A prospective, randomized study comparing morning to evening administration of gonadotropins in ART.

OBJECTIVE: We sought to determine whether administering the daily gonadotropin dose in the morning (AM) or in the evening (PM) affects cycle outcome in patients undergoing IVF. DESIGN: This is a prospective randomized study. SETTING: The study is performed in a private assisted reproductive technology (ART) clinic. PATIENT(S): The study included one hundred and twenty-seven women undergoing IVF. INTERVENTION(S): Morning (AM) and evening (PM) administration of gonadotropins (uFSH and hMG) was compared. MAIN OUTCOME MEASURE(S): Live birth rate was the main outcome measured. Secondary outcomes including total IU use, days of stimulation, peak E2, peak P4, endometrial thickness, number of oocytes retrieved, MII oocytes, fertilization rates, #ET, IR, and clinical PR were all assessed. RESULTS: A total of 127 cycles were included, 61 in the AM group and 67 in the PM group. Baseline and stimulation characteristics were similar in both groups. There was a trend for a higher implantation rate in the AM group vs. the PM group (60.3 vs. 47.2%, P = 0.066). The AM group had a higher chemical pregnancy rate compared to the PM group (81.7 vs. 65.6%, P = 0.024) and a higher clinical pregnancy rate (78.3 vs. 62.1%, P = 0.048), but the delivery rates were similar (68.3 vs. 56.1%, P = 0.16). The study was unfortunately prematurely terminated when uFSH (Bravelle©) was pulled out of the US market. CONCLUSIONS: AM administration of gonadotropins may be associated with a better ART outcome compared to PM administration. Larger studies are needed to confirm our findings.

Simulation to Prepare Graduate Nursing Students for Clinical Faculty Role.

Limited clinical sites and faculty to teach graduate students to be undergraduate clinical faculty have led to the exploration of innovative teaching strategies. This article describes and evaluates a simulation experience to supplement didactic learning about best clinical teaching practices within a graduate nursing course. Scenarios were created to simulate complex teaching situations with a patient, an undergraduate nursing student, and a clinical faculty member. Evaluations were positive regarding knowledge, performance, self-confidence, critical thinking, and satisfaction. Results of this project support the use of simulation in the preparation of graduate nursing students to become clinical faculty.

Developmental consequences of supplementing with matrix metallopeptidase-9 during in vitro maturation of heat-stressed bovine oocytes.

Because latent form of matrix metallopeptidase-9 (proMMP9) levels are positively related to blastocyst development, it was hypothesized that addition during maturation may improve development of heat-stressed oocytes. To test hypothesis, 0, 30 or 300 ng/ml human proMMP9 (hMMP9) was added at 18 h of in vitro maturation (hIVM) to cumulus-oocyte complexes matured at 38.5 or 41.0ºC (first 12 h only). Heat stress decreased 24 hIVM proMMP9 levels only in 0 and 30 ng/ml groups and increased progesterone in 0 and 300 ng/ml hMMP9 groups. Heat stress decreased cleavage and blastocyst development. Independent of maturation temperature, hMMP9 at 18 hIVM decreased blastocyst development. In a second study, cumulus-oocyte complexes were matured for 24 h at 38.5 or 41.0ºC (HS first 12 h only) with 0 or 300 ng/ml hMMP9 added at 12 hIVM. Without hMMP9, heat stress decreased 24 hIVM proMMP9 levels and increased progesterone production. Addition of 300 ng/ml of hMMP9 produced equivalent levels of proMMP9 at 24 hIVM (271 vs. 279 ± 77 for 38.5ºC and 41.0ºC treated oocytes, respectively). Heat stress did not affect ability of oocytes to cleave but reduced blastocyst development. Independent of temperature, hMMP9 decreased cleavage and blastocyst development. In summary, hMMP9 supplementation during IVM did not improve development of heat-stressed oocytes even when it was added for the entire maturation period. At doses tested, hMMP9 appeared detrimental to development when supplemented during the last 12 or 6 h of oocyte maturation.

Murine gammaherpesvirus 68 has evolved gamma interferon and stat1-repressible promoters for the lytic switch gene 50.

Cytokines regulate viral gene expression with important consequences for viral replication and pathogenesis. Gamma interferon (IFN-gamma) is a key regulator of chronic murine gammaherpesvirus 68 (gammaHV68) infection and a potent inhibitor of gammaHV68 reactivation from latency. Macrophages are the cell type that is responsive to the IFN-gamma-mediated control of gammaHV68 reactivation; however, the molecular mechanism of this IFN-gamma action is undefined. Here we report that IFN-gamma inhibits lytic replication of gammaHV68 in primary bone marrow-derived macrophages and decreases transcript levels for the essential lytic switch gene 50. Interestingly, IFN-gamma suppresses the activity of the two known gene 50 promoters, demonstrating that an inflammatory cytokine can directly regulate the promoters for the gammaHV68 lytic switch gene. Stat1, but not IFN-alpha/beta signaling, is required for IFN-gamma action. Moreover, Stat1 deficiency increases basal gammaHV68 replication, gene 50 expression, and promoter activity. Together, these data identify IFN-gamma and Stat1 as being negative regulators of the gammaHV68 lytic cycle and raise the possibility that gammaHV68 maintains IFN-gamma/Stat1-responsive gene 50 promoters to facilitate cell-extrinsic control over the interchange between the lytic and latent cycles.

Histone deacetylases and the nuclear receptor corepressor regulate lytic-latent switch gene 50 in murine gammaherpesvirus 68-infected macrophages.

Gammaherpesviruses are important oncogenic pathogens that transit between lytic and latent life cycles. Silencing the lytic gene expression program enables the establishment of latency and a lifelong chronic infection of the host. In murine gammaherpesvirus 68 (MHV68, γHV68), essential lytic switch gene 50 controls the interchange between lytic and latent gene expression programs. However, negative regulators of gene 50 expression remain largely undefined. We report that the MHV68 lytic cycle is silenced in infected macrophages but not fibroblasts and that histone deacetylases (HDACs) mediate silencing. The HDAC inhibitor trichostatin A (TSA) acts on the gene 50 promoter to induce lytic replication of MHV68. HDAC3, HDAC4, and the nuclear receptor corepressor (NCoR) are required for efficient silencing of gene 50 expression. NCoR is critical for transcriptional repression of cellular genes by unliganded nuclear receptors. Retinoic acid, a known ligand for the NCoR complex, derepresses gene 50 expression and enhances MHV68 lytic replication. Moreover, HDAC3, HDAC4, and NCoR act on the gene 50 promoter and are recruited to this promoter in a retinoic acid-responsive manner. We provide the first example of NCoR-mediated, HDAC-dependent regulation of viral gene expression.

Pharmacokinetics of posaconazole administered orally or by nasogastric tube in healthy volunteers.

The use of a nasogastric tube is one means of administering antifungal therapy to critically ill patients unable to receive medication via the oral route. This was a phase 1, open-label, single-center, randomized, crossover study of posaconazole administered via nasogastric tube in healthy volunteers. Each subject received two 400-mg single doses of posaconazole, one administered orally and one administered by nasogastric tube, with a 7-day washout period between each dose. Posaconazole was administered 5 to 10 min after subjects received a nutritional supplement. Blood samples for pharmacokinetic analysis were obtained up to 120 h postdose. The analysis of variance estimate of the study population suggests that the posaconazole nasogastric tube administration least-square mean values of observed maximum concentration (C(max)), area under the plasma concentration-time curve (AUC) to the last measurable concentration, and AUC to time infinity were 81%, 76%, and 77%, respectively, of the corresponding oral administration values. The reason for lower C(max) and AUC values when posaconazole is administered via the nasogastric tube route is not known. Oral and nasogastric tube administration of a single 400-mg dose of posaconazole suspension was safe and well tolerated in healthy adult subjects. The incidence and nature of treatment-emergent adverse events were similar with both administration routes, and no serious adverse events or clinically significant laboratory test or vital sign abnormalities were reported. Obtaining plasma posaconazole concentrations may be warranted when posaconazole is given to patients via a nasogastric tube to ensure adequate posaconazole exposure. Strategies that have been shown to enhance posaconazole exposure (such as splitting the dose and minimizing the use of proton pump inhibitors) may also be used.

Antifungal serum concentration monitoring: an update.

Invasive fungal infections (IFIs) are occurring with increasing incidence and are associated with significant morbidity and mortality. Understanding the relationship between the pharmacokinetic and pharmacodynamic properties of antifungals is essential to optimize the potential for favourable clinical and microbiological outcomes while minimizing risks of treatment-related toxicity. Antifungal serum concentrations may aid in the determination of appropriate dosing in select circumstances. The polyene and echinocandin classes of antifungals lack sufficient data to justify serum concentration monitoring in routine clinical practice. In contrast, serum concentration monitoring of flucytosine may help to reduce the risk of treatment-related haematological toxicity. Determination of itraconazole serum concentrations is advised in situations where the drug is used for prolonged periods to treat serious IFIs (such as invasive aspergillosis or histoplasmosis) because of variability in absorption following oral administration (most notable for the capsule formulation). The use of serum concentration monitoring during therapy with the extended-spectrum triazoles (i.e. voriconazole and posaconazole) is still evolving, due primarily to inter-patient variability in drug exposure combined with sparse data regarding relationships with efficacy (posaconazole) and both safety and efficacy (voriconazole).

MHV68 complement regulatory protein facilitates MHV68 replication in primary macrophages in a complement independent manner.

Murine gammaherpesvirus-68 (MHV68) is genetically related to human Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus and provides a tractable model to study gammaherpesvirus-host interactions in vivo and in vitro. The MHV68-encoded v-RCA product inhibits murine complement activation and shares sequence homology with other virus and host regulators of complement activation. Here we show that v-RCA is required for efficient MHV68 replication in primary murine macrophages, but not in murine embryonic fibroblasts. v-RCA-deficient MHV68 mutant viruses display defects in viral DNA synthesis in infected macrophages. Importantly, attenuated growth of v-RCA mutant viruses is not rescued in macrophages lacking critical components of the complement system including C3, indicating that the macrophage-specific role of v-RCA in MHV68 replication is complement-independent. This contrasts with the situation in vivo in which attenuated neurovirulence of v-RCA mutant viruses is rescued in C3-deficient mice. This study shows a novel, complement independent cell-type-specific function of a gammaherpesvirus RCA protein.

Autophagosome-independent essential function for the autophagy protein Atg5 in cellular immunity to intracellular pathogens.

The physiologic importance of autophagy proteins for control of mammalian bacterial and parasitic infection in vivo is unknown. Using mice with granulocyte- and macrophage-specific deletion of the essential autophagy protein Atg5, we show that Atg5 is required for in vivo resistance to the intracellular pathogens Listeria monocytogenes and Toxoplasma gondii. In primary macrophages, Atg5 was required for interferongamma (IFN-gamma)/LPS-induced damage to the T. gondii parasitophorous vacuole membrane and parasite clearance. While we did not detect classical hallmarks of autophagy, such as autophagosomes enveloping T. gondii, Atg5 was required for recruitment of IFN-gamma-inducible p47 GTPase IIGP1 (Irga6) to the vacuole membrane, an event that mediates IFN-gamma-mediated clearance of T. gondii. This work shows that Atg5 expression in phagocytic cells is essential for cellular immunity to intracellular pathogens in vivo, and that an autophagy protein can participate in immunity and intracellular killing of pathogens via autophagosome-independent processes such as GTPase trafficking.

A poxvirus-encoded pyrin domain protein interacts with ASC-1 to inhibit host inflammatory and apoptotic responses to infection.

Proinflammatory caspases play an essential role in innate immune responses to infection by regulating the cleavage and activation of proinflammatory cytokines. Activation of these enzymes requires the assembly of an intracellular molecular platform, termed the inflammasome, which is comprised of members of the pyrin domain (PYD)-containing superfamily of apoptosis and inflammation-regulatory proteins. We report here the identification and characterization of a poxvirus-encoded PYD-containing protein that interacts with the ASC-1 component of the inflammasome and inhibits caspase-1 activation and the processing of IL-1beta and IL-18 induced by diverse stimuli. Knockout viruses that do not express this protein are unable to productively infect monocytes and lymphocytes due to an abortive phenotype and are markedly attenuated in susceptible hosts due to decreased virus dissemination and enhanced inflammatory responses at sites of infection. Thus, modulation of inflammasome function constitutes an important immunomodulatory strategy employed by poxviruses to circumvent host antiviral responses.