Anticontractile Effect of Perivascular Adipose Tissue and Leptin are Reduced in Hypertension

Leptin causes vasodilatation both by endothelium-dependent and -independent mechanisms. Leptin is synthesized by perivascular adipose tissue (PVAT). The hypothesis of this study is that a decrease of leptin production in PVAT of spontaneously hypertensive rats (SHR) might contribute to a diminished paracrine anticontractile effect of the hormone. We have determined in aorta from Wistar-Kyoto (WKY) and SHR (i) leptin mRNA and protein levels in PVAT, (ii) the effect of leptin and PVAT on contractile responses, and (iii) leptin-induced relaxation and nitric oxide (NO) production. Leptin mRNA and protein expression were significantly lower in PVAT from SHR. Concentration-response curves to angiotensin II were significantly blunted in presence of PVAT as well as by exogenous leptin (10−9 M) only in WKY. This anticontractile effect was endothelium-dependent. Vasodilatation induced by leptin was smaller in SHR than in WKY, and was also endothelium-dependent. Moreover, release of endothelial NO in response to acute leptin was higher in WKY compared to SHR, but completely abolished in the absence of endothelium. In conclusion, the reduced anticontractile effect of PVAT in SHR might be attributed to a reduced PVAT-derived leptin and to an abrogated effect of leptin on endothelial NO release probably due to an impaired activation of endothelial NO synthase

Hundreds of dual-stage antimalarial molecules discovered by a functional gametocyte screen.

Plasmodium falciparum stage V gametocytes are responsible for parasite transmission, and drugs targeting this stage are needed to support malaria elimination. We here screen the Tres Cantos Antimalarial Set (TCAMS) using the previously developed P. falciparum female gametocyte activation assay (Pf FGAA), which assesses stage V female gametocyte viability and functionality using Pfs25 expression. We identify over 400 compounds with activities <2 μM, chemically classified into 57 clusters and 33 singletons. Up to 68% of the hits are chemotypes described for the first time as late-stage gametocyte-targeting molecules. In addition, the biological profile of 90 compounds representing the chemical diversity is assessed. We confirm in vitro transmission-blocking activity of four of the six selected molecules belonging to three distinct scaffold clusters. Overall, this TCAMS gametocyte screen provides 276 promising antimalarial molecules with dual asexual/sexual activity, representing starting points for target identification and candidate selection

Autologous stem-cell transplantation as consolidation of first-line chemotherapy in patients with peripheral T-cell lymphoma : a multicenter GELTAMO/FIL study

Peripheral T-cell lymphomas (PTCL) are a heterogeneous group of rare lymphoid malignancies that mostly have poor prognoses with currently available treatments. Upfront consolidation with autologous stem cell transplantation (ASCT) is frequently carried out, but its efficacy has never been investigated in randomized trials. We designed a multicenter, international, retrospective study with the main objective of comparing progression-free survival and overall survival of patients with PTCL who underwent ASCT in complete remission (CR) after first-line chemotherapy with a control group who did not undergo ASCT. From the initial population of 286 registered patients, 174 patients with PTCL other than anaplastic large cell lymphoma, ALK-positive, deemed fit for ASCT at the time of diagnosis, and who were in CR or uncertain CR after induction therapy (CR1) were included in our analysis. one hundred and three patients underwent ASCT, whereas 71 did not, in most cases (n=53) because the physician decided against it. With a median follow-up of 65.5 months, progression-free survival was significantly better in the transplanted patients than in the non-transplanted group: 63% versus 48% at 5 years (P =0.042). Overall survival was significantly longer for ASCT patients in the subgroup with advanced stage at diagnosis (5-year overall survival: 70% vs. 50%, P =0.028). In the multivariate analysis, first-line ASCT was associated with significantly prolonged progression-free survival (HR=0.57, 95% CI: 0.35-0.93) and overall survival (HR=0.57, 95% CI: 0.33-0.99). In conclusion, our study supports the use of ASCT as a consolidation strategy for patients with PTCL in CR1. These results should be confirmed in a prospective randomized study

Preventive medicine of von Hippel-Lindau disease-associated pancreatic neuroendocrine tumors

Pancreatic neuroendocrine tumors (PanNETs) are rare in von Hippel-Lindau disease (VHL) but cause serious morbidity and mortality. Management guidelines for VHL-PanNETs continue to be based on limited evidence, and survival data to guide surgical management are lacking. We established the European-American-Asian-VHL-PanNET-Registry to assess data for risks for metastases, survival and long-term outcomes to provide best management recommendations. Of 2330 VHL patients, 273 had a total of 484 PanNETs. Median age at diagnosis of PanNET was 35 years (range 10-75). Fifty-five (20%) patients had metastatic PanNETs. Metastatic PanNETs were significantly larger (median size 5 vs 2\u2009cm; P\u20091.5\u2009cm in diameter were operated. Ten-year survival was significantly longer in operated vs non-operated patients, in particular for PanNETs <2.8\u2009cm vs 652.8\u2009cm (94% vs 85% by 10 years; P\u2009=\u20090.020; 80% vs 50% at 10 years; P\u2009=\u20090.030). This study demonstrates that patients with PanNET approaching the cut-off diameter of 2.8\u2009cm should be operated. Mutations in exon 3, especially of codons 161/167 are at enhanced risk for metastatic PanNETs. Survival is significantly longer in operated non-metastatic VHL-PanNETs

Quantification of Anisakis simplex allergens in fresh, long-term frozen, and cooked fish muscle

Fish-borne parasitic zoonoses such as Anisakiasis were once limited to people living in countries where raw or undercooked fish is traditionally consumed. Nowadays, several factors, such as the growing international markets, the improved transportation systems, the population movements, and the expansion of ethnic ways of cooking in developed countries, have increased the population exposed to these parasites. Improved diagnosis technology and a better knowledge of the symptoms by clinicians have increased the Anisakiasis cases worldwide. Dietary recommendations to Anisakis-sensitized patients include the consumption of frozen or well-cooked fish, but these probably do not defend sensitized patients from allergen exposure. The aim of our work was to develop a sensitive and specific method to detect and quantify Anisakis simplex allergens in fish muscle and its derivatives. Protein extraction was made in saline buffer followed by preparation under acid conditions. A. simplex antigens were detected by IgG immunoblot and quantified by dot blot. The allergenic properties of the extracts were assessed by IgE immunoblotting and basophil activation test. We were able to detect less than 1 ppm of A. simplex antigens, among them the allergen Ani s 4, in fish muscle with no cross-reactions and with a recovery rate of 82.5%. A. simplex antigens were detected in hakes and anchovies but not in sardines, red mullets, or shellfish. We detected A. simplex allergens in cooked hakes and also in hake stock. We proved that A. simplex allergens are preserved in long-term frozen storage (-20°C±2°C for 11 months) of parasitized hakes. Basophil activation tests have proven the capability of the A. simplex-positive fish extracts to induce allergic symptoms. © 2010, Mary Ann Liebert, Inc.Peer Reviewe

Salmonid fish viruses and cell interactions at early steps of the infective cycle

12 páginas, 5 figuras, 1 tabla -- PAGS nros. 535-546A flow cytometric virus-binding assay that directly visualizes the binding and entry of infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) and virus haemorrhagic septicaemia virus (VHSV) to several cell lines was established. The highest efficiency of binding was shown by the BF-2 cell line and this was used to study, at the attachment level, the interactions of these cells with salmonid fish viruses in coinfections, and to further determine if the earliest stage of the viral growth cycle could explain the previously described loss of infectivity of IHNV when IPNV is present. Our results demonstrated that IPNV binds to around 88% of cells either in single or dual infections, whereas IHNV attachment always decreased in the presence of any of the other viruses. VHSV binding was not affected by IPNV, but coinfection with IHNV reduced the percentage of virus-binding cells, which suggests competition for viral receptors or co-receptors. Internalization of the adsorbed IHNV was not decreased by coinfection with IPNV, so the hypothetical competence could be restricted to the binding step. Treatment of the cells with antiviral agents, such as amantadine or chloroquine, did not affect the binding of IPNV and VHSV, but reduced IHNV binding by more than 30%. Tributylamine affected viral binding of the three viruses to different degrees and inhibited IPNV or IHNV entry in a large percentage of cells treated for 30 min. Tributylamine also inhibited IHNV cytopathic effects in a dose-dependent manner, decreasing the virus yield by 4 log of the 50% endpoint titre, at 10 mm concentration. IPNV was also inhibited, but at a lower level. The results of this study support the hypothesis that IHNV, in contrast to VHSV or IPNV, is less efficient at completing its growth cycle in cells with a simultaneous infection with IPNV. It can be affected at several stages of viral infection and is more sensitive to the action of antiviral compoundsThe present study was carried out with financial support from the Ministerio de Educación y Ciencia (MEC), grant AGL2004-01931Peer reviewe

In vitro and in vivo immune responses induced by a DNA vaccine encoding the VP2 gene of the infectious pancreatic necrosis virus

10 páginas, 7 figuras, 2 tablas -- PAGS nros. 120-129The work presented here describes the construction of a plasmid encoding the VP2 gene of the infectious pancreatic necrosis virus (IPNV), its expression in BF-2 cells and an evaluation of its activity in brown trout (Salmo trutta L) soon after injection. Preliminary experiments to evaluate the potential of the plasmid to induce neutralizing antibodies were also performed. We established a BF-2 cell line that expresses VP2 constitutively and we examined the infection of these VP2-transfected BF-2 cells with homologous and heterologous viruses. The expression kinetics of IFN, and of the IFN-induced genes Mx and ISG15, was also evaluated in brown trout over a 15 day interval, and quantified by real-time or semi-quantitative PCR. Type I IFN and Mx are markers of the non-specific innate immune response to viruses and they are involved in antiviral defences. Our results demonstrate that expression of the IPNV VP2 protein in BF-2 cells induces an antiviral state against IPNV and against the infectious haematopoietic necrosis virus (IHNV). In BF-2 infected cells, VP2 inhibited both the IPNV and IHNV-induced cytopathic effect to some extent, as well as the virus yield. In vivo, VP2 was expressed in haematopoietic tissues such as the head kidney of 7 month-old trout. In addition, it induced early immune responses and specific immunity 30 days after injection. IFN mRNA expression increased sharply on the 1st and 15th day post-injection and expression of other IFN-induced genes as Mx and ISG15 was also detected soon after vaccination of brown trout. Moreover, specific antibodies were detected 30 days after vaccination. These results suggest that the VP2 gene is a good candidate for the design of IPNV-DNA vaccines and to investigate the use of cytokines as co-stimulatory moleculesThe present study was carried out with financial support from the Ministerio de Educación y Ciencia (MEC, grants AGL2004-01931 and AGL-2007-60256/ACU)Peer reviewe

The persistence of infectious pancreatic necrosis virus and its influence on the early immune response

Persistent infection by IPNV was induced in RTG-2 and RTG-P1 cells in vitro and the influence of this phenomenon on viral infectivity, viral antigen expression and interference with homologous and heterologous viruses was characterized over successive passages. The induction of IFN was also assessed, as was the sequence of the VP2 viral capsid protein, the region believed to be responsible for virulence, attenuation or persistence. Viral antigen expression was recorded in cells with no evidence of cytopathic effects and in these conditions, flow cytometry was more sensitive than RT-PCR to demonstrate the presence of a non-lytic virus. Interference of homologous viral infection could be detected in cross-infection experiments and in RTG-P1 cells persistently infected with IPNV, the Mx1 promoter could still be activated for at least 5 successive passages. Indeed, although over-induction of luciferase was not observed by re-infection with homologous or heterologous viruses, a significant increase in luciferase was induced by poly I:C. IFN transcripts could be quantified by qRT-PCR in the persistent cells at several passages, suggesting that IFN plays a role in maintaining IPNV persistence. In addition, we observed the same determinants in the VP2 sequences from the persistent virus as those described previously for IPNV adaptation and persistence in cell culture.This study was supported by the Spanish Ministerio de Ciencia e Innovación MICIIN, grants AGL2004-01931 and AGL2007-60256/ACU. A. de las Heras was the recipient of a predoctoral fellowship from the MEC.Peer reviewe

Immunogenic and protective effects of an oral DNA vaccine against infectious pancreatic necrosis virus in fish

DNA vaccines and oral DNA-based immunotherapy against infectious pancreatic necrosis virus (IPNV) have scarcely been studied in salmonid fish. Here, a vector with the capsid VP2 gene inserted was encapsulated in alginate microspheres to avoid the aggressive gastrointestinal conditions experienced following oral administration. Alginate microspheres were effective to protect the pDNA encoding VP2, which was expressed early in different organs of the vaccinated trout and that persisted for at least 60 days. The vaccine induces innate immune responses, raising the expression of IFN more than 10-fold relative to the fish vaccinated with the empty plasmid, at 7 and 15 days post-vaccination. Likewise, maximal expression of the IFN-induced antiviral Mx protein was recorded 15 days post-vaccination and neutralizing antibodies were also detected after 15 days, although their titre rose further at 21 days post-vaccination. Protection was high in the immunized fish, which showed around an 80% relative survival when challenged 15 and 30 days after vaccine delivery. Very low viral load with respect to the control group was detected in the vaccinated fish that survived 45 days after challenge. Thus, this study demonstrates the potential of the encapsulation technique for IPNV-DNA vaccine delivery and the relevance of the IPNV-VP2 gene for future plasmid constructs.This research was supported by the Ministerio de Ciencia e Innovación (MICINN, Spain) grant AGL2007-60256/ACU. The authors would like to thank A. García from the Delegación Provincial de Medio Ambiente y Desarrollo Rural (CAClM, Cuenca, Spain) for providing the brown trout, and Mercedes Sánchez and Luis Guaita for their excellent technical assistance. A de las Heras is beneficiary of a pre-doctoral fellowship from the MICINN

Vacuna DNA oral frente al virus de la necrosis pancreática infecciosa de salmónidos cultivados

Trabajo presentado en el X Congreso Nacional de Virología, celebrado en Salamanca (España), del 21 al 24 de Junio de 200