Vitamin D receptor variants and the malignant melanoma risk: A population-based study

Background: There is continuing interest in identifying low-penetrance genes which are associated with an increased susceptibility to common types of cancer, including malignant melanoma. Methods: We sought to examine the association between four VDR common variants (rs1544410, rs731236, rs10735810, rs4516035) and the risk of melanoma in the Polish population. We also determined the prevalence of compound carriers of VDR and known MM genetic risk factors MC1R and CDKN2A (A148T) variants. We examined 763 unselected melanoma cases, 763 healthy adults matched for sex and age with the melanoma cases and 777 newborns. Results: None of the VDR variants alone or as compound carriers of two or more of the VDR genotypes were associated with MM risk. There were no major differences between the prevalences of the examined variants among patients with MM on UV-exposed and UV-non exposed skin areas, as well as among early-onset and late-onset cases. We found no association between VDR and MC1R or between VDR and CDKN2A common variants. A statistically significant over-representation of one VDR haplotype: rs731236_A + rs1544410_T (OR = 3.2, p = 0.02) was detected. Linkage disequilibrium of rs1544410 and rs731236 was confirmed. Conclusion: To answer the question, whether VDR can be regarded as melanoma susceptibility gene, additional, large multi-center association studies have to be performed.     

Short interfering RNA-mediated inhibition of herpes simplex virus type 1 gene expression and function during infection of human keratinocytes

RNA interference (RNAi) is an antiviral mechanism that is activated when double-stranded RNA is cleaved into fragments, called short interfering RNA (siRNA), that prime an inducible gene silencing enzyme complex. We applied RNAi against a herpes simplex virus type 1 (HSV-1) gene, glycoprotein E, which mediates cell-to-cell spread and immune evasion. In an in vitro model of infection, human keratinocytes were transfected with siRNA specific for glycoprotein E and then infected with wild-type HSV-1. RNAi-mediated gene silencing reproduced the small plaque phenotype of a gE-deletion mutant virus. The specificity of gene targeting was demonstrated by flow cytometry and Northern blot analyses. Exogenous siRNA can suppress HSV-1 glycoprotein E expression and function during active infection in vitro through RNAi. This work establishes RNAi as a genetic tool for the study of HSV and provides a foundation for development of RNAi as a novel antiviral therapy.     

Immunization strategies to block the herpes simplex virus type 1 immunoglobulin G Fc receptor

Herpes simplex virus type 1 (HSV-1) glycoprotein gE functions as an immunoglobulin G (IgG) Fc receptor (FcgammaR) that promotes immune evasion. When an IgG antibody binds by the F(ab')(2) domain to an HSV antigen, the Fc domain of some of the same antibody molecules binds to the FcgammaR, which blocks Fc-mediated functions. gE is a type 1 membrane glycoprotein with a large ectodomain that is expressed on the virion envelope and infected-cell surface. Our goal was to determine if immunizing with gE protein fragments could produce antibodies that bind by the F(ab')(2) domain to gE and block the FcgammaR, as measured by competitively inhibiting nonimmune human IgG binding to the FcgammaR. Three gE peptides were constructed in baculovirus spanning almost the entire ectodomain and used to immunize mice and rabbits. Two fragments were highly effective at producing antibodies that bind by the F(ab')(2) domain and block the FcgammaR. The most potent of these two antibodies was far more effective at blocking the FcgammaR than antibodies that are only capable of binding by the Fc domains to the FcgammaR, including anti-gC, anti-gD, and nonimmune IgG. These results suggest that immunizing with gE fragments has potential for preventing immune evasion by blocking activities mediated by the HSV-1 FcgammaR.     

Herpes simplex virus type 1 and 2 glycoprotein C prevents complement-mediated neutralization induced by natural immunoglobulin M antibody

Glycoprotein C (gC) of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) binds complement component C3b and protects virus from complement-mediated neutralization. Differences in complement interacting domains exist between gC of HSV-1 (gC1) and HSV-2 (gC2), since the amino terminus of gC1 blocks complement C5 from binding to C3b, while gC2 fails to interfere with this activity. We previously reported that neutralization of HSV-1 gC-null virus by HSV antibody-negative human serum requires activation of C5 but not of downstream components of the classical complement pathway. In this report, we evaluated whether activation of C5 is sufficient to neutralize HSV-2 gC-null virus, or whether formation of the membrane attack complex by C6 to C9 is required for neutralization. We found that activation of the classical complement pathway up to C5 was sufficient to neutralize HSV-2 gC-null virus by HSV antibody-negative human serum. We evaluated the mechanisms by which complement activation occurred in seronegative human serum. Interestingly, natural immunoglobulin M antibodies bound to virus, which triggered activation of C1q and the classical complement pathway. HSV antibody-negative sera obtained from four individuals differed over an approximately 10-fold range in their potency for complement-mediated virus neutralization. These findings indicate that humans differ in the ability of their innate immune systems to neutralize HSV-1 or HSV-2 gC-null virus and that a critical function of gC1 and gC2 is to prevent C5 activation.     

A replication-competent, neuronal spread-defective, live attenuated herpes simplex virus type 1 vaccine

Herpes simplex virus type 1 (HSV-1) produces oral lesions, encephalitis, keratitis, and severe infections in the immunocompromised host. HSV-1 is almost as common as HSV-2 in causing first episodes of genital herpes, a disease that is associated with an increased risk of human immunodeficiency virus acquisition and transmission. No approved vaccines are currently available to protect against HSV-1 or HSV-2 infection. We developed a novel HSV vaccine strategy that uses a replication-competent strain of HSV-1, NS-gEnull, which has a defect in anterograde and retrograde directional spread and cell-to-cell spread. Following scratch inoculation on the mouse flank, NS-gEnull replicated at the site of inoculation without causing disease. Importantly, the vaccine strain was not isolated from dorsal root ganglia (DRG). We used the flank model to challenge vaccinated mice and demonstrated that NS-gEnull was highly protective against wild-type HSV-1. The challenge virus replicated to low titers at the site of inoculation; therefore, the vaccine strain did not provide sterilizing immunity. Nevertheless, challenge by HSV-1 or HSV-2 resulted in less-severe disease at the inoculation site, and vaccinated mice were totally protected against zosteriform disease and death. After HSV-1 challenge, latent virus was recovered by DRG explant cocultures from <10% of vaccinated mice compared with 100% of mock-vaccinated mice. The vaccine provided protection against disease and death after intravaginal challenge and markedly lowered the titers of the challenge virus in the vagina. Therefore, the HSV-1 gEnull strain is an excellent candidate for further vaccine development.     

DNA repair gene polymorphisms and risk of early onset colorectal cancer in Lynch syndrome

DNA repair plays a pivotal role in maintaining genomic integrity with over 130 genes involved in various repair pathways that include base excision repair, nucleotide excision repair, double strand break repair and DNA mismatch repair. Polymorphisms within genes that are involved in these processes have been widely reported to be associated with cancer susceptibility in an extensive range of malignancies that include colorectal cancer (CRC). Lynch syndrome is caused by inherited germline mutations in DNA mismatch repair genes, predominantly in MLH1 and MSH2, that predispose to a variety of epithelial malignancies, most notably CRC. Despite being a relatively well understood hereditary cancer syndrome there remain several questions in relation to genetic influences on disease expression. Since Lynch syndrome is associated with a breakdown in DNA mismatch repair variation in other DNA repair genes may influence disease expression. In this report we have genotyped 424 Australian and Polish Lynch syndrome participants for eight common DNA repair gene polymorphisms to assess any association with the age of CRC onset. The DNA repair gene SNPs included in the study were: BRCA2 (rs11571653), MSH3 (rs26279), Lig4 (rs1805386), OGG1 (rs1052133), XRCC1 (rs25487), XRCC2 (rs3218536 and rs1799793) and XRCC3 (rs861539). Cox multi-variant regression modelling failed to provide any convincing evidence of an effect in any of the polymorphisms analysed. The data suggest that polymorphisms in DNA repair genes do not contribute to cancer risk in a population of CRC patients who are at increased risk of disease as a result in a deficiency of DNA mismatch repair.     

Purification of a soluble template-dependent rhinovirus RNA polymerase and its dependence on a host cell protein for viral RNA synthesis.

The soluble phase of the cytoplasm of human rhinovirus type 2-infected cells contains an enzymatic activity able to copy rhinovirion RNA without an added primer. This RNA-dependent RNA polymerase (replicase) makes a specific copy of the added rhinovirion RNA, as shown by hybridization of the product to its template RNA but not to other RNAs. The same replicase preparation also contains a virus-specific polyuridylic acid [poly(U)] polymerase activity which is dependent on added polyadenylic acid-oligouridylic acid template-primer. Both activities purify together until a step at which poly(U) polymerase but no replicase activity is recovered. Addition of a purified HeLa cell protein (host factor) to this poly(U) polymerase completely reconstitutes rhinovirus replicase activity. Host factor activity can be supplied by adding oligouridylic acid, suggesting that the host cell protein acts at the initiation step of rhinovirus RNA replication. A virus-specific 64,000-dalton protein purifies with both poly(U) polymerase and replicase activities.     

In Vivo Immune Evasion Mediated by the Herpes Simplex Virus Type 1 Immunoglobulin G Fc Receptor

Herpes simplex virus (HSV) glycoproteins gE and gI form an immunoglobulin G (IgG) Fc receptor (FcγR) that binds the Fc domain of human anti-HSV IgG and inhibits Fc-mediated immune functions in vitro. gE or gI deletion mutant viruses are avirulent, probably because gE and gI are also involved in cell-to-cell spread. In an effort to modify FcγR activity without affecting other gE functions, we constructed a mutant virus, NS-gE₃₃₉, that has four amino acids inserted into gE within the domain homologous to mammalian IgG FcγRs. NS-gE₃₃₉ expresses gE and gI, is FcγR⁻, and does not participate in antibody bipolar bridging since it does not block activities mediated by the Fc domain of anti-HSV IgG. In vivo studies were performed with mice because the HSV-1 FcγR does not bind murine IgG; therefore, the absence of an FcγR should not affect virulence in mice. NS-gE₃₃₉ causes disease at the skin inoculation site comparably to wild-type and rescued viruses, indicating that the FcγR⁻ mutant virus is pathogenic in animals. Mice were passively immunized with human anti-HSV IgG and then infected with mutant or wild-type virus. We postulated that the HSV-1 FcγR should protect wild-type virus from antibody attack. Human anti-HSV IgG greatly reduced viral titers and disease severity in NS-gE₃₃₉-infected animals while having little effect on wild-type or rescued virus. We conclude that the HSV-1 FcγR enables the virus to evade antibody attack in vivo, which likely explains why antibodies are relatively ineffective against HSV infection.