Unrecognized viral infections and chromosome abnormalities as a cause of fetal death – examination with fluorescence in situ hybridization,immunohistochemistry and polymerase chain reaction

Fifteen to 50% of fetal deaths remain unexplained after post‐mortem examination depending on inclusion criteria and classification systems. Our aim was to examine a selection of unexplained fetal deaths in order to investigate whether any common chromosome aberrations or viral infections were present. Reports from 351 fetal autopsies performed at the Department of Pathology and Medical Genetics at St. Olavs University Hospital from 2001 through 2010 were reviewed. Of these, 105 fetal deaths were classified as unexplained. Tissue samples from 30 cases were further examined with fluorescence in situ hybridization (FISH) to detect abnormalities in chromosomes 13, 18, and 21. The samples were also examined with immunohistochemistry (IHC) and polymerase chain reaction (PCR) to detect infections with cytomegalovirus, parvovirus B19, herpes simplex virus 1 and 2, enterovirus, and parechovirus. In two cases, a possible trisomy 13 mosaicism was found. No viruses were detected. In our selection of 30 unexplained cases, possible trisomy 13 mosaicism was found in two cases, and no viruses were detected. High degree of maceration and missing placental examination often complicate the investigation of fetal death, and extensive ancillary examinations do not necessarily contribute to a more specific diagnosis.     

Exercise rapidly increases eukaryotic elongation factor 2 phosphorylation in skeletal muscle of men

Protein synthesis in skeletal muscle is known to decrease during contractions but the underlying regulatory mechanisms are unknown. Here, the effect of exercise on skeletal muscle eukaryotic elongation factor 2 (eEF2) phosphorylation, a key component in protein translation machinery, was examined. Eight healthy men exercised on a cycle ergometer at a workload eliciting ∼67% peak pulmonary oxygen consumption     with skeletal muscle biopsies taken from the vastus lateralis muscle at rest as well as after 1, 10, 30, 60 and 90 min of exercise. In response to exercise, there was a rapid (i.e. < 1 min) 5- to 7-fold increase in eEF2 phosphorylation at Thr56 that was sustained for 90 min of continuous exercise. The in vitro activity of skeletal muscle eEF2 kinase was not altered by exercise indicating that the increased activity of eEF2 kinase to eEF2 is not mediated by covalent mechanisms. In support of this, the increase in AMPK activity was temporally unrelated to eEF2 phosphorylation. However, skeletal muscle eEF2 kinase was potently activated by Ca²⁺–calmodulin in vitro , suggesting that the higher eEF2 phosphorylation in working skeletal muscle is mediated by allosteric activation of eEF2 kinase by Ca²⁺ signalling via calmodulin. Given that eEF2 phosphorylation inhibits eEF2 activity and mRNA translation, these findings suggest that the inhibition of protein synthesis in contracting skeletal muscle is due to the Ca²⁺-induced stimulation of eEF2 kinase.     

Species identification of clinical isolates of anaerobic bacteria: a comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry systems

We compared two matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems (Shimadzu/SARAMIS and Bruker) on a collection of consecutive clinically important anaerobic bacteria (n = 290). The Bruker system had more correct identifications to the species level (67.2% versus 49.0%), but also more incorrect identifications (7.9% versus 1.4%). The system databases need to be optimized to increase identification levels. However, MALDI-TOF MS in its present version seems to be a fast and inexpensive method for identification of most clinically important anaerobic bacteria.     

Evaluation of an Enzyme Immunoassay for Detection of Histoplasma capsulatum Antigen from Urine Specimens

Detection of Histoplasma capsulatum urinary antigen (UAg) is important for the initial diagnosis of infection and for monitoring of patient responses to antifungal therapy. This study evaluated an analyte-specific reagent (ASR) enzyme immunoassay (EIA) for the detection of H. capsulatum UAg from Immuno Mycologics, Inc. (IMMY) (Norman, OK) in comparison with routine testing with the MiraVista (MVista) H. capsulatum quantitative EIA (MiraVista Diagnostics, Indianapolis, IN). Using prospectively collected urine specimens (n = 1,003), we observed an overall percent agreement between the two assays of 97.6% (979/1,003 samples). Compared with the MVista EIA, the sensitivity and specificity of the IMMY ASR EIA were 64.5% (40/62 samples) and 99.8% (939/941 samples), respectively, using a cutoff value of 0.5 ng/ml. Based on available clinical histories for 23/24 discordant samples, 5 IMMY assay-negative/MVista assay-positive samples were considered falsely positive. Furthermore, 10/23 discordant samples were positive by the MVista EIA but were below the limit of quantitation (<0.4 ng/ml). The clinical significance of these low positive results in the MVista EIA is unclear. In addition to the prospective study, we tested 11 urine specimens collected from patients with culture-confirmed Histoplasma infections, and 100% (11/11 samples) were positive by the IMMY ASR EIA. In conclusion, the IMMY ASR EIA may offer an alternative approach for the detection of Histoplasma UAg. Additional prospective studies are needed to better characterize the performance of the IMMY ASR EIA in conjunction with clinical and laboratory findings.     

Physical activity and plasma interleukin-6 in humans – effect of intensity of exercise

The present study included data from three marathon races to investigate the hypothesis that a relationship exists between running intensity and elevated concentrations of interleukin (IL)-6 in plasma. The study included a total of 53 subjects whose mean age was 30.6 [95% confidence interval (CI) 1.4] years, mean body mass 77.7 (95%CI 2.0) kg, mean maximal oxygen uptake (V˙O_2max) 59.3 (95%CI 1.4) ml · min⁻¹ · kg⁻¹, and who had participated in the Copenhagen Marathons of 1996, 1997 or 1998, achieving a mean running time of 206 (95%CI 7) min. Running intensity was calculated as running speed divided by V˙O_2max. The concentration of IL-6 in plasma peaked immediately after the run. There was a negative correlation between peak IL-6 concentration and running time (r=−0.30, P < 0.05) and a positive correlation between peak IL-6 concentration and running intensity (r=0.32, P < 0.05). The IL-1 receptor antagonist (IL-1ra) plasma concentration peaked 1.5 h after the run and there was a positive correlation between the peak plasma concentrations of IL-6 and IL-1ra (r=0.39, P < 0.01). Creatine kinase (CK) plasma concentration peaked on the 1st day after the run, but no association was found between peak concentrations of IL-6 and CK. In conclusion, the results confirmed the hypothesized association between plasma IL-6 concentration and running intensity, but did not confirm the previous finding of a connection between IL-6 plasma concentration and muscle damage. Accepted: 6 August 2000     

Rac1 Signaling Is Required for Insulin-Stimulated Glucose Uptake and Is Dysregulated in Insulin-Resistant Murine and Human Skeletal Muscle

The actin cytoskeleton–regulating GTPase Rac1 is required for insulin-stimulated GLUT4 translocation in cultured muscle cells. However, involvement of Rac1 and its downstream signaling in glucose transport in insulin-sensitive and insulin-resistant mature skeletal muscle has not previously been investigated. We hypothesized that Rac1 and its downstream target, p21-activated kinase (PAK), are regulators of insulin-stimulated glucose uptake in mouse and human skeletal muscle and are dysregulated in insulin-resistant states. Muscle-specific inducible Rac1 knockout (KO) mice and pharmacological inhibition of Rac1 were used to determine whether Rac1 regulates insulin-stimulated glucose transport in mature skeletal muscle. Furthermore, Rac1 and PAK1 expression and signaling were investigated in muscle of insulin-resistant mice and humans. Inhibition and KO of Rac1 decreased insulin-stimulated glucose transport in mouse soleus and extensor digitorum longus muscles ex vivo. Rac1 KO mice showed decreased insulin and glucose tolerance and trended toward higher plasma insulin concentrations after intraperitoneal glucose injection. Rac1 protein expression and insulin-stimulated PAK^Thr423 phosphorylation were decreased in muscles of high fat–fed mice. In humans, insulin-stimulated PAK activation was decreased in both acute insulin-resistant (intralipid infusion) and chronic insulin-resistant states (obesity and diabetes). These findings show that Rac1 is a regulator of insulin-stimulated glucose uptake and a novel candidate involved in skeletal muscle insulin resistance.Insulin increases glucose uptake in skeletal muscle by stimulating translocation of GLUT4 from intracellular compartments to the plasma membrane and transverse tubuli (1–4). Skeletal muscle accounts for up to 75% of postprandial glucose disposal in humans (5), and normal insulin action in skeletal muscle is therefore crucial for maintaining glucose homeostasis.The Rho family GTPase Rac1 has been shown to regulate insulin-stimulated GLUT4 translocation and glucose transport in cultured muscle cells (6–8). Insulin activates Rac1, which leads to reorganization of the cortical actin cytoskeleton. Downregulation of Rac1 by small interfering RNA prevents this process (7,9) and also abolishes insulin-stimulated glucose uptake and GLUT4 translocation in L6 myoblasts (6,7). In addition, expression of a constitutively active Rac1 increases GLUT4 translocation to the same level seen after maximal insulin stimulation in this cell line (6).Even though cultured muscle cell lines are powerful tools to understand intracellular mechanisms, they differ from mature skeletal muscle in the expression and reliance of various proteins in the regulation of insulin-stimulated glucose uptake (10). Cultured muscle myoblasts, although able to fuse into myotubes, do not reach the same end-stage differentiation (e.g., do not have cross striations and do not develop transverse tubules) as muscles in vivo and therefore do not fully mature into a system that mimics fully developed skeletal muscles (11,12). Furthermore, the location, expression, and insulin-stimulated GLUT4 translocation are very different in cultured cells compared with mature muscle and may not require the same trafficking steps (2,3,13,14). As a consequence, it is imperative to investigate the role of Rac1 in insulin-stimulated glucose uptake in fully matured skeletal muscle in order to understand its role in glucose metabolism. Furthermore, the importance of skeletal muscle Rac1 on whole-body glucose homeostasis has not been determined.Rac1 activates p21-activated kinase (PAK) by facilitating autophosphorylation of PAK on threonine 423 (p-PAK^Thr423), and this pathway induces actin remodeling of the actin cytoskeleton (15). Accordingly, disruption of the actin cytoskeleton by actin-depolymerizing agents, such as latrunculin B, inhibits insulin-stimulated GLUT4 translocation in L6 myotubes (16,17). Dynamic rearrangement of the actin cytoskeleton is thus necessary for insulin to induce GLUT4 translocation in these cells (18).These findings also apply to mature skeletal muscle, since latrunculin B inhibits insulin-stimulated glucose uptake in rat epitrochlearis muscle (19). Furthermore, Ueda et al. (20) recently showed that Rac1 is activated by insulin in mouse skeletal muscle and that insulin-stimulated GLUT4 translocation is decreased in muscle-specific Rac1 knockout (KO) mice. PAK1 was also recently shown to be implicated in the regulation of insulin-stimulated GLUT4 translocation in mouse skeletal muscle (21). However, GLUT4 translocation does not always mimic glucose uptake, and numerous studies have reported experimental conditions where GLUT4 translocation and transport can be clearly dissociated (22–27), suggesting that GLUT4 translocation is not always an adequate measure of the functional end point, glucose uptake. Thus, the involvement of Rac1 and its downstream signaling in insulin-stimulated glucose uptake in mature skeletal muscle has not yet been investigated, and Rac1-dependent signaling has not been characterized in animal or human models of insulin resistance.A decreased ability to rearrange the cortical actin cytoskeleton in response to insulin has been proposed as a central defect in insulin-resistant muscle cells (28–30). Although exposure to insulin resistance–inducing agents decreased Rac1 activation and GLUT4 translocation (7), only small reductions in Akt signaling were observed in L6 myotubes (8). It is therefore possible that Rac1 is a major regulator of glucose uptake in mature skeletal muscle, and its dysregulation might contribute to the phenotype of muscular insulin resistance and type 2 diabetes (T2D). In the current study, we hypothesized that activation of Rac1 and its downstream target, PAK, is crucial for insulin-induced glucose uptake in mature skeletal muscle and for maintaining whole-body glucose homeostasis. We further hypothesized that Rac1-dependent signaling is downregulated in insulin-resistant states.     

Effects of Odanacatib on Bone Structure and Quality in Postmenopausal Women With Osteoporosis: 5-Year Data From the Phase 3 Long-Term Odanacatib Fracture Trial (LOFT) and its Extension

Odanacatib (ODN), a selective oral inhibitor of cathepsin K, was an investigational agent previously in development for the treatment of osteoporosis. In this analysis, the effects of ODN on bone remodeling/modeling and structure were examined in the randomized, double-blind, placebo-controlled, event-driven, Phase 3, Long-term Odanacatib Fracture Trial (LOFT; NCT00529373) and planned double-blind extension in postmenopausal women with osteoporosis. A total of 386 transilial bone biopsies, obtained from consenting patients at baseline (ODN n = 17, placebo n = 23), month 24 (ODN n = 112, placebo n = 104), month 36 (ODN n = 42, placebo n = 41), and month 60 (ODN n = 27, placebo n = 20) were assessed by dynamic and static bone histomorphometry. Patient characteristics at baseline and BMD changes over 5 years for this subset were comparable to the overall LOFT population. Qualitative assessment of biopsies revealed no abnormalities. Consistent with the mechanism of ODN, osteoclast number was higher with ODN versus placebo over time. Regarding bone remodeling, dynamic bone formation indices in trabecular, intracortical, and endocortical surfaces were generally similar in ODN-treated versus placebo-treated patients after 2 years of treatment. Regarding periosteal modeling, the proportion of patients with periosteal double labels and the bone formation indices increased over time in the ODN-treated patients compared with placebo. This finding supported the observed numerical increase in cortical thickness at month 60 versus placebo. In conclusion, ODN treatment for 5 years did not reduce bone remodeling and increased the proportion of patients with periosteal bone formation. These results are consistent with the mechanism of action of ODN, and are associated with continued BMD increases and reduced risk of fractures compared with placebo in the LOFT Phase 3 fracture trial. © 2020 American Society for Bone and Mineral Research.     

Patient reported outcome measures in women undergoing surgery for urinary incontinence and pelvic organ prolapse in Denmark, 2006–2011

Introduction and hypothesis

The aim of this study was to evaluate the impact of urogynecological surgery on quality of life based on patient reported outcome measures (PROMs).

Methods

Data were retrieved from the Danish Urogynaecological Database. Inclusion criteria were Danish women undergoing surgery for urinary incontinence (UI) or pelvic organ prolapse (POP) from 2006 to 2011. Using frequency of symptoms and a visual analogue scale (VAS) both pre- and postoperatively, their severity of symptoms and quality of life were measured by questionnaires.

Results

During the study period, 20,629 urogynecological procedures were performed. The questionnaires on severity of symptoms and the VAS had been completed both pre- and postoperatively for approximately one third of women undergoing surgery. For UI surgery, 83 % had improved symptoms, 13 % were unchanged, and 4 % had worse symptoms postoperatively. For POP surgery, 80, 17, and 3 % were improved, unchanged, and worsened, respectively. The postoperative bother of symptoms and interference in everyday life evaluated by VAS were significantly reduced for both UI [preoperative median VAS score 9, postoperative median score 1 (p?<?0.001)] and POP [8 preoperatively and 0 postoperatively (p?<?0.001)].

Conclusions

Based on PROMs, surgery for UI and POP is effective in alleviating symptoms associated with UI or POP, and it can improve quality of life in symptomatic women. Pre- and postoperative questionnaires are useful tools in assessing symptomatic outcome measures after surgery.