infections worldwide are with the genotype 1 and 3 strains of the virus. Cellular immune responses are known to be important in the containment of HCV genotype 1 infection, and many genotype 1 T cell targets (epitopes) that are presented by host human leukocyte antigens (HLAs) have been identified. In contrast, there is almost no information known about the equivalent responses to genotype 3. Immune escape mechanisms used by HCV include the evolution of viral polymorphisms (adaptations) that abrogate this host-viral interaction. Evidence of HCV adaptation to HLA-restricted immune pressure on HCV can be observed at the population level as viral polymorphisms associated with specific HLA types. To evaluate the escape patterns of HCV genotypes 1 and 3, we assessed the associations between viral polymorphisms and specific HLA types from 187 individuals with genotype 1a and 136 individuals with genotype 3a infection. We identified 51 HLA-associated viral polymorphisms (32 for genotype 1a and 19 for genotype 3a). Of these putative viral adaptation sites, six fell within previously published epitopes. Only two HLA-associated viral polymorphisms were common to both genotypes. In the remaining sites with HLA-associated polymorphisms, there was either complete conservation or no significant HLA association with viral polymorphism in the alternative genotype. This study also highlights the diverse mechanisms by which viral evasion of immune responses may be achieved and the role of genotype variation in these processes. CONCLUSION: There is little overlap in HLA-associated polymorphisms in the nonstructural proteins of HCV for the two genotypes, implying differences in the cellular immune pressures acting on these viruses and different escape profiles. These findings have implications for future therapeutic strategies to combat HCV infection, including vaccine design.

Morphological and immunocytochemical analysis of Escherichia coli-specific surface antigens in wildtype strains and in recombinant Vibrio cholerae

Adhesion is the first step in the pathogenesis of enterotoxigenic Escherichia coli infections. The genes encoding the most prevalent adhesion factors CFA/I, CS3 and CS6 were cloned into Vibrio cholerae strain CVD 103-HgR and expression of fimbriae was investigated in wildtype and recombinant strains by transmission electron microscopy in conjunction with immunolabelling and negative staining. Negative staining was effective in revealing CFA/I and CS3, but not CS6. Although morphology of fimbriae differed between wildtype and recombinant strains, corresponding surface antigens were recognized by specific antibodies. The present study provides evidence that ETEC-specific fimbriae can adequately be expressed in an attenuated V. cholerae vaccine strain and that immunoelectron microscopy is a critical tool to validate the surface expression of antigens in view of their possible suitability for recombinant vaccines.