Correction to: Cesarean delivery to prevent anal incontinence: a systematic review and meta-analysis.

The original article can be found online.

Cesarean delivery to prevent anal incontinence: a systematic review and meta-analysis.

BACKGROUND: Cesarean delivery (CD), is increasingly recommended as a mode of delivery that prevents the anal incontinence (AI) that arises in some women after vaginal delivery (VD). The assessment of the efficacy of CD in this regard was the subject of this systematic review. METHODS: Searches were conducted in Medline, EMBASE and the Cochrane Library. Both randomized (RCTs) and non-randomized trials (NRTs) comparing the risk of sustained fecal and/or flatus incontinence after VD or CD were sought from 1966 to 1 January, 2019. Studies were eligible if they assessed AI more than 6 months after birth, and had statistical adjustment for at least one of the three major confounders for AI: age, maternal weight or parity. In addition, each study was required to contain more than 250 participants, more than 50 CDs and more than 25 cases of AI. Data after screening and selection were abstracted and entered into Revman for meta-analysis. Analyses were done for combined fecal and flatus incontinence (comAI), fecal incontinence (FI), gas incontinence (GI), CD before or during labor, time trend of incontinence after delivery, assessment of both statistical and clinical heterogeneity, parity and late incident AI. RESULTS: Out of the 2526 titles and abstracts found, 24 eligible studies were analyzed, 23 NRTs and one RCT. These included women with 29,597 VDs and women with 6821 CDs. Among the primary outcomes, VD was found not to be a significant predictor of postpartum comAI compared to CD in 6 studies, incorporating 18,951 deliveries (OR = 0.74; 0.54-1.02). VD was also not a significant predictor of FI in 14 studies, incorporating 29,367 deliveries, (OR = 0.89; 0.76-1.05). VD was not a significant predictor of GI in six studies, incorporating 6724 deliveries (OR = 0.96; 0.79-1.18). The strength of the grading of recommendations, assessment, development and evaluations (GRADE) evidence for each of these was low for comAI and moderate for FI and GI (upgrade for lack of expected effect). Time trend FI showed incontinence at 3 months often resolved at 1 year. Other secondary analyses assessing parity, delayed incidence of FI, clinical and statistical heterogeneity, spontaneous VD only, late risk of incidence of AI, and CD in or prior to labor all had similar results as in the primary outcomes. CONCLUSIONS: There are three components of pelvic floor dysfunction that are thought to be caused by VD and hopefully prevented by CD: AI, urinary incontinence and pelvic floor prolapse. Of these, AI was not found to be reliably prevented by CD in this review.

A giant thunderstorm on Saturn.

Lightning discharges in Saturn's atmosphere emit radio waves with intensities about 10,000 times stronger than those of their terrestrial counterparts. These radio waves are the characteristic features of lightning from thunderstorms on Saturn, which last for days to months. Convective storms about 2,000 kilometres in size have been observed in recent years at planetocentric latitude 35° south (corresponding to a planetographic latitude of 41° south). Here we report observations of a giant thunderstorm at planetocentric latitude 35° north that reached a latitudinal extension of 10,000 kilometres-comparable in size to a 'Great White Spot'-about three weeks after it started in early December 2010. The visible plume consists of high-altitude clouds that overshoot the outermost ammonia cloud layer owing to strong vertical convection, as is typical for thunderstorms. The flash rates of this storm are about an order of magnitude higher than previous ones, and peak rates larger than ten per second were recorded. This main storm developed an elongated eastward tail with additional but weaker storm cells that wrapped around the whole planet by February 2011. Unlike storms on Earth, the total power of this storm is comparable to Saturn's total emitted power. The appearance of such storms in the northern hemisphere could be related to the change of seasons, given that Saturn experienced vernal equinox in August 2009.

BRAF(V600E) Mutation is Associated with Decreased Disease-Free Survival in Papillary Thyroid Cancer.

BACKGROUND: The BRAF (V600E) mutation is a recognised molecular marker in papillary thyroid cancer (PTC), reported incidence from 30 to 80 %. BRAF(V600E) aberrantly activates the MAPK pathway, a central regulator of cell growth and proliferation. Previous studies have reported conflicting data regarding the impact of BRAF(V600E) on clinicopathological features of PTC. The study aims to determine whether BRAF(V600E) is useful as a prognostic biomarker in PTC. METHODS: A cohort study of patients undergoing surgery for PTC was undertaken. The primary outcome measure was disease-free survival. Secondary outcome measures were tumour size, nodal positivity and radioactive iodine ablation rate. All cases were re-examined to confirm PTC. Immunohistochemistry for BRAF(V600E) was performed on tissue microarrays. A single endocrine pathologist, blinded to clinicopathological data, interpreted staining. RESULTS: 496 patients with PTC were included, and 309 (62 %) were BRAF(V600E) positive. Tumour size was similar for BRAF(V600E)-positive and -negative tumours (21.3 vs. 23.2 mm, p = 0.23). BRAF(V600E)-positive patients were significantly older at first operation (mean age 45 versus 49 years, p = 0.003). BRAF(V600E)-positive PTCs had a higher rate of disease recurrence (12.9 vs. 5.6 %, p = 0.004), lymph node metastasis (44 vs. 29.4 %, p = 0.004) and extra-thyroidal extension (44 vs. 22 %, p < 0.001). Five-year disease-free survival was 89.6 % for BRAF(V600E) positive and 96.3 % for negative tumours, p < 0.001. There was no difference between groups for vascular invasion or multifocality. The mean follow-up was 57 months for both groups. CONCLUSION: BRAF(V600E) in PTC predicts an increased risk of lymph node metastasis, extra-thyroidal extension and reduced disease-free survival. It is an additional useful prognostic biomarker.

Elevating glucose and insulin secretion by carbohydrate formulation diets in late lactation to improve post-weaning fertility in primiparous sows.

Primiparous (P1) sows commonly lose excessive body reserves to meet energy requirements for maintenance and milk production during lactation, and consequently, post-weaning reproductive performance may be compromised. The present studies determined whether ad libitum feeding a glucogenic carbohydrate diet (CHO) during late lactation could stimulate insulin and glucose secretion (experiment 1) and improve subsequent litter size (experiment 2). For experiment 1, 15 P1 sows, and for experiment 2, 99 P1 sows (198.5 ± 2.7 kg) were allocated randomly according to suckled litter size (≥10 piglets), either to a CHO diet (14.3 MJ DE/kg, 19.8% crude protein) or a standard lactation diet (control; 14.2 DE MJ/kg, 19.5% crude protein) at 8 days before weaning. The CHO diet aimed to provide glucogenic content (extruded wheat, dextrose and sugar) as energy sources instead of fat sources without changing total dietary energy. Pre-prandial plasma glucose and insulin concentrations were not influenced by treatments. However, post-prandial plasma glucose and insulin concentrations and their peaks were both higher (p < .05) compared to the control treatment. Body weight loss during lactation was relatively low at 3%-4% for both treatments and did not differ between control and CHO treatments (-7.6 ± 1.6 vs -5.4 ± 1.2 kg; p > .05). Second litter size was not influenced by diet (p > .05), but the weaning-to-mating interval was shorter in CHO sows (p < .05). This study demonstrates that providing an enriched CHO diet in late lactation did influence post-weaning follicle growth but did not improve subsequent litter size. This may be due to the primiparous sows in this study not experiencing severe negative energy balance and there was no second litter syndrome in this farm which limited the ability of diet to improve sow fertility.

Depth of a strong jovian jet from a planetary-scale disturbance driven by storms.

The atmospheres of the gas giant planets (Jupiter and Saturn) contain jets that dominate the circulation at visible levels. The power source for these jets (solar radiation, internal heat, or both) and their vertical structure below the upper cloud are major open questions in the atmospheric circulation and meteorology of giant planets. Several observations and in situ measurements found intense winds at a depth of 24 bar, and have been interpreted as supporting an internal heat source. This issue remains controversial, in part because of effects from the local meteorology. Here we report observations and modelling of two plumes in Jupiter's atmosphere that erupted at the same latitude as the strongest jet (23 degrees N). The plumes reached a height of 30 km above the surrounding clouds, moved faster than any other feature (169 m s(-1)), and left in their wake a turbulent planetary-scale disturbance containing red aerosols. On the basis of dynamical modelling, we conclude that the data are consistent only with a wind that extends well below the level where solar radiation is deposited.

A SEER analysis of survival and prognostic factors in merkel cell carcinoma of the head and neck region.

While Merkel cell carcinoma (MCC) of the head and neck is highly malignant, it remains poorly characterized due to its rarity. The purpose of this study was to examine prognostic factors for overall survival (OS) and disease-specific survival (DSS) in patients with MCC of the head and neck region. The Surveillance, Epidemiology and End Results registry was reviewed for patients diagnosed between 1984 and 2016 with histologically confirmed, primary MCC of the head and neck region. A total of 2818 patients met the inclusion criteria, with a median age at diagnosis of 77 years. At five and 10 years, respectively, the OS was 42.4% and 25.1% and the DSS was 67.9% and 64.1%. Multivariate Cox analysis indicated that predictors of decreased DSS included age at diagnosis ≥75 years, white race, increasing tumor spread, lymph node involvement and either the lip or the scalp/neck as a primary site. When adjusting for the aforementioned factors, tumor depth was not found to be a prognostic factor for DSS. We anticipate these results will help clinicians to counsel patients regarding expectations and potential prognosis.

Resting-State Functional MRI for Determining Language Lateralization in Children with Drug-Resistant Epilepsy.

BACKGROUND AND PURPOSE: Task-based fMRI is a noninvasive method of determining language dominance; however, not all children can complete language tasks due to age, cognitive/intellectual, or language barriers. Task-free approaches such as resting-state fMRI offer an alternative method. This study evaluated resting-state fMRI for predicting language laterality in children with drug-resistant epilepsy. MATERIALS AND METHODS: A retrospective review of 43 children with drug-resistant epilepsy who had undergone resting-state fMRI and task-based fMRI during presurgical evaluation was conducted. Independent component analysis of resting-state fMRI was used to identify language networks by comparing the independent components with a language network template. Concordance rates in language laterality between resting-state fMRI and each of the 4 task-based fMRI language paradigms (auditory description decision, auditory category, verbal fluency, and silent word generation tasks) were calculated. RESULTS: Concordance ranged from 0.64 (95% CI, 0.48-0.65) to 0.73 (95% CI, 0.58-0.87), depending on the language paradigm, with the highest concordance found for the auditory description decision task. Most (78%-83%) patients identified as left-lateralized on task-based fMRI were correctly classified as left-lateralized on resting-state fMRI. No patients classified as right-lateralized or bilateral on task-based fMRI were correctly classified by resting-state fMRI. CONCLUSIONS: While resting-state fMRI correctly classified most patients who had typical (left) language dominance, its ability to correctly classify patients with atypical (right or bilateral) language dominance was poor. Further study is required before resting-state fMRI can be used clinically for language mapping in the context of epilepsy surgery evaluation in children with drug-resistant epilepsy.

Differential induction of transcription factors that regulate the interleukin 2 gene during anergy induction and restimulation.

T cell activation requires two distinct signals. The first is delivered through the antigen-specific T cell receptor (TCR), and the second is provided by costimulatory molecule(s) present on the surface of the antigen-presenting cell (APC). Stimulation of T helper type 1 T cell clones through the TCR in the absence of the costimulatory activity results in a lack of interleukin 2 (IL-2) secretion and proliferation, and the induction of a long-lived state of nonresponsiveness, termed anergy. In this study, we have examined the transcription factors involved in IL-2 gene expression that are expressed after stimulation of normal T cell clones through the TCR with and without engagement of the necessary costimulatory molecule(s). Antigen-specific activation of the clones results in the induction of a similar pattern of transcription factors that have been previously shown to regulate IL-2 expression. In contrast, antigen presentation by chemically fixed APC, a condition that results in T cell anergy, induces neither NF-AT nor one of the two NF-kappa B binding factors. Thus, the failure to express IL-2 during the induction of T cell anergy may be attributed to the absence of these two transcription factors. When anergized T cells are restimulated with antigen and conventional APC, they induce the transcription factors associated with IL-2 expression, but they fail to synthesize measurable IL-2. Taken together, these data indicate that the control of IL-2 gene expression during anergy induction and during normal stimulation of anergized cells are distinct, and suggest the presence of additional regulatory elements in the IL-2 gene.

Decreased maternal behavior and anxiety in ephrin-A5-/- mice.

During development of the nervous system, molecular signals mediating cell-cell interactions play critical roles in the guidance of axonal growth and establishment of synaptic functions. The Eph family of tyrosine kinase receptors and their ephrin ligands has been shown to mediate neuronal interactions in the development of topographic axon projection maps in several brain regions, and the loss of Eph activities result in defects in select axonal pathways. However, effects of deficiencies of the Eph signals on animal behavior have not been well documented. In this study, we showed that inactivation of a ligand of the Eph receptors, ephrin-A5, resulted in defects in maternal behavior and alterations in anxiety. Female ephrin-A5 -/- mice show significant defects in nest building and pup retrieval. In addition, lower levels of anxiety were observed in both male and female null mice. These changes were not due to deficiencies in estradiol, progesterone or corticosterone levels. Our observations suggest that ephrin-A5 plays a key role in the development and/or function of neural pathways mediating mouse maternal care and anxiety.

Blocking transforming growth factor beta signaling in transgenic epidermis accelerates chemical carcinogenesis: a mechanism associated with increased angiogenesis.

Mutations in the transforming growth factor beta type II receptor (TGF-betaRII) have been identified in human cancers, which suggests a causal role for the loss of TGF-betaRII in cancer development. To directly test this in vivo, we have generated transgenic mice expressing a dominant negative TGF-betaRII (delta betaRII) in the epidermis, using a truncated mouse loricrin promoter (ML). ML.delta betaRII transgenic mice exhibited a thickened skin due to epidermal hyperproliferation. When these mice were subjected to a standard two-stage chemical carcinogenesis protocol, they exhibited an increased sensitivity, with an earlier appearance and a 2-fold greater number of papillomas than control mice. In addition, papillomas in control mice regressed after termination of 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment; whereas ML.delta betaRII papillomas progressed to carcinomas. Furthermore, TPA promotion alone induced papilloma formation in ML.delta betaRII mice, which suggests an initiating role for delta betaRII in skin carcinogenesis. ML.delta betaRII tumors also exhibited increased neovascularization and progressed to metastases, although the primary tumors were still classified as carcinoma in situ or well-differentiated carcinomas. Increased expression of vascular endothelial growth factor, an angiogenesis factor, and decreased expression of thrombospondin-1, an angiogenesis inhibitor, were also observed in ML.delta betaRII tumors. The increased angiogenesis correlated with elevated endogenous TGF-beta1 in ML.delta betaRII tumors. These data provide in vivo evidence that inactivation of TGF-betaRII accelerates skin carcinogenesis at both earlier and later stages, and increased angiogenesis is one of the important mechanisms of accelerated tumor growth and metastasis.

Aberrant cell cycle progression contributes to the early-stage accelerated carcinogenesis in transgenic epidermis expressing the dominant negative TGFbetaRII.

Mutations in the transforming growth factor beta type II receptor (TGFbetaRII) have been found in various malignant tumors, suggesting that loss of TGFbeta signaling plays a causal role in late-stage cancer development. To test whether loss of TGFbetaRII is involved in early-stage carcinogenesis, we have generated transgenic mice expressing a dominant negative TGFbetaRII (deltabetaRII) in the epidermis. These mice exhibited an increased susceptibility to chemical carcinogenesis protocols at both early and late stages. In the current study, parameters for cell cycle progression and chromosome instability were analysed in deltabetaRII tumors. DeltabetaRII papillomas showed an increased S phase in flow cytometry. Bromodeoxyuridine (BrdU) labeling and mitotic indices in deltabetaRII papillomas also showed a threefold increase compared to papillomas developing in non-transgenic mice. When papillomas further progressed to squamous cell carcinomas (SCC), both control and deltabetaRII SCC showed similar BrdU labeling indices and percentages of S phase cells. However, deltabetaRII SCC cells showed a sixfold increase in the G2/M population. Mitotic indices in deltabetaRII SCC also showed a threefold increase compared to non-transgenic SCC. Consistent with a perturbed cell cycle, deltabetaRII papillomas and SCC showed reduced expression of the TGFbeta target genes p15 (INK4b), p21 (WAF-1) and p27 (Kip1), inhibitors of cyclin-dependent kinases (cdks). However, most deltabetaRII papilloma cells exhibited normal centrosome numbers, and deltabetaRII SCC exhibited a similar extent of centrosome abnormalities compared to control SCC (35-40% cells). Most of deltabetaRII SCC exhibited diploid chromosome profiles. These data indicate that inactivation of TGFbetaRII accelerates skin tumorigenesis at early stages by the acceleration of loss of cell cycle control, but not by increased chromosome instability.

Intrinsic connections of the rat amygdaloid complex: projections originating in the basal nucleus.

The amygdaloid complex is involved in associational processes, such as the formation of emotional memories about sensory stimuli. However, the anatomical connections through which the different amygdaloid nuclei process incoming information and communicate with the other amygdaloid nuclei, is poorly understood. As part of an ongoing project aimed at elucidating the intrinsic connections of the rat amygdaloid complex, we injected the anterograde tracer PHA-L (Phaseolus vulgaris-leucoagglutinin) into different rostrocaudal levels of the basal nucleus of the amygdala in 21 rats and analyzed the distribution of labeled fibers and terminals throughout the amygdaloid complex. The connectional analysis, together with cytoarchitectonic observations, suggested that contrary to previous notions the basal nucleus in the rat has three divisions: magnocellular, intermediate, and parvicellular. The magnocellular division has heavy reciprocal connections with the lateral portion of the parvicellular division and the intermediate division projects weakly to the parvicellular division, whereas the projection from the medial portion of the parvicellular division to the intermediate division is heavy and the lateral and medial portions of the parvicellular division are only weakly interconnected, as are the magnocellular and intermediate divisions. The main intraamygdaloid targets of the basal nucleus projections are the nucleus of the lateral olfactory tract, the anterior amygdaloid area, the medial and capsular divisions of the central nucleus, the anterior cortical nucleus, and the amygdalohippocampal area. Our findings provide the most detailed understanding of the intra-amygdala connections of the basal nucleus to date and show that the connections within the basal nucleus and between the basal nucleus and other amygdaloid areas are more widespread and topographically organized than previously recognized.

Intrinsic connections of the rat amygdaloid complex: projections originating in the accessory basal nucleus.

The amygdaloid complex plays an important role in the detection of emotional stimuli, the generation of emotional responses, the formation of emotional memories, and perhaps other complex associational processes. These functions depend upon the flow of information through intricate and poorly understood circuitries within the amygdala. As part of an ongoing project aimed at further elucidating these circuits, we examined the intra-amygdaloid connections of the accessory basal nucleus in the rat. In addition, we examined connections of the anterior cortical nucleus and amygdalahippocampal area to determine whether portions of these nuclei should be included in the accessory basal nucleus (as some earlier studies suggest). Phaseolus vulgaris leucogglutinin was injected into different rostrocaudal levels of the accessory basal nucleus (n = 12) or into the anterior cortical nucleus (n = 3) or amygdalahippocampal area (n = 2). The major intra-amygdaloid projections from the accessory basal nucleus were directed to the medial and capsular divisions of the central nucleus, the medial division of the amygdalohippocampal area, the medial division of the lateral nucleus, the central division of the medial nucleus, and the posterior cortical nucleus. The projections originating in the anterior cortical nucleus and the lateral division of the amygdalohippocampal area differed from those originating in the accessory basal nucleus, which suggests that these areas are not part of the accessory basal nucleus. The present findings and our previous data suggest that each of the deep amygdaloid nuclei have different intra-amygdaloid connections. The pattern of these various connections suggests that information entering the amygdala from different sources can be integrated only in certain amygdaloid regions.

Cellular and subcellular distribution of alpha 2A-adrenergic receptors in the visual cortex of neonatal and adult rats.

Activation of alpha 2-adrenergic receptors (alpha 2AR) in the cerebral cortex has been shown to modulate visually guided delayed response tasks as well as anxiety and depression. We used an antiserum directed specifically against the A subtype of alpha 2AR (alpha 2AAR) to determine the cell types and subcellular sites for noradrenergic reception mediated by this receptor in the adult and the developing rat visual cortices. Light microscopic examination of adult tissue revealed numerous labeled perikarya in layers II-VI, many of which appeared distinctly pyramidal. A few perikarya in layer I also were immunoreactive. In all layers, alpha 2AAR immunoreactivity (alpha 2AAR-ir) was present within proximal dendrites and fine processes. In neonatal tissue, there was an intense, distinct band of immunoreactivity spanning the layer composed of tightly packed immature cell bodies, i.e., the cortical plate. The band dissipated as this tier differentiated postnatally into the supragranular layers. Electron microscopy showed that the supragranular layers, which contain the highest density of noradrenergic fibers, also contain the highest areal density of labeled postsynaptic junctions beyond 2 weeks of age. Throughout the ages, the majority of immunoreactivity occurred at sites which, in single ultrathin sections, appeared to be nonjunctional sites of axons, dendrites, and in glial processes. Our observations indicate that (1) both pyramidal and nonpyramidal neurons are receptive to norepinephrine via alpha 2AAR, (2) alpha 2AAR synthesis is robust prior to synaptogenesis, and (3) alpha 2AAR operates both pre- and postsynaptically.

Cytoskeleton-dependent activation of the inducible nitric oxide synthase in cultured aortic smooth muscle cells.

1. Vascular endothelial and smooth muscle cells generate nitric oxide (NO) via different nitric oxide synthase (NOS) isozymes. Activation of the endothelial constitutive NOS (ecNOS) contributes to the maintenance of cardiovascular homeostasis, whereas expression of the endotoxin- and cytokine-inducible pathway (iNOS) within the vascular smooth muscle is thought to be responsible for the cardiovascular collapse which occurs during septic shock and antitumour therapy with cytokines. Since the cytoskeleton is involved in the activation of certain genes and in some effects of endotoxin in macrophages, we investigated the role of microtubules and microfilaments in the activation of the NO pathway in cultured vascular cells. 2. Depolymerization of microtubules by either nocodazole or colchicine prevented lipopolysaccharide (LPS)- and interleukin-1 beta-induction of NO-dependent cyclic GMP accumulation. Steady state levels of iNOS mRNA, assessed by Northern blot and RT-PCR, and iNOS protein, assessed by Western blotting, were also decreased by either colchicine or nocodazole treatment. 3. Taxol enhanced microtubule polymerization alone, and prevented microtubule depolymerization elicited by nocodazole and colchicine. Associated with its effect on microtubule assembly, taxol prevented the inhibitory effects of nocodazole and colchicine on cyclic GMP accumulation and iNOS mRNA levels. 4. Disruption of microfilaments by cytochalasins had no inhibitory effect on the activation of the inducible NO pathway. 5. In contrast to cytokine-stimulated smooth muscle cells, modulation of either microtubule or microfilament assembly did not affect the constitutive NO pathway in endothelial cells, as endothelial cell- and NO-dependent cyclic GMP accumulation in endothelial-smooth muscle co-cultures remained unchanged. 6. Our findings demonstrate that microtubules play a prominent role in the activation of the inducible NO pathway in response to inflammatory mediators in smooth muscle cells but not of the constitutive synthesis of NO in endothelial cells.

Mechanisms of tolerance to sodium nitroprusside in rat cultured aortic smooth muscle cells.

1. While exposure of smooth muscle cells to sodium nitroprusside (SNP) leads to the development of tolerance to soluble guanylate cyclase (sGC) activation, the mechanisms responsible for this phenomenon in intact cells remain unclear. In the present study, possible mechanisms of tolerance were investigated in a cell culture model where sGC activity was estimated from the accumulation of cyclic GMP in response to 10 microM SNP over a 15 min period in the presence of a phosphodiesterase (PDE) inhibitor. 2. Pretreatment of rat aortic smooth muscle cells with 10-500 microM SNP led to a dose-dependent downregulation of cyclic GMP accumulation upon subsequent SNP stimulation. This effect was evident as early as 2 h following incubation with 10 microM SNP, reached a plateau at 4 h and was blocked by co-incubation with 30 microM oxyhaemoglobin. 3. Pretreatment of smooth muscle cells with the PDE inhibitor, zaprinast, resulted in downregulation of the SNP-induced cyclic GMP accumulation in a time- and concentration-dependent manner, that was first evident after 12 h. Moreover, while the zaprinast-induced downregulation of cyclic GMP accumulation was completely inhibited by the protein kinase A (PKA) inhibitor, H89, tolerance to SNP was partially reversed by H89. 4. beta 1 sGC steady state mRNA levels of S-nitroso N-acetylpenicillamine (SNAP)- or 8Br-cyclic GMP-pretreated cells were unchanged, as indicated by Northern blot analysis. However, Western blot analysis revealed that alpha 1 protein levels were decreased in zaprinast, but not in SNP, SNAP or 8Br-cyclic GMP pretreated cells. 5. While thiol depletion did not prevent the development of tolerance, pretreatment of cells with SNP in the presence of reducing agents partially or completely restored the ability of cells to respond to SNP. 6. We conclude that tolerance to SNP results from two distinct mechanisms: an early onset, NO-mediated event that is reversed by reducing agents and a more delayed, PKA-sensitive process that is mediated through increases in cyclic GMP and a decrease in sGC protein levels.

Induction of nitric oxide synthase by protein synthesis inhibition in aortic smooth muscle cells.

1. The role of de novo protein synthesis in inducible NO synthase (iNOS) activation was investigated in vitro by evaluating the effects of protein synthesis inhibitors cycloheximide (CH) and anisomycin (ANI) on iNOS activity, protein and mRNA levels in rat aortic smooth muscle cells (RASMC). 2. As determined by cyclic GMP accumulation, substrate (L-arginine)- and inhibitor (N(G)-monomethyl-L-arginine, NMMA)-sensitive iNOS activity was significantly elevated in CH- or ANI-treated RASMC after 24 h. 3. Lipopolysaccharide (LPS) produced a time-dependent increase in cyclic GMP levels with maximal stimulation at 6 h and a decline to near baseline at 24 h. CH attenuated LPS-induced cyclic GMP accumulation at 3 and 6 h. However, cyclic GMP levels were superinduced at later times by CH. The concentration-dependence of cyclic GMP stimulation by cycloheximide was biphasic both in the absence and presence of LPS, with maximal stimulation at 10 microM and inhibition at higher concentrations. 4. Increased iNOS activity by CH was associated with elevated levels of immunoreactive iNOS protein as judged by Western blotting in LPS- and CH-treated cells. 5. CH-induced iNOS activity and superinduction of iNOS by CH in cells treated with LPS were both significantly inhibited by actinomycin D, a transcription inhibitor. 6. RT-PCR revealed elevated iNOS mRNA levels after 12 h of exposure to CH. The combination of LPS and CH caused a significant increase in iNOS gene expression relative to LPS- or CH stimulation alone. 7. These results show that partial protein synthesis inhibition by CH alone upregulates iNOS mRNA and superinduces iNOS mRNA in cytokine-treated RASMC, which is translated to the functional enzyme generating biologically active NO. Thus iNOS activation in these cells not only requires new protein synthesis but it also appears to be negatively regulated by newly synthesized proteins.

Regulation of the nitric oxide synthase-nitric oxide-cGMP pathway in rat mesenteric endothelial cells.

Most of the available data on the nitric oxide (NO) pathway in the vasculature is derived from studies performed with cells isolated from conduit arteries. We investigated the expression and regulation of components of the NO synthase (NOS)-NO-cGMP pathway in endothelial cells from the mesenteric vascular bed. Basally, or in response to bradykinin, cultured mesenteric endothelial cells (MEC) do not release NO and do not express endothelial NOS protein. MEC treated with cytokines, but not untreated cells, express inducible NOS (iNOS) mRNA and protein, increase nitrite release, and stimulate cGMP accumulation in reporter smooth muscle cells. Pretreatment of MEC with genistein abolished the cytokine-induced iNOS expression. On the other hand, exposure of MEC to the microtubule depolymerizing agent colchicine did not affect the cytokine-induced increase in nitrite formation and iNOS protein expression, whereas it inhibited the induction of iNOS in smooth muscle cells. Collectively, our findings demonstrate that MEC do not express endothelial NOS but respond to inflammatory stimuli by expressing iNOS, a process that is blocked by tyrosine kinase inhibition but not by microtubule depolymerization.

Infections associated with steroid use.

Patients receiving chronic steroids have an increased susceptibility to many different types of infections. The risk of infection is related to the dose of steroid and the duration of therapy. Although pyogenic bacteria are the most common pathogens, chronic steroid use increases the risk of infection with intracellular pathogens such as Listeria, many fungi, the herpes viruses, and certain parasites. Clinicians should consider both common and unusual opportunistic infections in patients receiving chronic steroids.