Incidence and prevalence of hepatitis B in patients with diabetes mellitus in the UK: A population-based cohort study using the UK Clinical Practice Research Datalink.

We assessed the incidence and prevalence of hepatitis B (Hep B) in patients with or without diabetes mellitus (DM) using the UK Clinical Practice Research Datalink (CPRD). This was a retrospective, observational study of diabetic and nondiabetic cohorts aged 0-80 years using CPRD (NCT02324218). Incidence rates (IR) for each cohort were calculated. Crude and adjusted (Poisson regression) IR ratios (IRR) were estimated with 95% confidence intervals (CI) to compare the cohorts. Hep B prevalence stratified by age, and hospitalization related to Hep B was also calculated. Of 7 712 043 subjects identified, 4 839 770 were included (DM: 160 760; non-DM: 4 679 010). Mean ages were 54.4 and 32.0 years, and 57.20% and 50.14% were male in the diabetic and nondiabetic cohorts, respectively. Hep B was identified in 29 diabetic and 845 nondiabetic subjects; IR was 4.03 per 100 000 person-years and 2.88 per 100 000 person-years, respectively. The adjusted IRR was 1.00 (95% CI: 0.70-1.50) between diabetic and nondiabetic cohorts. Hep B prevalence was higher in the diabetic cohort (0.01%-0.26%) than in the nondiabetic cohort (0.00%-0.03%) across the different age groups. Hep B-associated hospitalization IR was higher in the diabetic cohort (4.98-10.91) than the nondiabetic cohort (0.26-2.44). The Hep B IR, hospitalization and prevalence were higher in males in both cohorts. In conclusion, the risk of incident Hep B diagnosis in the diabetic cohort was not different from that in the nondiabetic cohort. However, prevalence of Hep B and Hep B-associated hospitalization rate was higher in the diabetic than in the nondiabetic cohort.

Randomised clinical trial: a placebo-controlled study of intravenous golimumab induction therapy for ulcerative colitis.

BACKGROUND: Tumour necrosis factor alpha (TNFα)-antagonism effectively treats ulcerative colitis (UC). The golimumab clinical programme evaluated subcutaneous (SC) and intravenous (IV) induction, and SC maintenance regimens, in TNFα-antagonist-naïve patients with moderate-to-severe active UC despite conventional treatment. AIM: To evaluate dose-response relationship, select IV golimumab induction doses for continued development, and evaluate the safety and efficacy of selected doses. METHODS: Adults with Mayo scores of 6-12 and endoscopic subscores ≥2 were enrolled into this multicentre, randomised, double-blind, placebo-controlled, integrated Phase 2/3 dose-finding/dose-confirming study. In Phase 2, 176 patients were randomised (1:1:1:1) to a single IV infusion of placebo, 1-, 2- or 4-mg/kg golimumab. While Phase 2 data were analysed to select doses for continued development, 71 additional patients were randomised. Phase 3 enrolment stopped after 44 additional patients were randomised (1:1:1) to placebo, 2- or 4-mg/kg golimumab. Due to insufficient power for the Phase 3 primary endpoint analysis (clinical response at week 6), efficacy analyses are considered exploratory and include all randomised patients. RESULTS: No dose-response was observed in Phase 2; however, higher serum golimumab exposure was associated with greater proportions of patients achieving more favourable clinical outcomes, clinical response and greater improvement in Mayo scores compared with placebo-treated patients and those with lower serum concentrations. Among all randomised patients, numerically greater proportions were in clinical response at week 6 in the 2- and 4-mg/kg golimumab groups compared with placebo [44.0% (33/75) and 41.6% (32/77) vs. 30.1% (22/73)]. CONCLUSIONS: Efficacy with single-dose golimumab IV induction was lower than expected and less than observed in the SC induction study. No new safety findings were observed. ClinicalTrials.gov Number, NCT00488774.

Systolic anterior motion causing hemodynamic instability and pulmonary edema during bleeding.

Systolic anterior motion (SAM) of mitral valve is the prolapse of a mitral leaflet into the left ventricle outflow tract (LVOT) during systole, causing LVOT obstruction and mitral valve regurgitation. We report the case of a patient who developed SAM-induced hemodynamic instability during bleeding with a clinical picture resembling pulmonary edema. A 77-year-old woman was admitted to our emergency room for abdominal bleeding in polycystic renal disease. Upon arrival, she was normotensive, despite being anuric and acidotic. After infusion of fluids and packed red blood cells (total 3 680 mL in 6 hours) she developed atrial fibrillation and clinical and radiological signs of pulmonary edema. Sedation and non-invasive ventilation brought to immediate severe hypotension. A transesophageal echocardiogram showed an "empty" hypertrophic hypercontractile left ventricle, SAM with LVOT obstruction (intraventricular gradient 154 mmHg) and moderate-to-severe mitral regurgitation. With further fluid infusion hemodynamic stability and sinus rhythm were recovered. SAM, LVOT obstruction and mitral regurgitation disappeared. SAM is a rare but dangerous cause of hemodynamic instability. It has been described in patients with and without left ventricular hypertrophy, in presence of hypovolemia and sympathetic stimulation. In our case it presented with a misleading clinical picture of pulmonary edema simulating fluid overload in an actually hypovolemic patient. In fact, SAM-associated mitral regurgitation together with diastolic dysfunction and tachycardia induced a pulmonary edema whose treatment worsened hypovolemia and precipitated LVOT obstruction and hypotension. Further fluid infusion was resolutive. Echocardiography was fundamental for diagnosis and treatment.

Mucosal gene signatures to predict response to infliximab in patients with ulcerative colitis.

BACKGROUND AND AIMS: Infliximab is an effective treatment for ulcerative colitis with over 60% of patients responding to treatment and up to 30% reaching remission. The mechanism of resistance to anti-tumour necrosis factor alpha (anti-TNFalpha) is unknown. This study used colonic mucosal gene expression to provide a predictive response signature for infliximab treatment in ulcerative colitis. METHODS: Two cohorts of patients who received their first treatment with infliximab for refractory ulcerative colitis were studied. Response to infliximab was defined as endoscopic and histological healing. Total RNA from pre-treatment colonic mucosal biopsies was analysed with Affymetrix Human Genome U133 Plus 2.0 Arrays. Quantitative RT-PCR was used to confirm microarray data. RESULTS: For predicting response to infliximab treatment, pre-treatment colonic mucosal expression profiles were compared for responders and non-responders. Comparative analysis identified 179 differentially expressed probe sets in cohort A and 361 in cohort B with an overlap of 74 probe sets, representing 53 known genes, between both analyses. Comparative analysis of both cohorts combined, yielded 212 differentially expressed probe sets. The top five differentially expressed genes in a combined analysis of both cohorts were osteoprotegerin, stanniocalcin-1, prostaglandin-endoperoxide synthase 2, interleukin 13 receptor alpha 2 and interleukin 11. All proteins encoded by these genes are involved in the adaptive immune response. These markers separated responders from non-responders with 95% sensitivity and 85% specificity. CONCLUSION: Gene array studies of ulcerative colitis mucosal biopsies identified predictive panels of genes for (non-)response to infliximab. Further study of the pathways involved should allow a better understanding of the mechanisms of resistance to infliximab therapy in ulcerative colitis. ClinicalTrials.gov number, NCT00639821.

Clinical trial: benefits and risks of immunomodulators and maintenance infliximab for IBD-subgroup analyses across four randomized trials.

BACKGROUND: Benefits and risks of concomitant immunomodulators and maintenance infliximab in inflammatory bowel disease (IBD) patients have not been adequately evaluated. AIM: To assess the effect of concomitant immunomodulator and infliximab maintenance therapy using data from four prospective, randomized Phase 3 trials in IBD patients. METHODS: Overall, 1383 patients from ACCENT I and ACCENT II [luminal and fistulizing Crohn's disease trials] and ACT 1 and ACT 2 [ulcerative colitis trials] were analysed. Patients were treated with placebo or infliximab 5 or 10 mg/kg at weeks 0, 2 and 6 followed by every-8-week maintenance therapy. Clinical response, clinical remission, fistula response, complete fistula response, infection and infusion reaction rates; serum infliximab concentrations and immunogenicity were summarized by baseline concomitant immunomodulator subgroup (use or non-use). RESULTS: Overall, almost 40% of evaluated IBD patients received concomitant immunomodulators. Efficacy, infection, and serious infection rates were generally similar in patients who received maintenance therapy with or without concomitant immunomodulators. There were no consistent differences in serum infliximab concentrations with or without immunomodulators in patients who received scheduled maintenance therapy. Concomitant immunomodulators reduced infusion reactions and immunogenicity. CONCLUSION: Concomitant immunomodulators did not improve efficacy or pharmacokinetics in IBD patients who received maintenance infliximab.

Good neurological recovery after cardiopulmonary resuscitation and thrombolysis in two old patients with pulmonary embolism.

The use of thrombolysis as an emergency treatment for cardiac arrest (CA) due to massive pulmonary embolism (MPE) has been described. However, there are no reports of successful treatment of MPE-associated CA in patients over 77 years of age. We report two cases of successful cardiopulmonary resuscitation for an MPE-associated CA in two very old women (87 and 86 years of age). In both cases, typical signs of MPE were documented using emergency echocardiography, which showed an acute right ventricle enlargement and a paradoxical movement of the interventricular septum. Emergency thrombolysis was administered during resuscitation, which lasted 45 and 21 min, respectively. Despite old age and prolonged resuscitation efforts, both patients had good neurological recovery and one of them was alive and neurologically intact 1 year later. Thrombolysis is a potentially useful therapy in MPE-associated CA. A good neurological outcome can be obtained even in very old patients and after prolonged resuscitation.

A histological investigation of the occurrence of non-reproductive female bluefin tuna Thunnus thynnus in the Mediterranean Sea.

The presence of non-reproductive Atlantic bluefin tuna Thunnus thynnus females in the Mediterranean Sea was investigated through histological analysis of the gonads. Three hundred and twenty-six ovary samples were collected from adults captured at different locations in the Mediterranean Sea during the reproductive seasons between 1998 and 2008. Only three specimens were considered to be in a non-reproductive state: two of them were in a reabsorbing state showing ovaries with early vitellogenic oocytes and extensive alpha and beta atresia of vitellogenic follicles; the third showed gonads with perinucleolar oocytes and was considered to be in a resting state. The low occurrence of non-reproductive individuals found in this study makes it unlikely that non-reproductive individuals aggregate with reproductive ones during their migration towards spawning grounds. Further research is suggested in order to investigate the potential presence of non-reproductive individuals on non-spawning grounds during the reproductive season.

A family-based study does not confirm the association of MYO9B with celiac disease in the Italian population.

Association between Myosin IXB (MYO9B) gene polymorphisms and celiac disease (CD) was recently detected by a case-control association study in the Dutch, but not confirmed in the British and Swedish/Norwegian populations. We tested the association between CD and the three most associated single nucleotide polymorphisms (SNPs) in the Dutch study by the transmission disequilibrium test in the Italian population. A total of 252 pediatric patients and 504 parents were genotyped. No transmission distortion was detected either for the single SNPs or for their haplotypic combinations. Control allele frequencies, calculated from untransmitted alleles, were significantly different from those of the Dutch control population. Conversely, allele frequencies were very similar in Italian, British, Swedish/Norwegian and Dutch patients. In conclusion, MYO9B is not involved in CD susceptibility in the Italian population. The difference with the Dutch result might be explained by an imperfect selection of the Dutch controls.

Trace elements in loggerhead turtles (Caretta caretta) from the eastern Mediterranean Sea: overview and evaluation.

Concentrations of trace elements (Hg, Cd, Pb, Zn, Cu, Fe, and Se) in different organs and tissues (liver, kidney, muscle tissue, spleen, heart, lung, and fat tissue) of loggerhead turtles Caretta caretta from eastern Mediterranean Sea were determined. The highest levels of mercury and cadmium were found in liver (Hg: 0.43 microg g(-1) wet weight; Cd: 3.36 microg g(-1) wet weight) and kidney (Hg: 0.16 microg g(-1) wet weight; Cd: 8.35 microg g(-1) wet weight). For lead the overall concentrations were low and often below the limit of detection. Copper and selenium tended to be higher in liver than in other tissues and organs, while for zinc the concentrations were quite homogenous in the different organs and tissues, except fat tissue (64.7 microg g(-1) wet weight) which showed a higher accumulation of this element. For iron the greatest concentrations were observed in liver (409 microg g(-1) wet weight) and spleen (221 microg g(-1) wet weight).

Phorbol ester treatment increases paracellular permeability across IEC-18 gastrointestinal epithelium in vitro.

The phorbol ester, TPA, transiently increases the transepithelial permeability across the gastrointestinal epithelium formed by IEC-18. There was a significant decrease in transepithelial resistance (R(T)) between 0 and 1.5 hr, accompanied by increased flux of polyethylene glycol (4000 MW), suggesting that the increase was across the tight junction. By 2 hr, the decrease in R(T) reversed and maintained control level. The transepithelial permeability increase was prevented by coincubation with the protein kinase C (PKC) inhibitor bisindolylmaleimide. There was a rapid (within 15 min) translocation of PKC-alpha from the cytosolic to the "membrane-associated" compartment, followed by a down-regulation that was detectable within 60 min of TPA treatment. The down-regulation of PKC-alpha from the membrane was prevented by either calpain inhibitor I or MG-132 and resulted in a sustained permeability increase. The permeability changes were not accompanied by significant effects on the amount or localization of the tight junctional proteins, occludin and ZO-1. However, occludin did show a reversible increase in phosphorylation with TPA treatment. Together these data support a role for PKC-alpha-mediated regulation of barrier permeability in an in vitro model of small intestinal epithelium, perhaps through modulation of the phosphorylation state of the tight junctional protein, occludin.

Plasma concentrations of soluble tumor necrosis factor receptor I and tumor necrosis factor during cardiopulmonary bypass.

BACKGROUND: Tumor necrosis factor-alpha (TNF) has been implicated in the development of postoperative morbidity after cardiopulmonary bypass for myocardial revascularization. Despite their postulated roles as modulators of TNF bioavailability, soluble TNF receptors have not been characterized in patients undergoing this procedure and is the focus of this study. METHODS: Soluble tumor necrosis factor receptor I (sTNFRI) and TNF were measured by immunoassay in plasma samples collected from 36 patients at events before, during, and after cardiopulmonary bypass. RESULTS: Plasma concentrations of sTNFRI averaged 1.39 ng/mL at the start of the operation. Preoperative sTNFRI concentrations were found to significantly correlate with a preoperative morbidity assessment score, age, duration of bypass, duration of supplemental oxygen, and length of hospital stay. Plasma sTNFRI increased in all of the patients during the procedure. Plasma concentrations of sTNFRI and TNF did not correlate at any time. CONCLUSIONS: Preoperative measurement of sTNFRI could potentially serve as a reliable indicator for prophylactic treatment with an anti-TNF therapy. Such a therapeutic approach might help attenuate inflammatory processes thought to underlie postoperative morbidity associated with cardiopulmonary bypass.

Modification of tight junction function by protein kinase C isoforms.

The regulation of tight junction permeability by a variety of signal transduction pathways is summarized. An emphasis is placed on regulation of paracellular permeability by the protein kinase C family of isoforms, which involves the reporting of a large number of studies using the phorbol ester family of protein kinase C activators. The ability of protein kinase C activation to open epithelial barriers to a very wide range of solutes is emphasized, but then countered with discussion of the role of phorbol esters and protein kinase C activation in epithelial carcinogenesis. The ability of protein kinase C activation to enable growth factors to leak from luminal fluid compartments of epithelial tissues into lateral intercellular and interstitial fluid spaces may play a role in this carcinogenic action. An examination of protein kinase C effects on the phosphorylation states of tight junctional proteins suggests that downstream kinases and/or phosphatases mediate protein kinase C's effect on tight junction permeability. A role for protein kinase C in transepithelial drug delivery is questioned herein. The tight junctional leakiness associated with protein kinase C activation and apparently intrinsic to transformed epithelia suggests a potentially useful role for tight junction leakiness as a marker for early cancer diagnosis.

Increased tight junction permeability can result from protein kinase C activation/translocation and act as a tumor promotional event in epithelial cancers.

Exposure of LLC-PK1 epithelial cell cultures to phorbol ester tumor promoters causes immediate translocation of protein kinase C-alpha (PKC-alpha) from cytosolic to membrane-associated compartments. With a very similar time course, a dramatic and sustained increase in tight junctional (paracellular) permeability occurs. This increased permeability extends not only to salts and sugars but macromolecules as well. Fortyfold increases of transepithelial fluxes of biologically active EGF and insulin occur. Recovery of tight junction barrier function coincides with proteasomal downregulation of PKC-alpha. The failure to downregulate activated membrane-associated PKC-alpha has correlated with the appearance of multilayered cell growth and persistent leakiness of tight junctions. Accelerated downregulation of PKC-alpha results in only a partial and transient increase in tight junction permeability. Transfection of a dominant/negative PKC-alpha results in a slower increase in tight junction permeability in response to phorbol esters. In a separate study using rat colon, dimethylhydrazine (DMH)-induced colon carcinogenesis has been preceded by linear increases in both the number of aberrant crypts and transepithelial permeability, as a function of weeks of DMH treatment. Adenocarcinomas of both rat and human colon have been found to have uniformly leaky tight junctions. Whereas most human colon hyperplastic and adenomatous polyps contain nonleaky tight junctions, adenomatous polyps with dysplastic changes did possess leaky tight junctions. Our overall hypothesis is that tight junctional leakiness is a late event in epithelial carcinogenesis but will allow for growth factors in luminal fluid compartments to enter the intercellular and interstitial fluid spaces for the first time, binding to receptors that are located on only the basal-lateral cell surface, and causing changes in epithelial cell kinetics. Tight junctional leakiness is therefore a promotional event that would be unique to epithelial cancers.

Activation of NF-kappaB is necessary for the restoration of the barrier function of an epithelium undergoing TNF-alpha-induced apoptosis.

Tumor necrosis factor-alpha (TNF) induces apoptosis in confluent LLC-PK1 epithelial cells, but also activates NF-kappaB, a negative regulator of apoptosis. The presence of increased TNF-induced apoptosis causes a transient increase in epithelial permeability, but the epithelial barrier function recovers, as assessed by measuring the transepithelial electrical resistance, the paracellular flux of mannitol and by the electron microscopic evaluation of the penetration of the electron-dense dye ruthenium red across the tight junctions. The integrity of the epithelial cell layer is maintained by rearrangement of non-apoptotic cells in the monolayer and by the phagocytosis of apoptotic fragments. To study the role of NF-kappaB in an epithelium exposed to TNF, NF-kappaB was inhibited in LLC-PK1 epithelial cells with either the dietary compound, curcumin, or by transfection with a dominant negative mutant inhibitor I kappaB alpha. Replacement of serine 32 and 36 by alanine has been shown to prevent its phosphorylation and degradation, blocking NF-kappaB activation. Inhibition of NF-kappaB altered the morphology of TNF-induced apoptotic cells, which showed lack of fragmentation and membrane blebbings, and absence of phagocytosis by neighboring cells. TNF treatment of NF-kappaB-inhibited cells also caused altered distribution of the tight junction-associated protein ZO-1, increased epithelial leakiness, and impaired the recovery of the epithelial barrier function, which normally occurs 6 hours after TNF treatment of LLC-PK1 cells. These data demonstrate that NF-kappaB activation is required for the maintenance of the barrier function of an epithelium undergoing TNF-induced apoptosis.

Tumor necrosis factor-alpha increases sodium and chloride conductance across the tight junction of CACO-2 BBE, a human intestinal epithelial cell line.

CACO-2 BBE was used to determine the response of a gastrointestinal epithelium to tumor necrosis factor-alpha (TNF). Incubation of CACO-2 BBE with TNF did not produce any effect on transepithelial resistance (TER) within the first 6 hr but resulted in a 40-50% reduction in TER and a 30% decrease in 1SC (short circuit current) relative to time-matched control at 24 hr. The decrease in TER was sustained up to 1 week following treatment with TNF and was not associated with a significant increase in the transepithelial flux of [14C]-D-mannitol or the penetration of ruthenium red into the lateral intercellular space. Dilution potential and transepithelial 22Na+ flux studies demonstrated that TNF-treatment of CACO-2 BBE cell sheets increased the paracellular permeability of the epithelium to Na+ and Cl-. The increased transepithelial permeability did not associate with an increase in the incidence of apoptosis. However, there was a TNF-dependent increase in [3H]-thymidine labeling that was not accompanied by a change in DNA content of the cell sheet. The increase in transepithelial permeability was concluded to be across the tight junction because: (i) 1 mM apical amiloride reduced the basolateral to apical flux of 22Na+, and (ii) dilution potential studies revealed a bidirectionally increased permeability to both Na+ and Cl-. These data suggest that the increase in transepithelial permeability across TNF-treated CACO-2 BBE cell sheets arises from an alteration in the charge selectivity of the paracellular conductive pathway that is not accompanied by a change in its size selectivity.

Different size limitations for increased transepithelial paracellular solute flux across phorbol ester and tumor necrosis factor-treated epithelial cell sheets.

By observing increases in the transepithelial paracellular permeability of a range of radiolabeled solutes and electron dense dyes, changes in molecular sieving caused by the cytokine, TNF (tumor necrosis factor), and the phorbol ester, TPA (12-0-tetra-decanoylphorbol-13-acetate), were characterized. Using 14C-labeled mannitol (mw 182), raffinose (mw 504), PEG (polyethylene glycol; mw 4000), and dextran (mw 10,000, 70,000 and 2,000,000), the transepithelial flux rates of these compounds were determined at the peak of the transepithelial electrical resistance (TER) changes caused by these two agents. TNF treatment resulted in increased permeability across LLC-PK1 epithelial cell sheets only to relatively small solutes, with an upper limit of approximately 4,000 mw. The low molecular weight "ceiling" for the TNF-treated epithelium is further evidence against TNF increasing transepithelial permeability by means of inducing nonspecific, microscopic "holes" in the epithelium, for which a "ceiling" would not exist. TPA treatment increases transepithelial paracellular permeability to a much broader range of solutes, extending well beyond 2 million mw. Transmission electron micrographs provide evidence that even the electron-dense dye complex, ruthenium red, can cross tight junctions of TPA-treated cell sheets. However, cationic ferritin cannot cross tight junctions of TPA-treated cell sheets. This shows that there is an upper limit to solutes able to cross TPA-treated cell sheets, but that this upper limit will include most proteins, which would then be able to cross tumor promoter-exposed (protein kinase C-activated) epithelial layers at accelerated rates. The biomedical implications for a high molecular weight cutoff in tumor promoter action in epithelial carcinogenesis, and for a low molecular weight cutoff in cytokine-induced epithelial apoptosis in inflammation, are discussed.

Tissue remodeling during tumor necrosis factor-induced apoptosis in LLC-PK1 renal epithelial cells.

The cytokine tumor necrosis factor-alpha (TNF) increases the frequency of apoptosis in confluent renal epithelial LLC-PK1 cells, an effect that can be blocked by an anti-TNFR1 monoclonal antibody. However, there were no visible "holes" in the cell sheet as a result of TNF-induced apoptosis. Instead a striking tissue remodeling occurred in response to the TNF-induced apoptosis. Apoptotic cells became surrounded and engulfed by repositioned neighboring cells distributed in a distinct "rosette" pattern. The cadherin-catenin cell-cell adhesion molecules, the tight junction-associated protein ZO-1, and actin accumulated at the sites of contact between apoptotic and neighboring cells. Pretreatment with cytochalasin B prevented the accumulation of cadherins-catenins and ZO-1 at the sites of apoptosis and resulted in microscopic holes in the TNF-treated cell sheet. Our results indicate that a renal epithelium can accommodate an increased frequency of apoptosis and still maintain its integrity by mechanisms of tissue remodeling involving the cadherin-catenin adhesion molecules, tight junctional proteins, and actin filaments.

Sodium-independent carrier-mediated inositol transport in cultured renal epithelial (LLC-PK1) cells.

In addition to the concentrative, Na(+)-dependent inositol transport system demonstrated in many cell types, carrier-mediated, Na(+)-independent inositol transport is also shown to exist in LLC-PK1 renal epithelia. Inhibition of inositol uptake in Na(+)-free saline by 0.1 mM phloretin, and self-inhibition by net concentrations of inositol exceeding 10 mM, demonstrate the carrier-mediation of the Na(+)-independent uptake and distinguish it from flux through anion channels. The Na(+)-dependent uptake exhibits higher affinity for inositol, as seen by the stronger self-inhibition at lower inositol concentrations in Na+ saline. Kinetic analyses indicate a Km of 178 microM and a Vmax of 2447 pmol/min per microgram DNA for the Na(+)-dependent system, whereas the lower affinity, lower capacity Na(+)-independent system manifests a Km of 5.2 mM and a Vmax of 249 pmol/min per microgram DNA. the Na(+)-independent uptake further differs from the Na(+)-dependent transport by the lack of inhibitory effect of 10 microM glucose, and the greater relative inhibition of phloretin compared to that of phlorizin. Both types of uptake appear to localize predominantly to the basal-lateral cell surface. The Na(+)-independent transport is bidirectional, functioning in efflux as well as influx of inositol.

The protein kinase C inhibitor, bisindolylmaleimide, inhibits the TPA-induced but not the TNF-induced increase in LLC-PK1 transepithelial permeability.

The transepithelial paracellular permeability of an epithelium formed by LLC-PK1 cells increases upon activation of protein kinase C (PKC) by the phorbol ester tumor promoter, TPA, or in response to the cytokine tumor necrosis factor-alpha (TNF). Until recently, however, we have not been able to inhibit the permeability effects of TPA or TNF using any of the currently available serine-threonine kinase inhibitors. In this study we report the treatment of epithelial cell sheets with the selective PKC inhibitor bisindolylmaleimide, GF109203X, completely prevents the TPA-induced but not the TNF-alpha induced increase in tight junction permeability. While PKC-alpha still translocates from the cytosol to the membrane of TPA-stimulated epithelial cells overall PKC activity in the membrane fraction is markedly reduced in the presence of GFX.

cAMP modulates transepithelial resistance response of LLC-PK1 renal epithelia to tumor necrosis factor.

For "leaky" epithelia the transepithelial resistance (Rt) is an electrophysiological measure of the paracellular pathway within the epithelial barrier. The Rt across a monolayer of LLC-PK1 porcine renal epithelial cells is specifically an inverse measure of paracellular transepithelial permeability and displays a multiphasic and reversible response to the cytokine tumor necrosis factor-alpha (TNF). The Rt response to TNF can be inhibited by the nonhydrolyzable adenosine 3',5'-cyclic monophosphate (cAMP) analogue, dibutyryl-cAMP. In addition, activation of adenylate cyclase (forskolin) or inhibition of phosphodiesterase (3-isobutyl-1-methylxanthine, Ro-20-1724, and pentoxifylline), each of which have been reported to elevate cellular cAMP levels, also inhibited the Rt response to TNF. Incubation of the LLC-PK1 cell sheet with N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide, an inhibitor of cAMP-dependent protein kinase (PKA), potentiated the Rt response to TNF. The Rt response to TNF was completely prevented by preincubation of the cultures with cholera toxin, whereas pertussis toxin pretreatment had a slight but significant potentiating effect on the response. Pretreatment with cholera toxin was associated with an approximately 18-fold elevation in cAMP levels in both control and TNF-treated cultures. Measurements of cellular cAMP content at selected intervals after TNF administration showed a significant elevation (P < 0.01) of 140% above time-matched controls at 1 h after the administration of TNF to the cell sheet. The level of cAMP then declined to approximate control level within 2.5 h of TNF administration.(ABSTRACT TRUNCATED AT 250 WORDS)