Identification of a Human Cyclin D1-Derived Peptide that Induces Human Cytotoxic CD4 T Cells

Cyclin D1 is over-expressed in various human tumors and therefore can be a potential oncogenic target antigen. However, only a limited number of T cell epitopes has been characterized. We aimed at identifying human cyclin D1-derived peptides that include both CD4 and CD8 T cell epitopes and to test if such multi-epitope peptides could yield improved cytotoxic CD8 T cell responses as well as cytotoxic CD4 T cells. Five HLA-DR.B1-binding peptides containing multiple overlapping CD4 epitopes and HLA-A0201-restricted CD8 T cell epitopes were predicted by computer algorithms. Immunogenicity of the synthetic peptides was assessed by stimulating T cells from healthy donors in vitro and the epitope recognition was measured by IFN-γ ELISPOT and ⁵¹Chromium release assays. A HLA-DR.B1 peptide, designed “DR-1”, in which a HLA-A0201-binding epitopes (D1-1) was imbedded, induced CD3 T cell responses against both DR-1 and D1-1 peptides in IFN-γ ELISPOT assay. This suggested processing of the shorter D1-1 epitope from the DR-1 sequence. However, only DR-1-stimulated CD4 or CD3 T cells possessed cytotoxicity against peptide-pulsed autologous DCs and a cancer cell line, that expresses a high level of cyclin D1. Monoclonal antibody to HLA-DR abrogated the epitope-specific responses of both CD3 and CD4 T cells, demonstrating class II-mediated killing. Our studies suggest a possible role of CD4 T cells in anti-tumor immunity as cytotoxic effectors against HLA-DR expressing cancers and provide a rationale for designing peptide vaccines that include CD4 epitopes.     

MAP dendrimer elicits antibodies for detecting rat and mouse GH‐binding proteins

The membrane‐bound rat GH‐R and an alternatively spliced isoform, the soluble rat GH‐BP, are comprised of identical N‐terminal GH‐binding domains; however, their C‐terminal sequences differ. Immunological reagents are needed to distinguish between the two isoforms in order to understand their respective roles in mediating the actions of GH. Accordingly, a tetravalent MAP dendrimer with four identical branches of a C‐terminal peptide sequence of the rat GH‐BP (GH‐BP_263–279) was synthesized and used as an immunogen in rabbits. Solid‐phase peptide synthesis of four GH‐BP_263–279 segments onto a tetravalent Lys₂‐Lys‐β‐Ala‐OH core peptide was carried out using Fmoc chemistry. The mass of the RP‐HPLC‐purified synthetic product, 8398 Da, determined by ESI‐MS, was identical to expected mass. Three anti‐rat GH‐BP_263–279 MAP antisera, BETO‐8039, BETO‐8040, and BETO‐8041, at dilutions of 10^?3, recognized both the rat GH‐BP_263–279 MAP and recombinant mouse GH‐BP with ED₅₀s within a range of 5–10 fmol, but did not cross‐react with BSA in dot blot analyses. BETO‐8041 antisera (10^?3 dilution) recognized GH‐BPs of rat serum and liver having M_rs ranging from 35 to 130 kDa, but did not recognize full‐length rat GH‐Rs. The antisera also detected recombinant mouse GH‐BPs. In summary, the tetravalent rat GH‐BP_263–279 MAP dendrimer served as an effective immunogenic antigen in eliciting high titer antisera specific for the C‐termini of both rat and mouse GH‐BPs. The antisera will facilitate studies aimed at improving our understanding of the biology of GH‐BPs. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

DNA-PKcs and ATM co-regulate DNA double-strand break repair

DNA double-strand breaks (DSBs) are repaired by nonhomologous end-joining (NHEJ) and homologous recombination (HR). The NHEJ/HR decision is under complex regulation and involves DNA-dependent protein kinase (DNA-PKcs). HR is elevated in DNA-PKcs null cells, but suppressed by DNA-PKcs kinase inhibitors, suggesting that kinase-inactive DNA-PKcs (DNA-PKcs-KR) would suppress HR. Here we use a direct repeat assay to monitor HR repair of DSBs induced by I-SceI nuclease. Surprisingly, DSB-induced HR in DNA-PKcs-KR cells was 2- to 3-fold above the elevated HR level of DNA-PKcs null cells, and ～4- to 7-fold above cells expressing wild-type DNA-PKcs. The hyperrecombination in DNA-PKcs-KR cells compared to DNA-PKcs null cells was also apparent as increased resistance to DNA crosslinks induced by mitomycin C. ATM phosphorylates many HR proteins, and ATM is expressed at a low level in cells lacking DNA-PKcs, but restored to wild-type level in cells expressing DNA-PKcs-KR. Several clusters of phosphorylation sites in DNA-PKcs, including the T2609 cluster, which is phosphorylated by DNA-PKcs and ATM, regulate access of repair factors to broken ends. Our results indicate that ATM-dependent phosphorylation of DNA-PKcs-KR contributes to the hyperrecombination phenotype. Interestingly, DNA-PKcs null cells showed more persistent ionizing radiation-induced RAD51 foci (but lower HR levels) compared to DNA-PKcs-KR cells, consistent with HR completion requiring RAD51 turnover. ATM may promote RAD51 turnover, suggesting a second (not mutually exclusive) mechanism by which restored ATM contributes to hyperrecombination in DNA-PKcs-KR cells. We propose a model in which DNA-PKcs and ATM coordinately regulate DSB repair by NHEJ and HR.     

Cross-National Analysis of the Associations among Mental Disorders and Suicidal Behavior: Findings from the WHO World Mental Health Surveys

Background

Suicide is a leading cause of death worldwide. Mental disorders are among the strongest predictors of suicide; however, little is known about which disorders are uniquely predictive of suicidal behavior, the extent to which disorders predict suicide attempts beyond their association with suicidal thoughts, and whether these associations are similar across developed and developing countries. This study was designed to test each of these questions with a focus on nonfatal suicide attempts.

Methods and Findings

Data on the lifetime presence and age-of-onset of Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV) mental disorders and nonfatal suicidal behaviors were collected via structured face-to-face interviews with 108,664 respondents from 21 countries participating in the WHO World Mental Health Surveys. The results show that each lifetime disorder examined significantly predicts the subsequent first onset of suicide attempt (odds ratios [ORs] = 2.9–8.9). After controlling for comorbidity, these associations decreased substantially (ORs = 1.5–5.6) but remained significant in most cases. Overall, mental disorders were equally predictive in developed and developing countries, with a key difference being that the strongest predictors of suicide attempts in developed countries were mood disorders, whereas in developing countries impulse-control, substance use, and post-traumatic stress disorders were most predictive. Disaggregation of the associations between mental disorders and nonfatal suicide attempts showed that these associations are largely due to disorders predicting the onset of suicidal thoughts rather than predicting progression from thoughts to attempts. In the few instances where mental disorders predicted the transition from suicidal thoughts to attempts, the significant disorders are characterized by anxiety and poor impulse-control. The limitations of this study include the use of retrospective self-reports of lifetime occurrence and age-of-onset of mental disorders and suicidal behaviors, as well as the narrow focus on mental disorders as predictors of nonfatal suicidal behaviors, each of which must be addressed in future studies.

Conclusions

This study found that a wide range of mental disorders increased the odds of experiencing suicide ideation. However, after controlling for psychiatric comorbidity, only disorders characterized by anxiety and poor impulse-control predict which people with suicide ideation act on such thoughts. These findings provide a more fine-grained understanding of the associations between mental disorders and subsequent suicidal behavior than previously available and indicate that mental disorders predict suicidal behaviors similarly in both developed and developing countries. Future research is needed to delineate the mechanisms through which people come to think about suicide and subsequently progress from ideation to attempts. Please see later in the article for Editors'' Summary     

Endogenous short RNAs generated by Dicer 2 and RNA-dependent RNA polymerase 1 regulate mRNAs in the basal fungus Mucor circinelloides

Endogenous short RNAs (esRNAs) play diverse roles in eukaryotes and usually are produced from double-stranded RNA (dsRNA) by Dicer. esRNAs are grouped into different classes based on biogenesis and function but not all classes are present in all three eukaryotic kingdoms. The esRNA register of fungi is poorly described compared to other eukaryotes and it is not clear what esRNA classes are present in this kingdom and whether they regulate the expression of protein coding genes. However, evidence that some dicer mutant fungi display altered phenotypes suggests that esRNAs play an important role in fungi. Here, we show that the basal fungus Mucor circinelloides produces new classes of esRNAs that map to exons and regulate the expression of many protein coding genes. The largest class of these exonic-siRNAs (ex-siRNAs) are generated by RNA-dependent RNA Polymerase 1 (RdRP1) and dicer-like 2 (DCL2) and target the mRNAs of protein coding genes from which they were produced. Our results expand the range of esRNAs in eukaryotes and reveal a new role for esRNAs in fungi.     

Assessment of synthetic chimeric multiple antigenic peptides for diagnosis of GB virus C infection

The use of synthetic peptides of both structural and nonstructural proteins of GB virus C (GBV-C) has been studied for the development of new systems to diagnose infection caused by this virus. In an attempt to increase the antigenicity of linear peptide sequences, chimeric multiple antigenic peptides (MAPs) containing epitopes from E2, NS4, and NS5 GBV-C proteins have been synthesized. The synthetic constructs were evaluated by ELISA to establish whether the epitopes in chimeric branched peptides are more efficiently recognized by the specific antibodies compared to the monomeric linear sequences. Moreover, we have investigated the application of a commercial biosensor instrument for the detection of antibodies against the GBV-C in human serum samples. The results of the immunoassays reported in this work highlight the usefulness of synthetic tetrameric branched peptides containing sequences from envelope and nonstructural GBV-C proteins for the diagnosis of GBV-C infection. The potential clinical value of the MAP₄(E2-NS5a) for the serodiagnosis of GBV-C infection was demonstrated, thus providing the basis for performing prevalence studies of the infection among the hemodialyzed and hepatitis C virus (HCV)-infected population.     

Autophagy in immune defense against Mycobacterium tuberculosis

Autophagy is a newly recognized innate and adaptive immunity defense against intracellular pathogens, in keeping with its role as a cytoplasmic maintenance pathway. Induction of autophagy by physiological, pharmacological or immunological means can eliminate intracellular Mycobacterium tuberculosis, providing one of the first examples of the immunological role of autophagy. Under normal circumstances, M. Tuberculosis survives in macrophages by inhibiting phagolysosome biogenesis. Induction of autophagy overcomes the mycobacterial phagosome maturation block, and delivers the tubercle bacilli to degradative compartments where they are eliminated.     

Liposome entrapment and immunogenic studies of a synthetic lipophilic multiple antigenic peptide bearing VP1 and VP3 domains of the hepatitis A virus: a robust method for vaccine design

Multiple antigen peptides (MAP) have been demonstrated to be efficient immunological reagents for the induction of immune responses to a variety of infectious agents. Several peptide domains of the hepatitis A virus (HAV) capsid proteins, mainly VP1 and VP3, are the immunodominant targets for a protective antibody response. In the present study we analyse the immunogenic properties of a tetrameric heterogeneous palmitoyl-derivatised MAP containing two defined HAV peptide sequences, VP1(11–25) and VP3(102–121), in rabbits immunised with either Freund’s adjuvant or multilamellar liposomes. The immune response was evaluated with a specific enzyme immunoassay using MAP[VP1+VP3], VP1 and VP3 as targets. The avidity of the immune response was measured by a non-competitive enzyme-linked immunosorbent assay and by the surface plasmon resonance technology. Antisera raised against the lipo-MAP peptide entrapped in liposomes demonstrated high avidity of binding with affinity rate constants approximately one order of magnitude greater than those obtained with the Freund’s protocol.