Genome-wide association study of relative telomere length

Telomere function is essential to maintaining the physical integrity of linear chromosomes and healthy human aging. The probability of forming proper telomere structures depends on the length of the telomeric DNA tract. We attempted to identify common genetic variants associated with log relative telomere length using genome-wide genotyping data on 3,554 individuals from the Nurses'' Health Study and the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial that took part in the National Cancer Institute Cancer Genetic Markers of Susceptibility initiative for breast and prostate cancer. After genotyping 64 independent SNPs selected for replication in additional Nurses'' Health Study and Women''s Genome Health Study participants, we did not identify genome-wide significant loci; however, we replicated the inverse association of log relative telomere length with the minor allele variant [C] of rs16847897 at the TERC locus (per allele β = −0.03, P = 0.003) identified by a previous genome-wide association study. We did not find evidence for an association with variants at the OBFC1 locus or other loci reported to be associated with telomere length. With this sample size we had >80% power to detect β estimates as small as ±0.10 for SNPs with minor allele frequencies of ≥0.15 at genome-wide significance. However, power is greatly reduced for β estimates smaller than ±0.10, such as those for variants at the TERC locus. In general, common genetic variants associated with telomere length homeostasis have been difficult to detect. Potential biological and technical issues are discussed.     

Comprehensive resequence analysis of a 97 kb region of chromosome 10q11.2 containing the MSMB gene associated with prostate cancer

Genome-wide association studies of prostate cancer have identified single nucleotide polymorphism (SNP) markers in a region of chromosome 10q11.2, harboring the microseminoprotein-β (MSMB) gene. Both the gene product of MSMB, the prostate secretory protein 94 (PSP94) and its binding protein (PSPBP), have been previously investigated as serum biomarkers for prostate cancer progression. Recent functional work has shown that different alleles of the significantly associated SNP in the promoter of MSMB found to be associated with prostate cancer risk, rs10993994, can influence its expression in tumors and in vitro studies. Since it is plausible that additional variants in this region contribute to the risk of prostate cancer, we have used next-generation sequencing technology to resequence a ~97-kb region that includes the area surrounding MSMB (chr10: 51,168,025–51,265,101) in 36 prostate cancer cases, 26 controls of European origin, and 8 unrelated CEPH individuals in order to identify additional variants to investigate in functional studies. We identified 241 novel polymorphisms within this region, including 142 in the 51-kb block of linkage disequilibrium (LD) that contains rs10993994 and the proximal promoter of MSMB. No sites were observed to be polymorphic within the exons of MSMB.     

Targeting of epidermal Langerhans cells with antigenic proteins: attempts to harness their properties for immunotherapy

Langerhans cells, a subset of skin dendritic cells in the epidermis, survey peripheral tissue for invading pathogens. In recent functional studies it was proven that Langerhans cells can present exogenous antigen not merely on major histocompatibility complexes (MHC)-class II molecules to CD4⁺ T cells, but also on MHC-class I molecules to CD8⁺ T cells. Immune responses against topically applied antigen could be measured in skin-draining lymph nodes. Skin barrier disruption or co-application of adjuvants was required for maximal induction of T cell responses. Cytotoxic T cells induced by topically applied antigen inhibited tumor growth in vivo, thus underlining the potential of Langerhans cells for immunotherapy. Here we review recent work and report novel observations relating to the potential use of Langerhans cells for immunotherapy. We investigated the potential of epicutaneous immunization strategies in which resident skin dendritic cells are loaded with tumor antigen in situ. This contrasts with current clinical approaches, where dendritic cells generated from progenitors in blood are loaded with tumor antigen ex vivo before injection into cancer patients. In the current study, we applied either fluorescently labeled protein antigen or targeting antibodies against DEC-205/CD205 and langerin/CD207 topically onto barrier-disrupted skin and examined antigen capture and transport by Langerhans cells. Protein antigen could be detected in Langerhans cells in situ, and they were the main skin dendritic cell subset transporting antigen during emigration from skin explants. Potent in vivo proliferative responses of CD4⁺ and CD8⁺ T cells were measured after epicutaneous immunization with low amounts of protein antigen. Targeting antibodies were mainly transported by langerin⁺ migratory dendritic cells of which the majority represented migratory Langerhans cells and a smaller subset the new langerin⁺ dermal dendritic cell population located in the upper dermis. The preferential capture of topically applied antigen by Langerhans cells and their ability to induce potent CD4⁺ and CD8⁺ T cell responses emphasizes their potential for epicutaneous immunization strategies. This article is a symposium paper from the conference “Immunotherapy—From Basic Research to Clinical Applications,” Symposium of the Collaborative Research Center (SFB) 685, held in Tübingen, Germany, 6–7 March 2008.     

Physical-chemical plant-derived signals induce differentiation in Ustilago maydis

Ustilago maydis is able to initiate pathogenic development after fusion of two haploid cells with different mating type. On the maize leaf surface, the resulting dikaryon switches to filamentous growth, differentiates appressoria and penetrates the host. Here, we report on the plant signals required for filament formation and appressorium development in U. maydis. In vitro , hydroxy-fatty acids stimulate filament formation via the induction of pheromone genes and this signal can be bypassed by genetically activating the downstream MAP kinase module. Hydrophobicity also induces filaments and these resemble the dikaryotic filaments formed on the plant surface. With the help of a marker gene that is specifically expressed in the tip cell of those hyphae that have formed an appressorium, hydrophobicity is shown to be essential for appressorium development in vitro . Hydroxy-fatty acids or a cutin monomer mixture isolated from maize leaves have a stimulatory role when a hydrophobic surface is provided. Our results suggest that the early phase of communication between U. maydis and its host plant is governed by two different stimuli.     

A gene-protein assay for human epidermal growth factor receptor 2 (HER2): brightfield tricolor visualization of HER2 protein, the HER2 gene, and chromosome 17 centromere (CEN17) in formalin-fixed, paraffin-embedded breast cancer tissue sections

ABSTRACT: BACKGROUND: The eligibility of breast cancer patients for human epidermal growth factor receptor 2 (HER2)-directed therapies is determined by the HER2 gene amplification and/or HER2 protein overexpression status of the breast tumor as determined by in situ hybridization (ISH) or immunohistochemistry (IHC), respectively. Our objective was to combine the US Food and Drug Administration (FDA)-approved HER2 & chromosome 17 centromere (CEN17) brightfield ISH (BISH) and HER2 IHC assays into a single automated HER2 gene-protein assay allowing simultaneous detection of all three targets in a single tissue section. METHODS: The HER2 gene-protein assay was optimized using formalin-fixed, paraffin-embedded (FFPE) samples of the xenograft tumors MCF7 [HER2 negative (non-amplified gene, protein negative)] and Calu-3 [HER2 positive (amplified gene, protein positive)]. HER2 IHC was performed using a rabbit monoclonal anti-HER2 antibody (clone 4B5) and a conventional 3,3' -diaminobenzidine IHC detection. The HER2 & CEN17 BISH signals were visualized using horseradish peroxidase-based silver and alkaline phosphatase-based red detection systems, respectively with a cocktail of 2,4-dinitrophenyl-labeled HER2 and digoxigeninlabeled CEN17 probes. The performance of the gene-protein assay on tissue microarray slides containing 189 randomly selected FFPE clinical breast cancer tissue cores was compared to that of the separate HER2 IHC and HER2 & CEN17 BISH assays. RESULTS: HER2 protein detection was optimal when the HER2 IHC protocol was used before (rather than after) the BISH protocol. The sequential use of HER2 IHC and HER2 & CEN17 BISH detection steps on FFPE xenograft tumor sections appropriately co-localized the HER2 protein, HER2 gene, and CEN17 signals after mitigating the silver background staining by using a naphthol phosphate-containing hybridization buffer for the hybridization step. The HER2 protein and HER2 gene status obtained using the multiplex HER2 gene-protein assay demonstrated high concordance with those obtained using the separate HER2 IHC and HER2 & CEN17 BISH assays, respectively. CONCLUSIONS: We have developed a protocol that allows simultaneous visualization of the HER2 IHC and HER2 & CEN17 BISH targets. This automated protocol facilitated the determination of HER2 protein and HER2 gene status in randomly selected breast cancer samples, particularly in cases that were equivocal or exhibited tumor heterogeneity. The HER2 gene-protein assay produced results virtually equivalent to those of the single FDA-approved HER2 IHC and HER2 & CEN17 BISH assays. Virtual slides The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2041964038705297.     

Identification of new genes involved in human adipogenesis and fat storage

Since the worldwide increase in obesity represents a growing challenge for health care systems, new approaches are needed to effectively treat obesity and its associated diseases. One prerequisite for advances in this field is the identification of genes involved in adipogenesis and/or lipid storage. To provide a systematic analysis of genes that regulate adipose tissue biology and to establish a target-oriented compound screening, we performed a high throughput siRNA screen with primary (pre)adipocytes, using a druggable siRNA library targeting 7,784 human genes. The primary screen showed that 459 genes affected adipogenesis and/or lipid accumulation after knock-down. Out of these hits, 333 could be validated in a secondary screen using independent siRNAs and 110 genes were further regulated on the gene expression level during adipogenesis. Assuming that these genes are involved in neutral lipid storage and/or adipocyte differentiation, we performed InCell-Western analysis for the most striking hits to distinguish between the two phenotypes. Beside well known regulators of adipogenesis and neutral lipid storage (i.e. PPARγ, RXR, Perilipin A) the screening revealed a large number of genes which have not been previously described in the context of fatty tissue biology such as axonemal dyneins. Five out of ten axonemal dyneins were identified in our screen and quantitative RT-PCR-analysis revealed that these genes are expressed in preadipocytes and/or maturing adipocytes. Finally, to show that the genes identified in our screen are per se druggable we performed a proof of principle experiment using an antagonist for HTR2B. The results showed a very similar phenotype compared to knock-down experiments proofing the "druggability". Thus, we identified new adipogenesis-associated genes and those involved in neutral lipid storage. Moreover, by using a druggable siRNA library the screen data provides a very attractive starting point to identify anti-obesity compounds targeting the adipose tissue.     

Identification and characterization of equine herpesvirus type 1 pUL56 and its role in virus-induced downregulation of major histocompatibility complex class I

Major histocompatibility complex class I (MHC-I) molecules play an important role in host immunity to infection by presenting antigenic peptides to cytotoxic T lymphocytes (CTLs), which recognize and destroy virus-infected cells. Members of the Herpesviridae have developed multiple mechanisms to avoid CTL recognition by virtue of downregulation of MHC-I on the cell surface. We report here on an immunomodulatory protein involved in this process, pUL56, which is encoded by ORF1 of equine herpesvirus type 1 (EHV-1), an alphaherpesvirus. We show that EHV-1 pUL56 is a phosphorylated early protein which is expressed as different forms and predominantly localizes to Golgi membranes. In addition, the transmembrane (TM) domain of the type II membrane protein was shown to be indispensable for correct subcellular localization and a proper function. pUL56 by itself is not functional with respect to interference with MHC-I and likely needs another unidentified viral protein(s) to perform this action. Surprisingly, pUL49.5, an inhibitor of the transporter associated with antigen processing (TAP) and encoded by EHV-1 and related viruses, appeared not to be required for pUL56-induced early MHC-I downmodulation in infected cells. In conclusion, our data identify a new immunomodulatory protein, pUL56, involved in MHC-I downregulation which is unable to perform its function outside the context of viral infection.