Increased renal responsiveness to vasopressin and enhanced V2 receptor signaling in RGS2-/- mice.

The antidiuretic effect of vasopressin is mediated by V2 receptors (V2R) that are located in kidney connecting tubules and collecting ducts. This study provides evidence that V2R signaling is negatively regulated by regulator of G protein signaling 2 (RGS2), a member of the family of RGS proteins. This study demonstrates that (1) RGS2 expression in the kidney is restricted to the vasopressin-sensitive part of the nephron (thick ascending limb, connecting tubule, and collecting duct); (2) expression of RGS2 is rapidly upregulated by vasopressin; (3) the vasopressin-dependent accumulation of cAMP, the principal messenger of V2R signaling, is significantly higher in collecting ducts that are microdissected from the RGS2(-/-) mice compared with their wild-type littermates; and (4) analysis of urine output of mice that were exposed to water restriction followed by acute water loading revealed that RGS2(-/-) mice exhibit an increased renal responsiveness to vasopressin. It is proposed that RGS2 is involved in negative feedback regulation of V2R signaling.

Induction of macrophage nitric oxide production by interferon-gamma and tumor necrosis factor-alpha is enhanced by interleukin-10.

Interleukin-10 (IL-10) has been reported to inhibit nitric oxide (NO) synthesis and microbicidal activity of interferon-gamma (IFN-gamma)-stimulated macrophages (M phi) by preventing the secretion of tumor necrosis factor-alpha (TNF-alpha) which serves as an autocrine activating signal. We have examined the effects of recombinant IL-10 on the capacity of IFN-gamma together with exogenous TNF-alpha to induce NO synthesis by bone marrow-derived M phi. Under these conditions and in contrast to its reported deactivating potential, IL-10 strongly enhanced NO synthesis measured as nitrite (NO2-) release (half maximal stimulation at approximately 10 U/ml). IL-10 further increased NO2- production by M phi stimulated in the presence of optimal concentrations of prostaglandin E2, a positive modulator of M phi activation by IFN-gamma/TNF-alpha. Increased steady state levels of NO synthase mRNA were observed in 4-h IFN-gamma/TNF-alpha cultures and enhanced NO2(-)-release was evident 24 h but not 48 h after stimulation. These results suggest that the effects of IL-10 on M phi function are more complex than previously recognized.

Enhanced recovery after surgery: are we ready, and can we afford not to implement these pathways for patients undergoing radical cystectomy?

Enhanced recovery after surgery (ERAS) for radical cystectomy seems logical, but our study has shown a paucity in the level of clinical evidence. As part of the ERAS Society, we welcome global collaboration to collect evidence that will improve patient outcomes.

Loss of mitochondrial functions associated with azole resistance in Candida glabrata results in enhanced virulence in mice.

Mitochondrial dysfunction is one of the possible mechanisms by which azole resistance can occur in Candida glabrata. Cells with mitochondrial DNA deficiency (so-called "petite mutants") upregulate ATP binding cassette (ABC) transporter genes and thus display increased resistance to azoles. Isolation of such C. glabrata mutants from patients receiving antifungal therapy or prophylaxis has been rarely reported. In this study, we characterized two sequential and related C. glabrata isolates recovered from the same patient undergoing azole therapy. The first isolate (BPY40) was azole susceptible (fluconazole MIC, 4 μg/ml), and the second (BPY41) was azole resistant (fluconazole MIC, >256 μg/ml). BPY41 exhibited mitochondrial dysfunction and upregulation of the ABC transporter genes C. glabrata CDR1 (CgCDR1), CgCDR2, and CgSNQ2. We next assessed whether mitochondrial dysfunction conferred a selective advantage during host infection by testing the virulence of BPY40 and BPY41 in mice. Surprisingly, even with in vitro growth deficiency compared to BPY40, BPY41 was more virulent (as judged by mortality and fungal tissue burden) than BPY40 in both systemic and vaginal murine infection models. The increased virulence of the petite mutant correlated with a drastic gain of fitness in mice compared to that of its parental isolate. To understand this unexpected feature, genome-wide changes in gene expression driven by the petite mutation were analyzed by use of microarrays during in vitro growth. Enrichment of specific biological processes (oxido-reductive metabolism and the stress response) was observed in BPY41, all of which was consistent with mitochondrial dysfunction. Finally, some genes involved in cell wall remodelling were upregulated in BPY41 compared to BPY40, which may partially explain the enhanced virulence of BPY41. In conclusion, this study shows for the first time that mitochondrial dysfunction selected in vivo under azole therapy, even if strongly affecting in vitro growth characteristics, can confer a selective advantage under host conditions, allowing the C. glabrata mutant to be more virulent than wild-type isolates.

Enhanced kangaroo mother care for heel lance in preterm neonates: a crossover trial.

OBJECTIVE: To test if enhancing maternal skin-to-skin contact, or kangaroo mother care (KMC) by adding rocking, singing and sucking is more efficacious than simple KMC for procedural pain in preterm neonates. STUDY DESIGN: Preterm neonates (n=90) between 32 0/7 and 36 0/7 weeks' gestational age participated in a single-blind randomized crossover design. The infant was held in KMC with the addition of rocking, singing and sucking or the infant was held in KMC without additional stimulation. The Premature Infant Pain Profile was the primary outcome with time to recover as the secondary outcome. A repeated-measures analysis of covariance was employed for analyses. RESULT: There were no significant differences in any of the 30 s time periods over the 2 min of blood sampling nor in time to return to baseline. Compared to historical controls of the same age in incubator, the pain scores were lower and comparable to other studies of KMC. There were site differences related to lower scores with the use of sucrose in one site and higher scores in younger, sicker infants in another site. CONCLUSION: The sensorial stimulations from skin-to-skin contact that include tactile, olfactory sensations from the mother are sufficient to decrease pain response in premature neonates. Other studies showing that rocking, sucking and music were efficacious were independent of skin-to-skin contact, which, when used alone has been shown to be effective as reported across studies.

Guidelines for perioperative care for pancreaticoduodenectomy: Enhanced Recovery After Surgery (ERAS®) Society recommendations.

BACKGROUND & AIMS: Protocols for enhanced recovery provide comprehensive and evidence-based guidelines for best perioperative care. Protocol implementation may reduce complication rates and enhance functional recovery and, as a result of this, also reduce length-of-stay in hospital. There is no comprehensive framework available for pancreaticoduodenectomy. METHODS: An international working group constructed within the Enhanced Recovery After Surgery (ERAS®) Society constructed a comprehensive and evidence-based framework for best perioperative care for pancreaticoduodenectomy patients. Data were retrieved from standard databases and personal archives. Evidence and recommendations were classified according to the GRADE system and reached through consensus in the group. The quality of evidence was rated "high", "moderate", "low" or "very low". Recommendations were graded as "strong" or "weak". RESULTS: Comprehensive guidelines are presented. Available evidence is summarised and recommendations given for 27 care items. The quality of evidence varies substantially and further research is needed for many issues to improve the strength of evidence and grade of recommendations. CONCLUSIONS: The present evidence-based guidelines provide the necessary platform upon which to base a unified protocol for perioperative care for pancreaticoduodenectomy. A unified protocol allows for comparison between centres and across national borders. It facilitates multi-institutional prospective cohort registries and adequately powered randomised trials.

Peptide vaccine induces enhanced tumor growth associated with apoptosis induction in CD8+ T cells.

CD8(+) CTLs play a critical role in antitumor immunity. However, vaccination with synthetic peptide containing CTL epitopes has not been generally effective in inducing protective antitumor immunity. In this study, we addressed the detailed mechanism(s) involved in this failure using a new tumor model of BALB/c transplanted tumors expressing NY-ESO-1, an extensively studied human cancer/testis Ag. Whereas peptide immunization with an H2-D(d)-restricted CTL epitope derived from NY-ESO-1 (NY-ESO-1 p81-88) induced NY-ESO-1(81-88)-specific CD8(+) T cells in draining lymph nodes and spleens, tumor growth was significantly enhanced. Single-cell analysis of specific CD8(+) T cells revealed that peptide immunization caused apoptosis of >80% of NY-ESO-1(81-88)-specific CD8(+) T cells at tumor sites and repetitive immunization further diminished the number of specific CD8(+) T cells. This phenomenon was associated with elevated surface expression of Fas and programmed death-1. When peptide vaccination was combined with an adjuvant, a TLR9 ligand CpG, the elevated Fas and programmed death-1 expression and apoptosis induction were not observed, and vaccine with peptide and CpG was associated with strong tumor growth inhibition. Selection of appropriate adjuvants is essential for development of effective cancer vaccines, with protection of effector T cells from peptide vaccine-induced apoptosis being a prime objective.

Enhanced excitation-coupled Ca(2+) entry induces nuclear translocation of NFAT and contributes to IL-6 release from myotubes from patients with central core disease.

Prolonged depolarization of skeletal muscle cells induces entry of extracellular calcium into muscle cells, an event referred to as excitation-coupled calcium entry. Skeletal muscle excitation-coupled calcium entry relies on the interaction between the 1,4-dihydropyridine receptor on the sarcolemma and the ryanodine receptor on the sarcoplasmic reticulum membrane. In this study, we directly measured excitation-coupled calcium entry by total internal reflection fluorescence microscopy in human skeletal muscle myotubes harbouring mutations in the RYR1 gene linked to malignant hyperthermia (MH) and central core disease (CCD). We found that excitation-coupled calcium entry is strongly enhanced in cells from patients with CCD compared with individuals with MH and controls. Furthermore, excitation-coupled calcium entry induces generation of reactive nitrogen species and enhances nuclear localization of NFATc1, which in turn may be responsible for the increased IL-6 released by myotubes from patients with CCD.

Enhanced oligonucleotide delivery to mouse retinal cells using iontophoresis.

PURPOSE: To study the combination of oligodeoxynucleotides (ODNs) intravitreous injection and saline transpalpebral iontophoresis on the delivery of ODNs to photoreceptors in the newborn rd1/rd1 mice. METHODS: Cathodal or anodal transpalpebral iontophoresis (1.43 mA/cm(2) for 5 min) was applied to eyes of postnatal day 7 (PN7) rd1/rd1 mice immediately before the intravitreous injection of ODNs. The effect of cathodal iontophoresis after ODNs injection was also evaluated. The influence of current intensity (0.5, 1.5, and 2.5 mA) was assayed with cathodal iontophoresis performed prior to ODNs injection. The duration of current-induced facilitation of ODNs delivery to photoreceptors was evaluated for 6 h following iontophoresis. One group of control eyes received cathodal iontophoresis prior to the intravitreous injection of phosphate buffered saline (PBS) or hexachlorofluorescein (Hex). The second control group received ODN or Hex intravitreous injection without iontophoresis. The penetration of fluorescent ODNs in the outer nuclear layer (ONL) was quantified by image analysis of the ONL fluorescence intensity on cryosection microphotographs. Integrity of ODN was assessed using acrylamide gel migration after its extraction from the retina of treated mice. The integrity of retinal structure, 1 and 24 h after iontophoresis, was analyzed using light and electron microscopy. RESULTS: Transpalpebral anodal or cathodal saline iontophoresis enhanced the penetration of ODNs in all retinal layers. Cathodal iontophoresis was more efficient than anodal iontophoresis in enhancing the tissue penetration of the injected ODN. Photoreceptor delivery of ODN was significantly higher when cathodal saline transpalpebral iontophoresis was applied prior than after the injection. The extent of enhanced tissue penetration decreased in parallel to the increased interval between iontophoresis application and the intravitreous injection. Current of 1.5 mA was safe and optimal for the delivery of ODNs to the ONL. One hour after iontophoresis followed by injection, ODN extracted from the retina of treated eyes remained intact. Histology and electron microscopy observations demonstrated that iontophoresis using the optimal parameters did not induce any permanent tissue alterations or structure damage. CONCLUSIONS: Saline transpalpebral iontophoresis facilitates the penetration of injected ODNs in photoreceptors for at least 3 h. This method may be considered for photoreceptor targeted gene therapy.

Heterologous prime-boost regimen adenovector 35-circumsporozoite protein vaccine/recombinant Bacillus Calmette-Guérin expressing the Plasmodium falciparum circumsporozoite induces enhanced long-term memory immunity in BALB/c mice.

BACKGROUND: Sustained antibody levels are a hallmark of immunity against many pathogens, and induction of long-term durable antibody titers is an essential feature of effective vaccines. Heterologous prime-boost approaches with vectors are optimal strategies to improve a broad and prolonged immunogenicity of malaria vaccines. RESULTS: In this study, we demonstrate that the heterologous prime-boost regimen Ad35-CS/BCG-CS induces stronger immune responses by enhancing type 1 cellular producing-cells with high levels of CSp-specific IFN-γ and cytophilic IgG2a antibodies as compared to a homologous BCG-CS and a heterologous BCG-CS/CSp prime-boost regimen. Moreover, the heterologous prime-boost regimen elicits the highest level of LLPC-mediated immune responses. CONCLUSION: The increased IFN-γ-producing cell responses induced by the combination of Ad35-CS/BCG-CS and sustained type 1 antibody profile together with high levels of LLPCs may be essential for the development of long-term protective immunity against liver-stage parasites.

Guidelines for perioperative care in elective colonic surgery: Enhanced Recovery After Surgery (ERAS®) Society recommendations.

BACKGROUND: This review aims to present a consensus for optimal perioperative care in colonic surgery and to provide graded recommendations for items for an evidenced-based enhanced perioperative protocol. METHODS: Studies were selected with particular attention paid to meta-analyses, randomised controlled trials and large prospective cohorts. For each item of the perioperative treatment pathway, available English-language literature was examined, reviewed and graded. A consensus recommendation was reached after critical appraisal of the literature by the group. RESULTS: For most of the protocol items, recommendations are based on good-quality trials or meta-analyses of good-quality trials (quality of evidence and recommendations according to the GRADE system). CONCLUSIONS: Based on the evidence available for each item of the multimodal perioperative-care pathway, the Enhanced Recovery After Surgery (ERAS) Society, International Association for Surgical Metabolism and Nutrition (IASMEN) and European Society for Clinical Nutrition and Metabolism (ESPEN) present a comprehensive evidence-based consensus review of perioperative care for colonic surgery.