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Insulin sensitivity of skeletal muscle was studied in male offspring of rat dams fed either a 20% (control) or 8% (low-protein) diet during pregnancy and lactation. Freshly isolated muscle strips took up more [3H]methylglucose from low-protein animals than from controls (19.2 +/- 2.5 and 4.26 +/- 0.45 nmol.min-1.mg muscle-1, respectively, P < 0.001). However, after a 60-min preincubation there was no significant difference in basal glucose transport (4.02 +/- 0.42 and 4.23 +/- 0.35 nmol.min-1.mg-1 for control and low-protein animals, respectively). Insulin (300 pM) had a significantly greater (P < 0.001) effect on stimulation of glucose transport into preincubated low-protein muscle strips than into controls (to 14.14 +/- 1.25 and 9.61 +/- 0.71 nmol.min-1.mg-1, respectively). There were no differences in total GLUT-4 protein content. However, subcellular fractionation revealed significantly (P < 0.001) more GLUT-4 in muscle plasma membranes of low-protein animals compared with controls. Insulin increased (P < 0.001) the GLUT-4 content of control plasma membranes but had no effect in low-protein animals. There were twofold more insulin receptors in low-protein muscle membranes compared with controls (2.35 +/- 0.17 x 10(11) and 1.28 +/- 0.10 x 10(11) and insulin receptors/mg muscle membrane protein, respectively, P < 0.01). These results suggest that programming of muscle insulin sensitivity can occur during fetal life.

HPV late gene expression is initiated as an infected basal cell migrates through the differentiating layers of the epidermis, resulting in the onset of vegetative viral DNA replication and the expression of viral late proteins. We have used a large synthetic immunoglobulin library displayed on phage (diversity 6.5 x 10(10) phage) to isolate three Fabs (TVG405, 406, and 407) which recognize distinct epitopes on the E4 late protein of HPV16. A C-terminal monoclonal (TVG404) was generated by hybridoma technology, and N-terminal polyclonal antiserum was prepared by peptide immunization (alpha N-term). The most potent antibody (TVG405) had an affinity for E4 of approximately 1.0 nM. All antibodies recognized the protein in paraffin-embedded archival material, allowing us to map events in the late stages of virus infection. Expression of E4 in vivo does not coincide with synthesis of the major virus coat protein L1, but precedes it by 1 or 2 cell layers in premalignant lesions caused by HPV16 and by up to 20 cell layers in HPV63-induced warts. In higher grade lesions associated with HPV16, E4 is produced in the absence of L1. By contrast, vegetative viral DNA replication and E4 expression correlate exactly and in some lesions begin as the infected epithelial cell leaves the basal layer. Differentiation markers such as filaggrin, loricrin, and certain keratins are not detectable in E4-positive cells, and nuclear degeneration is delayed. HPV16 E4 has a filamentous distribution in the lower epithelial layers, but associates with solitary perinuclear structures in more differentiated cells. Antibodies to the N-terminus of the protein stained these structures poorly. Our findings are compatible with a role for the HPV16 E4 protein in vegetative DNA replication or in modifying the phenotype of the infected cell to favor virus synthesis or virus release. The Fabs will be of value in the evaluation of model systems for mimicking HPV infection in vitro.

IMPORTANCEIn patients who require mechanical ventilation for acute hypoxemic respiratory failure, further reduction in tidal volumes, compared with conventional low tidal volume ventilation, may improve outcomes. OBJECTIVE To determine whether lower tidal volume mechanical ventilation using extracorporeal carbon dioxide removal improves outcomes in patients with acute hypoxemic respiratory failure. DESIGN, SETTING, AND PARTICIPANTS This multicenter, randomized, allocation-concealed, open-label, pragmatic clinical trial enrolled 412 adult patients receiving mechanical ventilation for acute hypoxemic respiratory failure, of a planned sample size of 1120, between May 2016 and December 2019 from 51 intensive care units in the UK. Follow-up ended on March 11, 2020. INTERVENTIONS Participants were randomized to receive lower tidal volume ventilation facilitated by extracorporeal carbon dioxide removal for at least 48 hours (n = 202) or standard care with conventional low tidal volume ventilation (n = 210). MAIN OUTCOMES AND MEASURESThe primary outcome was all-cause mortality 90 days after randomization. Prespecified secondary outcomes included ventilator-free days at day 28 and adverse event rates. RESULTS Among 412 patients who were randomized (mean age, 59 years; 143 [35%] women), 405 (98%) completed the trial. The trial was stopped early because of futility and feasibility following recommendations from the data monitoring and ethics committee. The 90-day mortality rate was 41.5% in the lower tidal volume ventilation with extracorporeal carbon dioxide removal group vs 39.5% in the standard care group (risk ratio, 1.05 [95% CI, 0.83-1.33]; difference, 2.0% [95% CI, −7.6% to 11.5%]; P = .68). There were significantly fewer mean ventilator-free days in the extracorporeal carbon dioxide removal group compared with the standard care group (7.1 [95% CI, 5.9-8.3] vs 9.2 [95% CI, 7.9-10.4] days; mean difference, −2.1 [95% CI, −3.8 to −0.3]; P = .02). Serious adverse events were reported for 62 patients (31%) in the extracorporeal carbon dioxide removal group and 18 (9%) in the standard care group, including intracranial hemorrhage in 9 patients (4.5%) vs 0 (0%) and bleeding at other sites in 6 (3.0%) vs 1 (0.5%) in the extracorporeal carbon dioxide removal group vs the control group. Overall, 21 patients experienced 22 serious adverse events related to the study device.CONCLUSIONS AND RELEVANCE Among patients with acute hypoxemic respiratory failure, the use of extracorporeal carbon dioxide removal to facilitate lower tidal volume mechanical ventilation, compared with conventional low tidal volume mechanical ventilation, did not significantly reduce 90-day mortality. However, due to early termination, the study may have been underpowered to detect a clinically important difference.

The complete androgen insensitivity syndrome (CAIS), caused by mutations in the androgen receptor (AR) gene, is associated with abnormal testicular development and an increased risk of germ cell malignancy. Previous histological studies in CAIS have selected patients purely on the basis of clinical diagnosis and were mostly based on small numbers, many of whom were post-pubertal. Here, we present 44 cases of CAIS, each with molecular pathological confirmation of an AR mutation. The median age at gonadectomy was 5.5 years (5.5; IQR 1-13). We have been able, therefore, to investigate testicular development in infancy, childhood and puberty, and estimate the incidence of premalignant change in this series. In addition, we have investigated whether the presence of epididymides and/or vasa deferentia in CAIS, previously shown to be associated with residual activity of mutant ARs, is related to a particular testicular phenotype. Epididymides/vasa deferentia were present in 36% of cases and these patients showed varying degrees of seminiferous tubule maturation at puberty above those without epididymides/vasa deferentia (p = 0.003). There were no other histological differences between these patient groups. In both groups, features of testicular degeneration and dysgenesis were present and germ cell development was delayed, with prolonged expression of the gonocyte markers, placental-like alkaline phosphatase and activator protein-2gamma. Germ cell numbers rapidly declined after the first year of life (R(2) = 0.42). Only two cases of carcinoma in situ were identified in our study and both patients were postpubertal (17 and 53 years). From these results and the literature, we conclude that the risk of premalignant change in germ cells is low before and during puberty. Patients can be advised, therefore, that gonadectomy can be delayed to allow for a natural puberty, with low risk of malignant transformation. Our study only included one patient over 18 years, so we cannot comment on the risk of malignant transformation in later life.

H igh-risk human papillomaviruses (HPV), such as HPV type 16 (HPV-16), are associated with a spectrum of precancerous neoplastic changes which occur within the locally infected epithelium. HPV infections that exhibit mild neoplastic changes are classified as low-grade squamous intraepithelial lesions (LSIL), whereas infections showing more severe neoplastic changes are classified as high-grade squamous intraepithelial lesions (HSIL). LSIL and HSIL occur in mucosal epithelia, such as the cervix, and equivalent lesion grades can occur in cutaneous epithelia, such as the vulva (3).How the virus influences the pathological progression from LSIL to HSIL is not completely understood. Recent studies of both cutaneous and mucosal epithelial lesions have shown that the numbers of cells expressing cell cycle proteins, such as the E7 surrogate marker, minichromosome maintenance protein 7 (MCM-7), are increased in HSIL (2,15,22). Furthermore, the prolonged expression of E7 and MCM-7 in cells of the upper epithelial layers coincides with a delay in HPV-16 late gene expression, including that of the genes coding for the E4 and L1 capsid proteins (15). Regions of HSIL often do not support a productive virus life cycle, even though the majority of cells within the lesion still maintain intact viral episomes (8, 11). These observations suggest a model in which the deregulated expression of the HPV-16 early E7 and/or E6 oncogene from intact viral episomes allows infected cells to remain in cycle throughout the upper epithelial layers, thus resulting in an HSIL and an abortive virus life cycle.While studying viral gene expression patterns in organotypic raft cultures of an HPV-16 episome-containing normal immortalized human keratinocyte line (NIKS), we noticed that in marked contrast to what is seen with cell populations, individual HPV-16 cell line clones of the same early passage number were heterogeneous when propagated in raft culture and could mimic either LSIL or HSIL phenotypes with respect to viral gene expression patterns (i.e., E7/MCM, E4, and L1) and cellular pathology. In both the LSIL-like and HSIL-like clones, the viral oncogenes were expressed exclusively from intact viral episomes rather than from integrated sequences, with expression at confluence correlating closely with both the phenotype in raft culture and the extent of suprabasal E7/MCM-7 expression. This work supports the recent vaccine trial results showing that LSIL and HSIL may sometimes arise within a similar time frame and that, in some instances, a cervical intraepithelial neoplasia grade of 2ϩ (CIN2ϩ) can be detected within months or even weeks of first infection (16,17,20). The use of episomal cell lines provides a novel model of early-stage cervical disease and has revealed a correlation between the extent of expression of viral oncogenes and the severity of neoplasia prior to the acquisition of the cancer phenotype.Characterization of HPV16 LSIL-and HSIL-like phenotypes following life cycle reconstruction in organotypic raft culture. To study episo...

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Angiogenesis is essential for the replacement of cartilage by bone during growth and repair. In order to obtain a better understanding of the mechanisms regulating vascular invasion at sites of endochondral ossification we have investigated the expression of the endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), by chondrocytes in human neonatal growth plates. VEGF was absent from chondrocytes in the resting zone and only weakly expressed by occasional chondrocytes in the proliferating region. In the hypertrophic zone the number of chondrocytes stained and the intensity of staining for VEGF increased with chondrocyte hypertrophy, maximum expression of VEGF being observed in chondrocytes in the lower hypertrophic and mineralised regions of the cartilage. These observations provide the first demonstration of the presence of VEGF in situ in developing human bone and are consistent with in vitro observations demonstrating the upregulation of proangiogenic growth factor production with increasing chondrocyte hypertrophy. The presence of numerous small blood vessels and vascular structures in the subchondral region where VEGF expression was maximal indicates that VEGF produced by hypertrophic chondrocytes may play a key role in the regulation of vascular invasion of the growth plate.