Association between intrauterine fetal growth restriction and ABO blood groups at King Abdulaziz University Hospital, Saudi Arabia: Retrospective study.

OBJECTIVES: To investigate the potential association between ABO blood groups and intrauterine fetal growth restriction (IUGR) among pregnant women who delivered at King Abdulaziz University Hospital (KAUH), Jeddah, Saudi Arabia. METHODS: This is a retrospective cohort study analyzed the medical records of pregnant women who delivered at KAUH and had postnatal follow-up visits. Missing data were completed by conducting phone interviews with patients. RESULTS: A total of 436 patients with a mean age of 31.2±5.5 years were included in the study. 50.7% of the women had blood type O, 28.4% had blood type A, 16.5% had blood type B, and 4.4% had blood type AB. The majority (94.7%) tested positive for the Rhesus antigen. Although statistically significant, women with blood groups AB and B exhibited higher rates of IUGR (31.6%, and 27.8%, respectively) compared to those with blood groups A and O, who had lower rates of IUGR (26.6%, and 24%, respectively). CONCLUSION: Our study results showed that women with blood groups AB and B had slightly higher rates of IUGR than those with A and O, who had lower rates of IUGR. A larger study comparing blood group O to other groups may provide more insight into the relationship between ABO blood groups and IUGR.

Psychometric properties of the Obstacles and Curb tests and their discriminative ability across functional levels in ambulatory children with spastic cerebral palsy.

The Obstacles and Curb tests are timed walking assessments that have emerged from the Spinal Cord Injury Functional Ambulation Profile and have been modified for children; however, their psychometric properties have not been adequately investigated. The aim of this research was to examine the psychometric properties of the Obstacles and Curb tests for children with cerebral palsy (CP). This cross-sectional study included 68 children aged 6-12 years; there were 34 children with CP and 34 age- and sex-matched typically developing children. Validity was examined by correlation with the 10-m Walk Test (10-MWT), Modified Time Up and Go test (mTUG), and Pediatric Balance Scale (PBS). Differences in the Obstacle and Curb test scores were calculated between children with CP and typically developing children and within different Gross Motor Function Classification System (GMFCS) levels. Children with CP completed the tests twice within a 30-min interval in the same session. The tests showed significant strong to very strong correlations with the 10-MWT, mTUG, and PBS. The within-session reliability was excellent, typically developing children were significantly faster than children with CP with high sensitivity and specificity, and the time differed significantly within the GMFCS level. Thus, the Obstacles and Curb tests can be considered valid, reliable, and sensitive walking tests for ambulatory children with CP.

Normative reference values for Obstacles Test and Curb Test and their correlation with demographic characteristics: a cross-sectional study in Saudi children.

The Obstacles Test and Curb Test have been used to measure gait speed and functional balance in adults. Recently, they have been modified for use in children but the normative values have not been established. This requires correlating the sex, age, height, weight, and BMI% of children with the test results and developing prediction equations. In this cross-sectional study, the Obstacles Test and Curb Test were administered to a convenience sample of 240 typically developing children aged 6-11 years. The factors associated with the time to complete each test were studied and prediction equations were established. The completion times were 5.27 ± 0.81 s for the Obstacles Test and 2.82 ± 0.45 s for the Curb Test. The Obstacles Test showed a fair negative relationship with height (Pearson's r = -0.41, P < 0.001), age ( r = -0.35, P < 0.001), and weight ( r = -0.32, P < 0.05). The Curb Test also had fair negative correlations with height ( r = -0.42, P < 0.001), age ( r = -0.39, P < 0.001), and weight ( r = -0.31, P < 0.001). Both tests showed poor correlations with the sex [eta ( η ) = 0.15 and 0.12, respectively]. Nonetheless, age and sex emerged as the main predictors of both test scores, accounting for 14% and 17% of the total variance in the Obstacles Test and Curb Test times, respectively. Normative values and prediction equations for both tests in typically developing children may be used for individual comparisons and in clinical research for the evaluation of interventions targeting disabled children.

Chorioamnionitis and Its Complications: A Cross-Sectional Study to Assess the Awareness of Married Women in Jeddah.

BACKGROUND:  Chorioamnionitis (CA) is a common pregnancy complication characterized by inflammation of the placental membranes and chorion. To our knowledge, there are limited studies evaluating the awareness of CA and its complications among women in Jeddah, Saudi Arabia. This study aimed to determine the awareness of married women in Jeddah toward CA and its complications. MATERIALS AND METHODS:  This cross-sectional study was conducted between March 2021 and August 2021. It involved 406 women who were or have been married in Jeddah, Saudi Arabia. Data were obtained via an online survey and analyzed using IBM SPSS Statistics version 24 (IBM Corp., Armonk, NY). Different statistical tests were used for data analysis, including percentages, mean, frequency, and chi-square. Content validity and reliability were checked. Based on a woman's knowledge score, the score was classified into three levels: good knowledge level (score: 9-12), fair knowledge level (score: 5-8), and poor knowledge level (score: 0-4). RESULTS: Of the total number of women who participated in the study, most of them had a poor knowledge score about CA complications (49.95%), and only 8.1% had good knowledge. Among the women, 25% had previously heard about CA, while only 2.5% were diagnosed with CA, and 50% of these women delivered by cesarean section. Analysis showed a significant relationship between women who had CA and their birth method (p = 0.000). However, there was a nonsignificant difference between the females' knowledge and their age (p = 0.297), or their level of education (p = 0.099). CONCLUSION: The study concluded that there was a poor level of knowledge regarding CA and its complications among women who experienced pregnancy.

The pattern and use of Twitter among dental schools in Saudi Arabia.

OBJECTIVE: Twitter as a social media platform has revolutionized the way we interact with others and receive information. The presence of dental schools in Twitter facilitates the engagement of students, educators, dental professionals, and the community. Given the explosive popularity of Twitter as a social media platform and its potential use in the areas of education and branding, the questions of why and how dental schools use these services warrant comprehensive research. Thus, the aim of this study was to analyze the pattern and use of Twitter as a social media platform for dental schools in Saudi Arabia. METHODS: The tweets were extracted within the timeframe from July 15, 2019, to July 15, 2020. The Twitter data collected included: full text content, the count of retweets, quotes, replies and likes. Extracted tweets were categorized into five main themes: news and announcement, dental professional communication, general communication, oral health education, and promoting participation. Tweets in each main theme were further categorized according to the dental schools' academic roles namely; education, research and community service. In addition, tweets were classified according to originality of the tweet, language used, nature of the tweet and the use of hashtags and mentions. Descriptive analysis presented in the form of frequency tables with percentages and mean (SD) as well as graphical presentation of the pattern and use of Twitter for Saudi dental schools in the form of bar, pie and line charts. Categorical data were analyzed using chi square test, while continuous data were analyzed using ANOVA. Statistical significance was set at p ≤ 0.05. RESULTS: A total of 15 Saudi dental schools with Twitter accounts were included in the analysis. King Saud University (KSU) had the largest number of followers with 17,200. Within the time frame of this study, a total of 1,889 original tweets from dental schools were found. Imam Abdulrahman Bin Faisal University (IAU) had the highest number of posted tweets (n = 647, 34.3%). The distribution of tweets was highest in September 2019 (n = 239) and lowest in July 2020 (n = 22). Majority of the tweets (81.9%) belonged to five out of the 15 dental schools. News and announcements were the most tweeted thematic subject with 1,034 tweets (55%). While community service was the most tweeted academic role with 803 tweets (42%). The top five active dental schools' performance for both thematic and academic role classifications were significantly different based on the chi square test (p < 0.001). CONCLUSION: This study highlights the importance of Twitter as a social media platform, in dental education especially when it comes to presence and branding for dental schools. Twitter is a helpful platform to expose dental schools to the community, this can be seen by their academic achievements as well as their active role with community service.

Development of a Novel Multi-Epitope Vaccine Candidate against Streptococcus Iniae Infection in Fish: An Immunoinformatics Study.

Streptococcus Iniae infection is recognized as a disease with substantial economic losses, infecting a wide range of fish species. The limitations of current vaccines and strategies have led to the identification of new methods to control this disease. Multi-epitope vaccines which employ various immunogenic proteins can be promising. The current research project aimed to design an efficient multi-epitope vaccine against Streptococcus Iniae infection in fish. To this end, six immunogenic proteins of Streptococcus Iniae, including FBA, ENO, Sip11, GAPDH, MtsB, and SCPI proteins, were applied for epitope prediction. The best B cell, T cell, and IFNγ epitopes of the immunogenic proteins, as well as interleukin-8, were used to construct a multi-epitope vaccine. Thereafter, different parameters of the designed vaccine, including physicochemical features, antigenicity, secondary structure, and tertiary structure, were evaluated. Moreover, the interaction of the interleukin-8 domain of the designed vaccine and its receptor was investigated by molecular docking strategy. Finally, nucleotide sequence of the vaccine was adapted to express in Escherichia coli. The results of the present study pointed out that the designed vaccine was a stable vaccine with molecular weight and antigenicity score of 45 kDa and 0.936, respectively. Furthermore, the structure analysis results revealed that the designed vaccine contained 23.49% alpha helix, with 90.5% residues in favored region. Finally, it was demonstrated that the interleukin-8 domain of the designed vaccine could be successfully docked to its receptor with the lowest energy of -1020.9. Based on the obtained results, it seems that the designed vaccine can be an efficient candidate to prevent Streptococcus Iniae infection in fish.

Efficacy and safety of paranasal sinus balloon catheter dilation in pediatric chronic rhinosinusitis: a systematic review.

OBJECTIVE: Chronic rhinosinusitis (CRS) negatively affects quality of life (QoL), and balloon catheter sinuplasty (BCS) has shown good outcomes in adult patients. However, there has not been much research on the effects of BCS on pediatric patients. The objective of this review is to systematically assess the literature for studies demonstrating the effectiveness and safety of BCS in pediatric CRS patients. DATA SOURCES: PubMed, Embase and Cochrane Library. STUDY SELECTION: We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis recommendations (PRISMA) to conduct our study. Observational- and interventional-based studies reporting efficacy and/or side effects of BCS among pediatric populations were included. Efficacy was evaluated by clinically reliable measures including Sino-Nasal 5 (SN-5) QoL scale. Antibiotic usage and revision surgery were also evaluated. DATA EXTRACTION: Articles were screened, and data were obtained. Study design, sample size and demographics, treated sinuses, criteria of inclusion, adjunct procedure(s), follow-up time, and outcomes measured were reported. MAIN FINDINGS: Out of 112 articles identified, 10 articles were included: two interventional controlled trials and eight observational studies. All studies evaluating QoL by SN-5 showed a remarkable reduction in SN-5 score postoperatively. Improvement in the computed tomography (CT) and endoscopic findings for up to 1 year after operation was reported. Furthermore, the majority of patinets treated with BCS did not recieve any course of sinusitis-indicated antibiotics during long-term follow-up, and they had low surgical revision rates. Minor side effects were reported, most commonly synechia. CONCLUSION: Available evidence suggests that BCS is safe and effective for the treatment of CRS in pediatric patients. Future randomized controlled studies with large sample size are warranted. Such studies can further determine the efficacy of BCS in managing children with CRS.

GnRH Antagonist Cetrorelix Administration Before hCG for Protection of Ovarian Hyperstimulation Syndrome.

OBJECTIVE: Studying the effect of GnRH antagonist administration on the day of hCG to cases of IVF/ICSI with estradiol level above 5000 ng/dl for protection of ovarian hyperstimulation syndrome. DESIGN: Prospective study. MATERIALS AND METHODS: Sixty patients undergoing controlled hyperstimulation COH, for IVF/ICSI using long agonist and E2 level on the day of hCG, are above 5000 ng/dl, 52 patients received single dose of cetrorelix 0.25 mg on the day of hCG, and 8 patients received two doses of 0.25 mg/day cetrorelix started one day before the day of hCG. RESULTS: There was no significant difference regarding patients BMI, number of stimulation days, recombinant FSH dose, and number of retrieved oocytes. Clinical pregnancy rate was 76.6% (46/60), in patients received single dose of antagonist PR were significantly higher 80.7% (42/52) versus 50% (4/8) in patients received two doses p = 0.047. Live birth rate was 50% (30/60), abortion rate was 20% (12/60), and preterm delivery was 20% (12/60). Mean E2 was 6853.2 ng/dl. Six patients developed moderate ovarian hyperstimulation OHSS (6/60) 10% and no cases of severe OHSS. CONCLUSIONS: GnRH antagonist administration on the day of hCG in cases undergoing IVF/ICSI with long agonist protocol is effective in protection of OHSS and does not affect the clinical pregnancy rate nor live birth rate.

Rise in Group W Meningococcal Carriage in University Students, United Kingdom.

MenACWY conjugate vaccination was recently introduced in the United Kingdom for adolescents and young adults to reduce disease from infection by Neisseria meningitidis group W. We conducted a cross-sectional meningococcal carriage study in first-year UK university students. Despite 71% MenACWY vaccine coverage, carriage of group W increased substantially.

Reliable collection of Caspian brown trout (Salmo trutta caspius) sperm using a catheter.

The traditional stripping procedure for collecting fish semen is associated with the risk of urine contamination, which may significantly affect semen quality and quantity. The use of a catheter as an alternative method for semen collection may overcome this problem. Therefore, this study compared Caspian brown trout (Salmo trutta caspius) semen parameters (i.e. sperm density, seminal plasma osmolality, motility parameters of spermatozoa analysed using computer-assisted sperm analysis and fertility) between the traditional stripping method and the use of a catheter. All parameter values of the semen collected with a catheter were significantly higher (p < .05; density = 7.67 ± 1.02 × 10(9)  ml(-1) and osmolality = 279.28 ± 32.84 mOsm kg(-1) ) than those collected with stripping method (density = 4.85 ± 0.47 × 10(9)  ml(-1) and osmolality = 216.42 ± 20.75 mOsm kg(-1) ). Semen collected with a catheter was characterized by higher spermatozoa motility compared with sperm collected via stripping. Similarly, the fertilization ability of sperm collected with a catheter was significantly greater (p < .05) than sperm collected with the traditional stripping method. In conclusion, collection of sperm with a catheter was shown to effectively reduce urine contamination and is therefore recommended for the collection of Caspian brown trout sperm.

Impact of human cytomegalovirus infection UL55-nested polymerase chain reaction method in hematopoietic stem cell transplant donors and recipients.

Human cytomegalovirus (HCMV) is one of the most important and critical viral causes of graft rejection among hematopoietic stem cell transplant (HSCT) recipients. Monitoring of this viral infection has a critical role in the management of HSCT clinical complications. In this retrospective cohort, blood (plasma and buffy coat) and urine samples were collected from 110 HSCT patients and 95 donors pretransplantation and weekly for 100 days posttransplantation. An HCMV-optimized UL55-nested polymerase chain reaction (PCR) method was used to detect HCMV infection. Genotyping of the HCMV UL55 gene was performed for all UL55-nested, PCR-positive samples. HSCT donor and recipient laboratory and clinical data were statistically analyzed using SPSS version 15 software. UL55-nested, PCR-positive results were obtained in 3540/4950 (71.5%), 3634/4950 (73.4%), and 3292/4950 (66.5%) of these plasma, buffy coat, and urine samples, respectively. Twenty-five percent of transplant donors were infected with HCMV. An increase in HCMV infection was observed from pre- to post-HSCT conditions. Detection of the gB2 UL55 genotype in most transplant patient samples suggested the need to examine the possible impact of HCMV UL55 genotypes and HCMV infections among stem cell transplant recipients.

NF-kappaB activation as a key mechanism in ethanol-induced disruption of the F-actin cytoskeleton and monolayer barrier integrity in intestinal epithelium.

Intestinal barrier disruption has been implicated in several intestinal and systemic disorders including alcoholic liver disease (ALD). Using monolayers of intestinal (Caco-2) cells, we showed that ethanol (EtOH) disrupts the barrier integrity via destabilization of the cytoskeleton. Because proinflammatory conditions are associated with activation of NF-kappa B (NF-kappaB), we hypothesized that EtOH induces disruption of cytoskeletal assembly and barrier integrity by activating NF-kappaB. Parental cells were pretreated with pharmacological modulators of NF-kappaB. Other cells were stably transfected with a dominant negative mutant for the NF-kappaB inhibitor, I-kappaBalpha. Monolayers of each cell type were exposed to EtOH and we then monitored monolayer barrier integrity (permeability); cytoskeletal stability and molecular dynamics (confocal microscopy and immunoblotting); intracellular levels of the I-kappaBalpha (immunoblotting); subcellular distribution and activity of NF-kappaB (immunoblotting and sensitive ELISA); and intracellular alterations in the 43kDa protein of the actin cytoskeleton, polymerized F-actin, and monomeric G-actin (SDS-PAGE fractionation). EtOH caused destabilizing alterations, including I-kappaBalpha degradation, NF-kappaB nuclear translocation, NF-kappaB subunit (p50 and p65) activation, actin disassembly (upward arrow G-, downward arrow F-), actin cytoskeleton instability, and barrier disruption. Inhibitors of NF-kappaB and stabilizers of I-kappaBalpha (e.g., MG-132, lactacystin, etc) prevented NF-kappaB activation while protecting against EtOH-induced injury. In transfected I-kappaBalpha mutant clones, stabilization of I-kappaBalpha to inactivate NF-kappaB protected against all measures of EtOH-induced injury. Our data support several novel mechanisms where NF-kappaB can affect the molecular dynamics of the F-actin cytoskeleton and intestinal barrier integrity under conditions of EtOH injury. (1) EtOH induces disruption of the F-actin cytoskeleton and of intestinal barrier integrity, in part, through I-kappaBalpha degradation and NF-kappaB activation; (2) The mechanism underlying this pathophysiological effect of the NF-kappaB appears to involve instability of the assembly of the subunit components of actin network.

Regulation of oxidant-induced intestinal permeability by metalloprotease-dependent epidermal growth factor receptor signaling.

Inflammatory bowel disease (IBD) affects more than 1 million Americans with more than 30,000 new cases diagnosed each year. IBD increases patient morbidity and susceptibility to colorectal cancer, yet its etiology remains unknown. Current models identify two key determinants of IBD pathogenesis: hyperpermeability of the gut epithelial barrier to bacterial products and an abnormal immune response to these products. Two factors seem critical for hyperpermeability: oxidant-induced stress and proinflammatory cytokines (e.g., tumor necrosis factor-alpha). The aim of this study was to investigate the role of oxidant stress-mediated transactivation of the epidermal growth factor receptor (EGFR) in intestinal hyperpermeability. This study used the Caco-2 human colonic epithelial cell in vitro model of intestinal epithelium. Cells were grown on inserts for permeability and signaling studies and glass coverslips for microscopy studies. show that oxidant-induced intestinal hyperpermeability can be blocked by specific inhibitors of the EGFR, tumor necrosis factor convertase (TACE) metalloprotease, transforming growth factor (TGF)-alpha, and mitogen-activated protein kinases, especially extracellular signal-regulated kinase 1/2. We also show that oxidant initiates these signaling events, in part by causing translocation of TACE to cell-cell contact zones. In this study, our data identify a novel mechanism for oxidant-induced intestinal hyperpermeability relevant to IBD. We propose a new intestinal permeability model in which oxidant transactivates EGFR signaling by activation of TACE and cleavage of precursor TGF-alpha. These data could have a significant effect on our view of IBD pathogenesis and provide new therapeutic targets for IBD treatment.

The role of protein kinase C isoforms in modulating injury and repair of the intestinal barrier.

Gastrointestinal cells express a diverse group of protein kinase C (PKC) isoforms that play critical roles in a number of cell functions, including intracellular signaling and barrier integrity. PKC isoforms expressed by gastrointestinal epithelial cells consist of three major PKC subfamilies: conventional isoforms (alpha, beta1, beta2, and gamma), novel isoforms (delta, epsilon, theta, eta, and mu), and atypical isoforms (lambda, tau, and zeta). This review highlights recent discoveries, including our own, that some PKC isoforms in gastrointestinal epithelia monolayer cell culture are involved in injury to, whereas others are involved in protection of, intestinal barrier integrity. For example, certain PKC isoforms aggravate oxidative damage, whereas others protect against it. These findings suggest that the development of agents that selectively activate or inhibit specific PKC isoforms may lead to new therapeutic modalities for important gastrointestinal disorders such as cancer and inflammatory bowel disease.

theta Isoform of protein kinase C alters barrier function in intestinal epithelium through modulation of distinct claudin isotypes: a novel mechanism for regulation of permeability.

Using monolayers of intestinal Caco-2 cells, we discovered that the isoform of protein kinase C (PKC), a member of the "novel" subfamily of PKC isoforms, is required for monolayer barrier function. However, the mechanisms underlying this novel effect remain largely unknown. Here, we sought to determine whether the mechanism by which PKC- disrupts monolayer permeability and dynamics in intestinal epithelium involves PKC--induced alterations in claudin isotypes. We used cell clones that we recently developed, clones that were transfected with varying levels of plasmid to either stably suppress endogenous PKC- activity (antisense, dominant-negative constructs) or to ectopically express PKC- activity (sense constructs). We then determined barrier function, claudin isotype integrity, PKC- subcellular activity, claudin isotype subcellular pools, and claudin phosphorylation. Antisense transfection to underexpress the PKC- led to monolayer instability as shown by reduced 1) endogenous PKC- activity, 2) claudin isotypes in the membrane and cytoskeletal pools ( downward arrowclaud-1, downward arrowclaud-4 assembly), 3) claudin isotype phosphorylation ( downward arrow phospho-serine, downward arrow phospho-threonine), 4) architectural stability of the claudin-1 and claudin-4 rings, and 5) monolayer barrier function. In these antisense clones, PKC- activity was also substantially reduced in the membrane and cytoskeletal cell fractions. In wild-type (WT) cells, PKC- (82 kDa) was both constitutively active and coassociated with claudin-1 (22 kDa) and claudin-4 (25 kDa), forming endogenous PKC-/claudin complexes. In a second series of studies, dominant-negative inhibition of the endogenous PKC- caused similar destabilizing effects on monolayer barrier dynamics, including claudin-1 and -4 hypophosphorylation, disassembly, and architectural instability as well as monolayer disruption. In a third series of studies, sense overexpression of the PKC- caused not only a mostly cytosolic distribution of this isoform (i.e., <12% in the membrane + cytoskeletal fractions, indicating PKC- inactivity) but also led to disruption of claudin assembly and barrier function of the monolayer. The conclusions of this study are that PKC- activity is required for normal claudin assembly and the integrity of the intestinal epithelial barrier. These effects of PKC- are mediated at the molecular level by changes in phosphorylation, membrane assembly, and/or organization of the subunit components of two barrier function proteins: claudin-1 and claudin-4 isotypes. The ability of PKC- to alter the dynamics of permeability protein claudins is a new function not previously ascribed to the novel subfamily of PKC isoforms.

Critical role of the atypical {lambda} isoform of protein kinase C (PKC-{lambda}) in oxidant-induced disruption of the microtubule cytoskeleton and barrier function of intestinal epithelium.

Oxidant injury to epithelial cells and gut barrier disruption are key factors in the pathogenesis of inflammatory bowel disease. Studying monolayers of intestinal (Caco-2) cells, we reported that oxidants disrupt the cytoskeleton and cause barrier dysfunction (hyperpermeability). Because the lambda isoform of protein kinase C (PKC-lambda), an atypical diacylglycerol-independent isozyme, is abundant in parental (wild type) Caco-2 cells and is translocated to the particulate fractions upon oxidant exposure, we hypothesized that PKC-lambda is critical to oxidative injury to the assembly and architecture of cytoskeleton and the intestinal barrier function. To this end, Caco-2 cells were transfected with an inducible plasmid, a tetracycline-responsive system, to create novel clones stably overexpressing native PKC-lambda. Other cells were transfected with a dominant-negative plasmid to stably inhibit the activity of native PKC-lambda. Cells were exposed to oxidant (H(2)O(2)) +/- modulators. Parental Caco-2 cells were treated similarly. We then monitored barrier function (fluorescein sulfonic acid clearance), microtubule cytoskeletal stability (confocal microscopy, immunoblotting), subcellular distribution of PKC-lambda (immunofluorescence, immunoblotting, immunoprecipitation), and PKC-lambda isoform activity (in vitro kinase assay). Monolayers were also processed to assess alterations in tubulin assembly, polymerized tubulin (S2, an index of cytoskeletal integrity), and monomeric tubulin (S1, an index of cytoskeletal disassembly) (polyacrylamide gel electrophoresis fractionation and immunoblotting. In parental cells, oxidant caused: 1) translocation of PKC-lambda from the cytosol to the particulate (membrane + cytoskeletal) fractions, 2) activation of native PKC-lambda, 3) tubulin pool instability (increased monomeric S1 and decreased polymerized S2), 4) disruption of cytoskeletal architecture, and 5) barrier dysfunction (hyperpermeability). In transfected clones, overexpression of the atypical (74 kDa) PKC-lambda isoform by itself ( approximately 3.2-fold increase) led to oxidant-like disruptive effects, including cytoskeletal and barrier hyperpermeability. Overexpressed PKC-lambda was mostly found in particulate cell fractions (with a smaller cytosolic distribution) indicating its activation. Disruption by PKC-lambda overexpression was also potentiated by oxidant challenge. Stable inactivation of endogenous PKC-lambda ( approximately 99.6%) by a dominant-negative protected against all measures of oxidant-induced disruption. We conclude that: 1) oxidant induces disruption of epithelial barrier integrity by disassembling the cytoskeleton, in large part, through the activation of PKC-lambda isoform; and 2) activation of PKC-lambda by itself appears to be sufficient for disruption of cellular cytoskeleton and monolayer barrier permeability. The unique ability to mediate an oxidant-like injury and cytoskeletal depolymerization and instability is a novel mechanism not previously attributed to the atypical subfamily of PKC isoforms.

Novel effect of NF-kappaB activation: carbonylation and nitration injury to cytoskeleton and disruption of monolayer barrier in intestinal epithelium.

Using monolayers of intestinal cells, we reported that upregulation of inducible nitric oxide synthase (iNOS) is required for oxidative injury and that activation of NF-kappaB is key to cytoskeletal instability. In the present study, we hypothesized that NF-kappaB activation is crucial to oxidant-induced iNOS upregulation and its injurious consequences: cytoskeletal oxidation and nitration and monolayer dysfunction. Wild-type (WT) cells were pretreated with inhibitors of NF-kappaB, with or without exposure to oxidant (H(2)O(2)). Other cells were transfected with an IkappaBalpha mutant (an inhibitor of NF-kappaB). Relative to WT cells exposed to vehicle, oxidant exposure caused increases in IkappaBalpha instability, NF-kappaB subunit activation, iNOS-related activity (NO, oxidative stress, tubulin nitration), microtubule disassembly and instability (increased monomeric and decreased polymeric tubulin), and monolayer disruption. Monolayers pretreated with NF-kappaB inhibitors (MG-132, lactacystin) were protected against oxidation, showing decreases in all measures of the NF-kappaB --> iNOS --> NO pathway. Dominant mutant stabilization of IkappaBalpha to inactivate NF-kappaB suppressed all measures of the iNOS/NO upregulation while protecting monolayers against oxidant insult. In these mutants, we found prevention of tubulin nitration and oxidation and enhancement of cytoskeletal and monolayer stability. We concluded that 1) NF-kappaB is required for oxidant-induced iNOS upregulation and for the consequent nitration and oxidation of cytoskeleton; 2) NF-kappaB activation causes cytoskeletal injury following upregulation of NO-driven processes; and 3) the molecular event underlying the destabilizing effects of NF-kappaB appears to be increases in carbonylation and nitrotyrosination of the subunit components of cytoskeleton. The ability to promote NO overproduction and cytoskeletal nitration/oxidation is a novel mechanism not previously attributed to NF-kappaB in cells.

Theta-isoform of PKC is required for alterations in cytoskeletal dynamics and barrier permeability in intestinal epithelium: a novel function for PKC-theta.

Using intestinal Caco-2 cells, we previously showed that assembly of cytoskeleton is required for monolayer barrier function, but the underlying mechanisms remain poorly understood. Because the theta-isoform of PKC is present in wild-type (WT) intestinal cells, we hypothesized that PKC-theta is crucial for changes in cytoskeletal and barrier dynamics. We have created the first multiple sets of gastrointestinal cell clones transfected with varying levels of cDNA to stably inhibit native PKC-theta (antisense, AS; dominant negative, DN) or to express its activity (sense). We studied transfected and WT Caco-2 cells. First, relative to WT cells, AS clones underexpressing PKC-theta showed monolayer injury as indicated by decreased native PKC-theta activity, reduced tubulin phosphorylation, increased tubulin disassembly (decreased polymerized and increased monomeric pools), reduced architectural integrity of microtubules, reduced stability of occludin, and increased barrier hyperpermeability. In these AS clones, PKC-theta was substantially reduced in the particulate fractions, indicating its inactivation. In WT cells, 82-kDa PKC-theta was constitutively active and coassociated with 50-kDa tubulin, forming an endogenous PKC-theta/tubulin complex. Second, DN transfection to inhibit the endogenous PKC-theta led to similar destabilizing effects on monolayers, including cytoskeletal hypophosphorylation, depolymerization, and instability as well as barrier disruption. Third, stable overexpression of PKC-theta led to a mostly cytosolic distribution of theta-isoform (<10% in particulate fractions), indicating its inactivation. In these sense clones, we also found disruption of occludin and microtubule assembly and increased barrier dysfunction. In conclusion, 1). PKC-theta isoform is required for changes in the cytoskeletal assembly and barrier permeability in intestinal monolayers, and 2). the molecular event underlying this novel biological effect of PKC-theta involves changes in phosphorylation and/or assembly of the subunit components of the cytoskeleton. The ability to alter the cytoskeletal and barrier dynamics is a unique function not previously attributed to PKC-theta.

Inhibition of oxidant-induced nuclear factor-kappaB activation and inhibitory-kappaBalpha degradation and instability of F-actin cytoskeletal dynamics and barrier function by epidermal growth factor: key role of phospholipase-gamma isoform.

Using monolayers of intestinal (Caco-2) cells as a model for studying inflammatory bowel disease (IBD), we previously showed that nuclear factor-kappaB (NF-kappaB) activation is required for oxidant-induced disruption of cytoskeletal and barrier integrity. Epidermal growth factor (EGF) stabilizes the F-actin cytoskeleton and protects against oxidant damage, but the mechanism remains unclear. We hypothesized that the mechanism involves activation of phospholipase C-gamma (PLC-gamma), which prevents NF-kappaB activation and the consequences of this activation, namely, cytoskeletal and barrier disruption. We studied wild-type and transfected cells. The latter were transfected with varying levels (1-5 microg) of cDNA to either stably overexpress PLC-gamma or to inhibit its activation. Cells were pretreated with EGF before exposure to oxidant (H(2)O(2)). Stably overexpressing PLC-gamma (+2.0-fold) or preincubating with EGF protected against oxidant injury as indicated by 1) decreases in several NF-kappaB-related variables [NF-kappaB (p50/p65 subunit) nuclear translocation, NF-kappaB subunit activity, inhibitory-kappaBalpha (I-kappaBalpha) phosphorylation and degradation]; 2) increases in F-actin and decreases in G-actin; 3) stabilization of the actin cytoskeletal architecture; and 4) enhancement of barrier function. Overexpression induced inactivation of NF-kappaB was potentiated by EGF. PLC-gamma was found mostly in membrane and cytoskeletal fractions (<9% in the cytosolic fractions), indicating its activation. Dominant negative inhibition of endogenous PLC-gamma (-99%) substantially prevented all measures of EGF protection against NF-kappaB activation. We concluded 1) EGF protects against oxidant-induced barrier disruption through PLC-gamma activation, which inactivates NF-kappaB; 2) Activation of PLC-gamma by itself is protective against NF-kappaB activation; 3) the ability to modulate the dynamics of NF-kappaB/I-kappa Balpha is a novel mechanism not previously attributed to the PLC family of isoforms in cells; and 4) development of PLC-gamma mimetics represents a possible new therapeutic strategy for IBD.

PKC-beta1 isoform activation is required for EGF-induced NF-kappaB inactivation and IkappaBalpha stabilization and protection of F-actin assembly and barrier function in enterocyte monolayers.

Using monolayers of intestinal Caco-2 cells, we reported that activation of NF-kappaB is required for oxidative disruption and that EGF protects against this injury but the mechanism remains unclear. Activation of the PKC-beta1 isoform is key to monolayer barrier integrity. We hypothesized that EGF-induced activation of PKC-beta1 prevents oxidant-induced activation of NF-kappaB and the consequences of NF-kappaB activation, F-actin, and barrier dysfunction. We used wild-type (WT) and transfected cells. The latter were transfected with varying levels of cDNA to overexpress or underexpress PKC-beta1. Cells were pretreated with EGF or PKC modulators +/- oxidant. Pretreatment with EGF protected monolayers by increasing native PKC-beta1 activity, decreasing IkappaBalpha phosphorylation/degradation, suppressing NF-kappaB activation (p50/p65 subunit nuclear translocation/activity), enhancing stable actin (increased F-actin-to-G-actin ratio), increasing stability of actin cytoskeleton, and reducing barrier hyperpermeability. Cells stably overexpressing PKC-beta1 were protected by low, previously nonprotective doses of EGF or modulators. In these clones, we found enhanced IkappaBalpha stabilization, NF-kappaB inactivation, actin stability, and barrier function. Low doses of the modulators led to increases in PKC-beta1 in the particulate fractions, indicating activation. Stably inhibiting endogenous PKC-beta1 substantially prevented all measures of EGF's protection against NF-kappaB activation. We conclude that EGF-mediated protection against oxidant disruption of the intestinal barrier function requires PKC-beta1 activation and NF-kappaB suppression. The molecular event underlying this unique effect of PKC-beta1 involves inhibition of phosphorylation and increases in stabilization of IkappaBalpha. The ability to inhibit the dynamics of NF-kappaB/IkappaBalpha and F-actin disassembly is a novel mechanism not previously attributed to the classic subfamily of PKC isoforms.