Gene sequence variations of the platelet P2Y12 receptor are associated with coronary artery disease

Background

By performing extensive scanning of whole coding and flanking sequences of the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene, we had previously identified point mutations in 167 out of 182 (91.7%) males with isolated congenital bilateral absence of the vas deferens (CBAVD). Conventional PCR-based methods of mutation analysis do not detect gross DNA lesions. In this study, we looked for large rearrangements within the whole CFTR locus in the 32 CBAVD patients with only one or no mutation.

Methods

We developed a semi-quantitative fluorescent PCR assay (SQF-PCR), which relies on the comparison of the fluorescent profiles of multiplex PCR fragments obtained from different DNA samples. We confirmed the gross alterations by junction fragment amplification and identified their breakpoints by direct sequencing.

Results

We detected two large genomic heterozygous deletions, one encompassing exon 2 (c.54-5811_c.164+2186del8108ins182) [or CFTRdele2], the other removing exons 22 to 24 (c.3964-3890_c.4443+3143del9454ins5) [or CFTRdele 22_24], in two males carrying a typical CBAVD mutation on the other parental CFTR allele. We present the first bioinformatic tool for exon phasing of the CFTR gene, which can help to rename the exons and the nomenclature of small mutations according to international recommendations and to predict the consequence of large rearrangements on the open reading frame.

Conclusion

Identification of large rearrangements further expands the CFTR mutational spectrum in CBAVD and should now be systematically investigated. We have designed a simple test to specifically detect the presence or absence of the two rearrangements identified in this study.     

T cell costimulation by the hepatitis C virus envelope protein E2 binding to CD81 is mediated by Lck

Binding of the hepatitis C virus (HCV) envelope protein E2 to CD81 provides a costimulatory signal for human T cells. This phenomenon may play a role in liver damage and autoimmune manifestations associated with HCV infection. Here we show that cross-linking of CD81 by HCV E2 induced a calcium flux in T cells that depends on Lck since it was blocked by PP1 and absent in Lck-deficient Jurkat T cells. In wild-type Jurkat cells, Lck was activated by CD81 cross-linking, and CD81, like Lck, was found in lipid rafts. Indeed, the integrity of the raft compartment was required for the induction of a calcium flux by E2, since methyl-beta-cyclodextrin abolished this response. A requirement for TCR/CD3 expression was indicated by the absence of a calcium flux following E2 stimulation of TCR/CD3-deficient Jurkat cells. CD81 cross-linking increased and prolonged the anti-CD3-induced tyrosine phosphorylation of TCR1 and of other proteins, indicating that the CD81-mediated signal converges with the TCR/CD3 signaling cascade at its most upstream step. In conclusion, we propose that the costimulatory effects of HCV E2 on T cells depend on CD81 cross-linking that activates Lck through raft aggregation and thus leads to enhanced TCR signaling.     

The ribosomal basis of Diamond-Blackfan Anemia: mutation and database update

Diamond-Blackfan Anemia (DBA) is characterized by a defect of erythroid progenitors and, clinically, by anemia and malformations. DBA exhibits an autosomal dominant pattern of inheritance with incomplete penetrance. Currently nine genes, all encoding ribosomal proteins (RP), have been found mutated in approximately 50% of patients. Experimental evidence supports the hypothesis that DBA is primarily the result of defective ribosome synthesis. By means of a large collaboration among six centers, we report here a mutation update that includes nine genes and 220 distinct mutations, 56 of which are new. The DBA Mutation Database now includes data from 355 patients. Of those where inheritance has been examined, 125 patients carry a de novo mutation and 72 an inherited mutation. Mutagenesis may be ascribed to slippage in 65.5% of indels, whereas CpG dinucleotides are involved in 23% of transitions. Using bioinformatic tools we show that gene conversion mechanism is not common in RP genes mutagenesis, notwithstanding the abundance of RP pseudogenes. Genotype-phenotype analysis reveals that malformations are more frequently associated with mutations in RPL5 and RPL11 than in the other genes. All currently reported DBA mutations together with their functional and clinical data are included in the DBA Mutation Database.     

JAK/STAT/PKCδ molecular pathways in synovial fluid T lymphocytes reflect the in vivo T helper-17 expansion in psoriatic arthritis

Looking to the sustained psoriatic arthritis (PsA) joint as a model of local human inflammation, this study was designed to assess the T lymphocyte signal transduction pathways potentially involved in this chronic immune-mediated inflammatory process, as characterized by direct ex vivo analysis of T helper (Th)-17 T effector (Teff) cell phenotypes in synovial fluid (SF) and peripheral blood (PB) of clinically active PsA patients. The reverse-phase protein arrays (RPPA) technique was employed to identify STAT3, STAT1, JAK1, JAK2, PKCδ and ERK1/2 phosphoprotein levels on total T cell lysates in SF samples of PsA patients. Frequencies of T CD4⁺IL-17A-F⁺ and T CD4⁺IL-23R⁺ Th17 cells were quantified in SF and matched PB of PsA patients by flow cytometry and compared with PB of healthy controls (HC). Increased levels of JAK1, STAT3, STAT1 and PKCδ phosphoproteins were found in SF T cells of PsA patients, compared with PB of HC. The expansion of T CD4⁺IL-17A-F⁺ cells, as well as of T CD4⁺ cells expressing IL-23Rp19 (T CD4⁺ IL-23R⁺), considered as the pathogenic phenotype of effector Th17 cells, was found to be confined to the joints of PsA patients, as the frequencies of both populations were significantly higher in SF than in matched PB, or in PB of HC. In conclusion, T lymphocyte signal transduction pathway mapping revealed an enhanced activation of JAK1/STAT3/STAT1 and PKCδ phosphoproteins that may drive the local inflammatory process, characterized by the in vivo expansion of T CD4⁺IL-17A-F⁺ and T CD4⁺IL-23R⁺ Th17 Teff cells in SF of clinically active joints of PsA patients.     

R0 resection in the treatment of gastric cancer: Room for improvement

Gastric carcinoma is one of the most frequent malignancies in the world and its clinical behavior especially depends on the metastatic potential of the tumor.In particular,lymphatic metastasis is one of the main predictors of tumor recurrence and survival,and current pathological staging systems reflect the concept that lymphatic spread is the most relevant prognostic factor in patients undergoing curative resection.This is compounded by the observation that two-thirds of gastric cancer in the Western world...     

A small molecule Smac mimic potentiates TRAIL-mediated cell death of ovarian cancer cells

OBJECTIVES: Ovarian cancer remains a leading cause of death in women and development of new therapies is essential. Second mitochondria derived activator of caspase (Smac) has been described to sensitize for apoptosis. We have explored the proapoptotic activity of a small molecule mimic of Smac/DIABLO on ovarian cancer cell lines (A2780 cells and its chemoresistant derivatives A2780/ADR and A2780/DDP), cancer cell lines and in primary ovarian cancer cells. METHODS: The effects of a small molecule mimic of Smac/DIABLO on ovarian cancer cell lines and primary ovarian cancer cells were determined by cell proliferation, apoptosis and biochemical assays. RESULTS: This compound added alone elicited only a weak proapoptotic effect; however, it strongly synergizes with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or agonistic TRAILR2 antibody (Lexatumumab) in inducing apoptosis of ovarian cancer cells. CONCLUSIONS: These observations suggest that small molecule mimic of Smac/DIABLO could be useful for the development of experimental strategies aiming to treat ovarian cancer. Interestingly, in addition to its well known proapoptotic effects, Smac/DIABLO elicited a significant increase of pro-caspase-3 levels.     

Polylactide/polyglycolide copolymer in bone defect healing in humans

This pilot study aims to evaluate the healing of a large defects in the human jawbone filled with a Poly-Lactide-co-Glycolide (PLG) polymer (Fisiograft) by means of clinical, radiological and histological methods and to compare the results with those of platelet-rich plasma (PRP) clot or autologous bone (AB) fillings. Bone cysts, where previous non-surgical treatments failed to promote healing, underwent surgery. Nineteen consenting male patients were randomly split into three groups, packed with PRP, AB or PLG. A core biopsy was performed 4 and 6 months after surgery. All treated defects showed clinical, radiological and histological progresses over time. AB provided the best clinical and histological performance and PLG had overlapping outcomes; PRP filling was statistically different. Six months after surgery, bone activities were enhanced in sites treated with PLG and fairly good with PRP. Additionally, PLG showed some new lamellar formations. In conclusion, outcomes were best with AB graft, but suitable results were achieved using PLG to promote healing of severe bone defects. PLG shows only a delayed regenerative capability but does not require a secondary donor site.     

Postprandial lipemia as an early predictor of cardiovascular complications in childhood obesity

Abstract. The growing trend of childhood overweight and obesity is a major health concern worldwide. Although obesity is a key risk factor for cardiovascular disease, the etiologic link between obesity and the progression of vascular disease remains unknown. Traditionally, lowering fasting blood cholesterol concentration has been the main interventional target for decreasing the risk of heart disease. However, there is increasing evidence that elevated concentrations of intestinally-derived chylomicron particles are associated with cardiovascular disease risk and that this is particularly evident in insulin-resistance and obesity in adulthood. In this review we comment on recent evidence suggesting that overweight children have fasting chylomicron concentrations equivalent to that found in adults diagnosed with cardiovascular disease. Further, we consider the hypothesis that fasting and postprandial chylomicron metabolism has a central role in the genesis of cardiovascular disease during childhood obesity.