RESUMEN
OBJECTIVES: There is clinical uncertainty over the optimal treatment for penicillin-susceptible Staphylococcus aureus (PSSA) infections. Furthermore, there is concern that phenotypic penicillin susceptibility testing methods are not reliably able to detect some blaZ-positive S. aureus. METHODS: Nine S. aureus isolates, including six genetically diverse strains harbouring blaZ, were sent in triplicate to 34 participating laboratories from Australia (nâ=â14), New Zealand (nâ=â6), Canada (nâ=â12), Singapore (nâ=â1) and Israel (nâ=â1). We used blaZ PCR as the gold standard to assess susceptibility testing performance of CLSI (P10 disc) and EUCAST (P1 disc) methods. Very major errors (VMEs), major error (MEs) and categorical agreement were calculated. RESULTS: Twenty-two laboratories reported 593 results according to CLSI methodology (P10 disc). Nineteen laboratories reported 513 results according to the EUCAST (P1 disc) method. For CLSI laboratories, the categorical agreement and calculated VME and ME rates were 85% (508/593), 21% (84/396) and 1.5% (3/198), respectively. For EUCAST laboratories, the categorical agreement and calculated VME and ME rates were 93% (475/513), 11% (84/396) and 1% (3/198), respectively. Seven laboratories reported results for both methods, with VME rates of 24% for CLSI and 12% for EUCAST. CONCLUSIONS: The EUCAST method with a P1 disc resulted in a lower VME rate compared with the CLSI methods with a P10 disc. These results should be considered in the context that among collections of PSSA isolates, as determined by automated MIC testing, less than 10% harbour blaZ. Furthermore, the clinical relevance of phenotypically susceptible, but blaZ-positive S. aureus, remains unclear.
Asunto(s)
Antibacterianos , Infecciones Estafilocócicas , Humanos , Antibacterianos/farmacología , Staphylococcus aureus/genética , Penicilinas/farmacología , Pruebas de Sensibilidad Microbiana , Toma de Decisiones Clínicas , IncertidumbreRESUMEN
BACKGROUND: Nafamostat mesylate is a potent in vitro antiviral agent that inhibits the host transmembrane protease serine 2 enzyme used by severe acute respiratory syndrome coronavirus 2 for cell entry. METHODS: This open-label, pragmatic, randomized clinical trial in Australia, New Zealand, and Nepal included noncritically ill hospitalized patients with coronavirus disease 2019 (Covid-19). Participants were randomly assigned to usual care or usual care plus nafamostat. The primary end point was death (any cause) or receipt of new invasive or noninvasive ventilation or vasopressor support within 28 days after randomization. Analysis was with a Bayesian logistic model in which an adjusted odds ratio <1.0 indicates improved outcomes with nafamostat. Enrollment was closed due to falling numbers of eligible patients. RESULTS: We screened 647 patients in 21 hospitals (15 in Australia, 4 in New Zealand, and 2 in Nepal) and enrolled 160 participants from May 2021 to August 2022. In the intention-to-treat population, the primary end point occurred in 8 (11%) of 73 patients with usual care and 4 (5%) of 82 with nafamostat. The median adjusted odds ratio for the primary end point for nafamostat was 0.40 (95% credible interval, 0.12 to 1.34) with a posterior probability of effectiveness (adjusted odds ratio <1.0) of 93%. For usual care compared with nafamostat, hyperkalemia occurred in 1 (1%) of 67 and 7 (9%) of 78 participants, respectively, and clinically relevant bleeding occurred in 1 (1%) of 73 and 7 (8%) of 82 participants. CONCLUSIONS: Among hospitalized patients with Covid-19, there was a 93% posterior probability that nafamostat reduced the odds of death or organ support. Prespecified stopping criteria were not met, precluding definitive conclusions. Hyperkalemia and bleeding were more common with nafamostat. (Funded by ASCOT and others; ClinicalTrials.gov number, NCT04483960.)
Asunto(s)
COVID-19 , Humanos , SARS-CoV-2 , Guanidinas/farmacología , BenzamidinasRESUMEN
Maintenance and activation of the limited supply of primordial follicles in the ovary are important determinants of reproductive lifespan. Currently, the molecular programme that maintains the primordial phenotype and the early events associated with follicle activation are not well defined. Here, we have systematically analysed these events using microscopy and detailed image analysis. Using the immature mouse ovary as a model, we demonstrate that the onset of granulosa cell (GC) proliferation results in increased packing density on the oocyte surface and consequent GC cuboidalization. These events precede oocyte growth and nuclear translocation of FOXO3a, a transcription factor important in follicle activation. Immunolabelling of the TGFß signalling mediators and transcription factors SMAD2/3 revealed a striking expression pattern specific to GCs of small follicles. SMAD2/3 were expressed in the nuclei of primordial GCs but were mostly excluded in early growing follicles. In activated follicles, GC nuclei lacking SMAD2/3 generally expressed Ki67. These findings suggest that the first phenotypic changes during follicle activation are observed in GCs, and that TGFß signalling is fundamental for regulating GC arrest and the onset of proliferation.
Asunto(s)
Núcleo Celular/metabolismo , Células de la Granulosa/metabolismo , Folículo Ovárico/metabolismo , Ovario/metabolismo , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Animales , Núcleo Celular/genética , Proliferación Celular , Femenino , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Células de la Granulosa/citología , Ratones , Ratones Endogámicos C57BL , Oocitos/citología , Oocitos/metabolismo , Folículo Ovárico/crecimiento & desarrollo , Ovario/crecimiento & desarrollo , Transporte de Proteínas , Transducción de Señal , Proteína Smad2/genética , Proteína smad3/genética , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are 2 closely related TGF-ß ligands implicated as key regulators of follicle development and fertility. Animals harboring mutations of these factors often exhibit a blockage in follicle development beyond the primary stage and therefore little is known about the role of these ligands during subsequent (preantral) stages. Preantral follicles isolated from immature mice were cultured with combinations of BMP15, GDF9, and activin receptor-like kinase (ALK) inhibitors. Individually, GDF9 and BMP15 promoted follicle growth during the first 24 hours, whereas BMP15 subsequently (48-72 h) caused follicle shrinkage and atresia with increased granulosa cell apoptosis. Inhibition of ALK6 prevented the BMP15-induced reduction in follicle size and under basal conditions promoted a rapid increase in granulosa cell proliferation, suggesting BMP15 signals through ALK6, which in turn acts to restrain follicle growth. In the presence of GDF9, BMP15 no longer promoted atresia and in fact follicle growth was increased significantly more than with either ligand alone. This cooperative effect was accompanied by differential expression of Id1-3, Smad6-7, and Has2 and was blocked by the same ALK5 inhibitor used to block GDF9 signaling. Immunostaining for SMAD2/3 and SMAD1/5/8, representing the 2 main branches of TGF-ß signaling, supported the fact that both canonical pathways have the potential to be active in growing follicles, whereas primordial follicles only express SMAD2/3. Overall results highlight differential effects of the 2 main TGF-ß signaling pathways during preantral follicle growth.
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Proteína Morfogenética Ósea 15/metabolismo , Folículo Ovárico/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta1/metabolismo , Receptores de Activinas Tipo I/antagonistas & inhibidores , Receptores de Activinas Tipo I/metabolismo , Receptores de Activinas Tipo II , Animales , Apoptosis , Proteína Morfogenética Ósea 15/genética , Femenino , Atresia Folicular , Regulación de la Expresión Génica/efectos de los fármacos , Células de la Granulosa/citología , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Factor 9 de Diferenciación de Crecimiento/genética , Factor 9 de Diferenciación de Crecimiento/metabolismo , Humanos , Cinética , Ligandos , Ratones , Ratones Endogámicos C57BL , Folículo Ovárico/citología , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/crecimiento & desarrollo , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Técnicas de Cultivo de TejidosRESUMEN
In the ovary, initiation of follicle growth is marked by cuboidalization of flattened granulosa cells (GCs). The regulation and cell biology of this shape change remains poorly understood. We propose that characterization of intercellular junctions and associated proteins is key to identifying as yet unknown regulators of this important transition. As GCs are conventionally described as epithelial cells, this study used mouse ovaries and isolated follicles to investigate epithelial junctional complexes (tight junctions [TJ], adherens junctions [AJ], and desmosomes) and associated molecules, as well as classic epithelial markers, by quantitative PCR and immunofluorescence. These junctions were further characterized using ultrastructural, calcium depletion and biotin tracer studies. Junctions observed by transmission electron microscopy between GCs and between GCs and oocyte were identified as AJs by expression of N-cadherin and nectin 2 and by the lack of TJ and desmosome-associated proteins. Follicles were also permeable to biotin, confirming a lack of functional TJs. Surprisingly, GCs lacked all epithelial markers analyzed, including E-cadherin, cytokeratin 8, and zonula occludens (ZO)-1alpha+. Furthermore, vimentin was expressed by GCs, suggesting a more mesenchymal phenotype. Under calcium-free conditions, small follicles maintained oocyte-GC contact, confirming the importance of calcium-independent nectin at this stage. However, in primary and multilayered follicles, lack of calcium resulted in loss of contact between GCs and oocyte, showing that nectin alone cannot maintain attachment between these two cell types. Lack of classic markers suggests that GCs are not epithelial. Identification of AJs during GC cuboidalization highlights the importance of AJs in regulating initiation of follicle growth.
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Moléculas de Adhesión Celular/fisiología , Conexinas/fisiología , Folículo Ovárico/fisiología , Uniones Adherentes/fisiología , Uniones Adherentes/ultraestructura , Animales , Calcio/fisiología , Moléculas de Adhesión Celular/ultraestructura , Conexinas/ultraestructura , Células Epiteliales/fisiología , Células Epiteliales/ultraestructura , Femenino , Uniones Comunicantes/fisiología , Uniones Comunicantes/ultraestructura , Ratones , Folículo Ovárico/ultraestructura , Uniones Estrechas/fisiología , Uniones Estrechas/ultraestructuraRESUMEN
The postnatal mouse ovary is rich in quiescent and early-growing oocytes, each one surrounded by a layer of somatic granulosa cells (GCs) on a basal lamina. As oocytes start to grow the GCs change shape from flattened to cuboidal, increase their proliferation and form multiple layers, providing a unique model for studying the relationship between cell shape, proliferation and multilayering within the context of two different intercommunicating cell types: somatic and germ cells. Proliferation of GCs was quantified using immunohistochemistry for Ki67 and demonstrated that, unusually, cuboidal cells divided more than flat cells. As a second layer of GCs started to appear, cells on the basal lamina reached maximum packing density and the axes of their mitoses became perpendicular to the basal lamina, resulting in cells dividing inwards to form second and subsequent layers. Proliferation of basal GCs was less than that of inner cells. Ultrastructurally, collagen fibrils outside the basal lamina became more numerous as follicles developed. We propose that the basement membrane and/or theca cells that surround the follicle provide an important confinement for rapidly dividing columnar cells so that they attain maximum packing density, which restricts lateral mitosis and promotes inwardly oriented cell divisions and subsequent multilayering.
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Proliferación Celular , Células de la Granulosa/citología , Folículo Ovárico/crecimiento & desarrollo , Animales , Forma de la Célula , Femenino , Células de la Granulosa/ultraestructura , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Ratones , Microscopía Electrónica de Transmisión , Mitosis , Oocitos/crecimiento & desarrollo , Folículo Ovárico/ultraestructura , Ovario/metabolismo , Ovario/ultraestructura , Células Tecales/metabolismo , Células Tecales/ultraestructuraRESUMEN
The absolute requirement for reproduction implies that the hypothalamo-pituitary-gonadal axis, controlling fertility, is an evolutionary robust mechanism. The GnRH neurons of the hypothalamus represent the key cell type within the body dictating fertility. However, the level of functional redundancy within the GnRH neuron population is unknown. As a result of a fortuitous transgene insertion event, GNR23 mice exhibit a marked allele-dependent reduction in GnRH neuron number within their brain. Wild-type mice have approximately 600 GnRH neurons, compared with approximately 200 (34%) and approximately 70 (12%) in GNR23(+/-) and GNR23(-/-) mice, respectively. Using these mice, we examined the minimal GnRH neuron requirements for fertility. Male GNR23(-/-) mice exhibited normal fertility. In contrast, female GNR23(-/-) mice were markedly subfertile, failing to produce normal litters, have estrous cycles, or ovulate. The failure of ovulation resulted from an inability of the few existing GnRH neurons to generate the LH surge. This was not the case, however, for the first cycle at puberty that appeared normal. Together, these observations demonstrate that 12% of the GnRH neuron population is sufficient for pulsatile gonadotropin secretion and puberty onset, whereas between 12 and 34% are required for cyclical control in adult female mice. This indicates that substantial redundancy exists within the GnRH neuronal population and suggests that the great majority of GnRH neurons must be dysfunctional before fertility is affected.
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Fertilidad/fisiología , Hormona Liberadora de Gonadotropina/metabolismo , Infertilidad Femenina/fisiopatología , Ovulación/fisiología , Maduración Sexual/fisiología , Animales , Recuento de Células , Ciclo Estral/fisiología , Femenino , Hormona Liberadora de Gonadotropina/genética , Infertilidad Femenina/patología , Hormona Luteinizante/sangre , Eminencia Media/patología , Eminencia Media/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Neuronas/patología , Neuronas/fisiología , Ovariectomía , Área Preóptica/patología , Área Preóptica/fisiologíaRESUMEN
BACKGROUND: The aim of this study was to investigate evidence for cell death by apoptosis in small unilaminar ovarian follicles of adult humans. METHODS: Cortical biopsies from 13 healthy donors were either frozen and protein extracted for western blots or fixed for immunohistochemistry (IH) to localize procaspase-3 and active-caspase-3, to detect DNA fragmentation in situ and undertake routine transmission electron microscopy (TEM). RESULTS: Blots identified the presence of the inactive pro-form of caspase-3, and IH localized this in all follicles studied. In contrast, the active form of caspase-3, a major effector of apoptosis, was only detected in large antral follicles that also had microscopic signs of atresia. Active caspase-3 was not detected in primordial (n = 87), primary (n = 8) or secondary follicles. The atretic follicles were also the only ovarian structures with positive evidence of DNA fragmentation after terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) treatment. Confocal microscopy showed dual labelling for both active caspase-3 and TUNEL in individual granulosa cells in large atretic follicles, but no such labelling was evident in any other follicles. No apoptotic bodies were seen by TEM in sections of 39 small follicles from seven patients. CONCLUSION: This study found evidence for TUNEL and active caspase-3 only in human ovarian antral follicles.