Shear stress inhibition of H<Subscript>2</Subscript>O<Subscript>2</Subscript> induced p66<Superscript>Shc</Superscript> phosphorylation by ASK1–JNK inactivation in endothelium

Shear stress protects endothelium from a variety of risk factors for vascular disease. Here, we demonstrate a novel mechanism whereby shear stress inhibited reactive oxygen species (ROS)-triggered signaling cascades in endothelial cells. Stimulation of bovine aortic endothelial cells (BAECs) with H₂O₂ induced a 3.07-fold increase in p66^Shc phosphorylation. This response was fully blocked by pretreatment of cells with specific JNK but not p38 or ERK MAP kinase inhibitor. Further study showed that knocking down of apoptosis signal-regulating kinase 1 (ASK1) by siRNA transfection in cells dramatically inhibited phosphorylation of JNK and p66^Shc elicited by H₂O₂. Pre-perfusion of BAECs cultured in silastic tubes with laminar flow generated by a servo-pump system for 30 min also significantly suppressed H₂O₂-induced phosphorylation of p66^Shc. This was accompanied by quantitatively similar inhibition of ASK1 and JNK phosphorylation and activation. These results suggested that shear stress protects endothelium against oxidant stress by suppression of ASK1–JNK-mediated p66^Shc phosphorylation.     

HERG K<Superscript>+</Superscript> channel expression in CD34<Superscript>+</Superscript>/CD38<Superscript>−</Superscript>/CD123<Superscript>high</Superscript> cells and primary leukemia cells and analysis of its regulation in leukemia cells

The human ether-a-go-go-related (herg) gene encoding K⁺ channels (HERG) belongs to an evolutionarily conserved multigene family of voltage activated K⁺ channels. The functional properties of HERG K⁺ channels are complex and their contribution to the repolarization of the cardiac action potential are well understood. Recent studies revealed that HERG K⁺ channels are preferentially expressed in different histogenesis of tumor cells. Leukemia is a cancer that originates in the bone marrow hematopoietic stem cells (HSCs). Leukemia stem cells (LSCs) are critical in the perpetuation of the disease. A better understanding of LSCs and molecular biology will allow the design of more effective therapies. We report in this study that herg was expressed in CD34⁺/CD38⁻/CD123^high LSCs but not expressed in normal bone marrow CD34⁺/CD38⁻ HSCs. In addtion, herg is also expressed in leukemia cell lines K562 and HL-60 and almost all the primary leukemia cells whereas not in the normal bone marrow cells. In addition, the expression of herg mRNA was not associated with the clinical and cytogenetic feature of leukemia. Moreover, HERG K⁺ channels can regulate leukemia cells proliferation and cell cycle. These data provide evidence for the oncogenic potential of HERG K⁺ channels and it may be a novel, potential pharmacological target for leukemia therapy in the future.     

Adsorptive Depletion of α4 Integrin<Superscript>hi</Superscript>- and CX<Subscript>3</Subscript>CR1<Superscript>hi</Superscript>-Expressing Proinflammatory Monocytes in Patients with Ulcerative Colitis

Background  

Two main functionally distinct monocytes phenotypes are known: the CD14^hiCD16⁻ “classical” and the CD14⁺CD16⁺ “proinflammatory” phenotypes. The latter phenotype is elevated in patients with ulcerative colitis (UC) and is suspected to have a major role in the immunopathogenesis of UC.     

Comparison of serological markers between ACPA<Superscript>+</Superscript> and ACPA<Superscript>−</Superscript> of RA patients

Anti-citrullinated protein/peptide antibodies (ACPA) have recently emerged as sensitive and specific serological markers of rheumatoid arthritis (RA), providing superior alternative of the rheumatoid factor (RF) test in the laboratory diagnostics of RA. We compare the change of serum RF, CRP, IgG, IgM, IgA, total complement, C3 and C4. The sera sample was collected from 123 patients with RA. ACPA were detected with ELISA, and RF, CRP and total complement (Ct), C3 and C4 were examined by automatic biochemical analyzer. Serum RF and total complement concentrations were significantly higher in ACPA+ than in ACPA−, but there were no correlation between ACPA and RF and Ct. Between ACPA+ and ACPA−, there were no significant difference of CRP, IgG, IgM, IgA, total complement, C3 and C4. While there were significant correlation between the concentration of C3 and IgM and ACPA in ACPA+. Conclusion: This is the first study to show that ACPA concentration in ACPA+ patients with RA is positively related to serum IgM and C3 levels.     

Marginal expression of CXCR4 on c-kit<Superscript>+</Superscript>Sca-1<Superscript>+</Superscript>Lineage<Superscript>?</Superscript> hematopoietic stem/progenitor cells

Stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 are the key regulatory molecules of hematopoietic stem cell (HSC) migration and engraftment to the bone marrow (BM) microenvironment. However, the significance of the ligand–receptor complex on HSC in steady-state BM is not clear. There is currently a lack of information as to how CXCR4 is expressed on HSCs. We herein demonstrate that c-kit⁺Sca-1⁺Lineage⁻ (KSL) cells freshly isolated from BM expressed very low to undetectable levels of CXCR4. Two hours of incubation at 37°C quickly up-modulated the receptor expression on KSL cells. Protein synthesis was not required for this early stage up-regulation, thus suggesting the emergence of intracellularly pooled receptors to the cell surface. However, protein synthesis was involved at the later stage of up-regulation. The up-regulated CXCR4 was functional, as evidenced by the fact that the incubated KSL cells more efficiently migrated to the SDF-1 gradient in vitro. Therefore, although KSL cells are able to express functional CXCR4, the receptors are only marginally expressed in the steady-state BM microenvironment. These observations therefore indicate the limited role of the SDF-1-CXCR4 axis on HSC functionality in a static BM environment.     

Val/Leu<Superscript>247</Superscript> and Trp/Ser<Superscript>316</Superscript> polymorphisms in β<Subscript>2</Subscript> glycoprotein I and their association with thrombosis in unselected Chilean patients

It is known that polymorphisms of β ₂-glycoprotein I (β ₂GPI) in exon 7 affect interaction between the phospholipid binding site and the antibodies, and that other polymorphisms in exon 8 increase the generation of antibodies. In this study, we analyzed genetic polymorphisms of β ₂GPI in unselected Chilean patients to determine the prevalence of β ₂GPI polymorphisms in the phospholipid domain in patients with venous and arterial thrombosis and the clinical correlation with thromboembolic complications. This study comprised 149 patients with venous and arterial thrombosis (62 with venous thrombosis and 87 with arterial thrombosis) and 160 healthy controls with no previous history of thrombosis. Polymorphisms of exons 7 and 8 of β ₂GPI, which encode for its fifth domain, were determined by PCR-RFLP. The presence of aPL or anti-β ₂GPI in the patients was detected by ELISA. Anti-β ₂GPI were present in 8/149 patients (5.4%); of these, five had aCL antibodies of low titer. The allele containing Val/Leu²⁴⁷ and Trp/Ser³¹⁶ was significantly more frequent in patients with thrombosis than in the control group (OR=3.1, CI 1.6–6.0, p=0.0003; OR=2.9, CI 1.1–8.6, p=0.027, respectively). These polymorphisms did not correlate with aPL or anti-β ₂GPI but significant differences were observed with venous thrombosis (p=<0.0001) and arterial thrombosis (p=0.026). In conclusion, the β ₂GPI polymorphisms Val/Leu²⁴⁷ and Trp/Ser³¹⁶ are not related to the presence of anti-β ₂GPI antibodies in unselected Chilean patients with venous and arterial thrombosis, but they are significantly associated with venous and arterial thrombosis.     

α<Subscript>1</Subscript>-Antitrypsinmangel

α₁-antitrypsin deficiency is characterized by a pathologic reduction of the serum concentration of α₁-antitrypsin, the most important antiprotease in man. It is one of the most common hereditary diseases in Caucasians. Approximately 2% of obstructive airway diseases are caused by α₁-antitrypsin deficiency. Patients above 35 years may develop lung emphysema, especially in the lower lobes. Symptoms are those of chronic obstructive pulmonary disease such as cough, sputum expectoration, and progressive dyspnoea. Patients with homozygous defect often develop cholestatic hepatitis in the neonatal period. However, only few adult patients develop chronic liver disease up to liver cirrhosis with an elevated risk for malignant liver tumors. The diagnostic hallmark is the reduced serum concentration of α₁-antitrypsin while genetic testing proves the defect. An early recognition of the disease is decisive for prophylactic and therapeutic measures. Smoking should be stopped immediately. Treatment of lung disease includes physiotherapy, antiobstructive and antiinflammatory medication, augmentation with human α₁-antitrypsin and lung surgery including lung transplantation. Liver toxins should be avoided. Besides experimental therapeutic approaches, liver disease can only be treated by liver transplantation.     

Pharmacological Stimulation of β <Subscript>2</Subscript>-adrenergic Receptors (β <Subscript>2</Subscript>AR) Enhances Therapeutic Effectiveness of β <Subscript>1</Subscript>AR Blockade in Rodent Dilated Ischemic Cardiomyopathy

Background. We have reported that β ₂ adrenoreceptor (β ₂AR) stimulation is anti-apoptotic, and has strong beneficial effect on cardiac remodeling in an experimental model of post myocardial infarction chronic heart failure (CHF) in rats. Here we investigate whether the addition of chronic pharmacological β ₂AR stimulation enhances the therapeutic effects of β ₁AR blockade on cardiac remodeling in the same model. Methods and Results. Metoprolol, a β ₁AR blocker, given alone (β ₁) or in combination with β ₂AR agonist, fenoterol (β ₁β ₂) were administered to rats via drinking water for 6 weeks, beginning 2 weeks following permanent coronary ligation. Progressive left ventricular (LV) remodeling of untreated animals, assessed by repeated echocardiography, occurred during the observation time, i.e., 42% and 25% increases in end-systolic and end-diastolic LV volumes respectively, 27% fall in ejection fraction, and 35% infarct expansion. Pressure-volume loop analyses at 2d and 8th post infarction weeks showed continuous deterioration of systolic and diastolic functions and arterio-ventricular mismatch. Histological evaluation at the end of 8 weeks revealed the MI expansion and hypertrophy of cardiomyocytes. β ₁β ₂ prevented LV remodeling, MI expansion and cardiomyocytes hypertrophy to a greater degree than β ₁, due, in large part, to a vasodilatory effect of β ₂AR stimulation and thus improvement of arterio-ventricular mismatch. The abnormal diastolic performance improved only in β ₁β ₂. β ₁β ₂ treatment reduced myocardial apoptosis throughout myocardium, but β ₁ reduced apoptopsis only in the areas remote from MI. Conclusion. The therapeutic effects of chronic β ₁AR blockade on cardiac remodeling of heart failure are enhanced and extended when supplemented with β ₂AR stimulation. This research was supported by the Intramural Research Program of the National Institute on Aging, NIH.     

Disequilibrium in the CD8<Superscript>+</Superscript>CD28<Superscript>+</Superscript>/CD8<Superscript>+</Superscript>CD28<Superscript>−</Superscript> T Lymphocyte Balance Is Related to Prognosis in Rats with Trinitrobenzenesulfonic Acid-Induced Colitis

Purpose

The CD8⁺CD28⁺/CD8⁺CD28^? T lymphocyte balance is vital for human ulcerative colitis (UC) but has not been defined in experimental colitis. This investigation will try to identify the changes that occur in the CD8⁺CD28⁺/CD8⁺CD28^? T lymphocyte balance during the progression of trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats.

Methods

The frequencies of blood CD8⁺CD28⁺ and CD8⁺CD28^? T lymphocytes were detected in the rats belonging to the normal, model, and treated groups on five days using flow cytometry. The treated rats were administered with mesalazine and were euthanized after a 14-day treatment, as were the normal and model rats. The sensitivity and specificity of the CD8⁺CD28⁺/CD8⁺CD28^? T lymphocyte balance in diagnosing early colitis were analyzed by receiver operating characteristics (ROC) curves. The frequencies of CD8⁺CD28⁺ and CD8⁺CD28^? T lymphocytes in the colon tissue were tested via immunofluorescence. ELISA was used to measure the levels of the cytokines. Immunohistochemistry and Western blotting were used to detect the colonic expression of JAK3, STAT6, NFATc2, and GATA3.

Results

We found that the ratio of CD8⁺CD28⁺/CD8⁺CD28^? T lymphocytes decreased, as did the level of interleukin-7, but not IL-12p40, IL-13, or IL-15, in the blood; however, the ratio increased along with JAK3, STAT6, NFATc2, and GATA3 in the colon of the rats with colitis. The changes were effectively reversed through the administration of mesalazine for 13 days. Surprisingly, the balance in the blood could sensitively distinguish rats with early colitis from normal rats.

Conclusion

These data show that increase in CD8⁺CD28⁺ T cells in blood and decrease in CD8⁺CD28^? T cells in colon are associated with experimental colitis.

     

Carbohydrate-Electrolyte (E-Lyte<Superscript>®</Superscript>) Solution Enhances Bowel Preparation With Oral Fleet<Superscript>®</Superscript> Phospho-soda<Superscript>®</Superscript>

PURPOSE: Bowel preparation with oral sodium phosphate can cause symptomatic dehydration and electrolyte disturbances. This randomized, controlled trial was designed to evaluate whether carbohydrate-electrolyte (E-Lyte^®) solution enhanced bowel preparation and improved patient acceptance with oral sodium phosphate.METHODS: A total of 187 consecutive adults undergoing colonoscopy by two endoscopists were randomized to receive two packets of oral sodium phosphate (Fleet^® Phospho-soda^®) with or without additional supplement of a carbohydrate-electrolyte (E-Lyte^®) solution. All patients and endoscopists completed a standardized questionnaire. Urine-specific gravity and serum biochemistry were randomly performed in 150 and 50 patients, respectively.RESULTS: Ninety patients were randomized to have oral sodium phosphate with E-Lyte^® supplements (Group 1) and 94 patients to sodium phosphate without E-Lyte^® supplements (Group 2). The groups were similar in age and gender, indication for colonoscopy, and previous colonic surgery. Patients taking E-Lyte^® supplement had significantly less dizziness (none, 80 vs. 56 percent; P < 0.001) and a trend toward less nausea (none, 70 vs. 56 percent; P = 0.05). All patients in Group 1 completed the bowel preparation as opposed to 3 percent of Group 2 being unable to complete the preparation. Hypokalemia was significantly more frequent (P = 0.008) in Group 2 patients without E-Lyte^® supplements. More patients in Group 2 needed intravenous rehydration (11 vs. 4 percent). Differences in serum creatinine and urine-specific gravity suggested possibly a lesser degree of hypovolemia in patients taking E-Lyte^® supplements. The quality of bowel cleansing in patients taking E-Lyte^® supplements was considered better by both the endoscopists and patients.CONCLUSIONS: Carbohydrate-electrolyte (E-Lyte^®) solution protects against hypokalemia, improves patient tolerability, and may enhance use of oral sodium phosphate as a bowel-preparation agent.Presented at the meeting of The American Society of Colon and Rectal Surgeons, New Orleans, Louisiana, June 21 to 26, 2003.Fleet^® Phospho-soda^® and E-Lyte^® were supplied by CB Fleet Co., Inc., Lynchburg, VA.     

Low Ambient [Cl<Superscript>−</Superscript>] Increases Ca<Superscript>2+</Superscript> Mobilization and Stimulates Nitric Oxide and Prostaglandin E<Subscript>2</Subscript> Production in Human Bronchial Epithelial Cells

Changes in ionic composition of airway surface fluid may modulate airway epithelial functions. We tested the hypothesis that fluctuations of ambient ionic composition could affect airway epithelial Ca²⁺ dynamics and Ca²⁺-dependent cellular functions, including NO release and PGE₂ production in vitro. The responses of intracellular Ca²⁺ concentration ([Ca²⁺]_i) to changes in extracellular Cl⁻ and Na⁺ concentrations ([Cl⁻]_e, [Na⁺]_e) in the human bronchial epithelial cell line, 16HBE cells, were measured by the fura-2 method. The NO release to the medium after lowering [Cl⁻]_e was measured by an amperometric NO sensor. PGE₂ production was measured by radioimmunoassay. Changing to isotonic low [Cl⁻]_e solution by substitution with gluconate caused a sustained increase in [Ca²⁺]_i in a concentration-dependent manner, with the maximal [Ca²⁺]_i increase from the baseline level being 243 ± 110 nM with Cl-free solution. The effect was not altered by thapsigargin but abolished by EGTA and by Cl channel blockers, including diphenylamine-2-carboxylate, disodium 4,4′-diisothiocyanatostilbene-2,2′-disulfonate, and disodium cromoglycate. In contrast, the effect of reduction of [Na⁺]_e by substitution with N-methyl-D-glucamine⁺ on [Ca²⁺]_i was less than that of reduction of [Cl⁻]_e. The reduction of [Cl⁻]_e caused a concentration-dependent rise in NO contents in the medium and PGE₂ production. This release of NO was inhibited by EGTA but not by dexamethasone pretreatment. These results suggest that the decrease in ambient [Cl⁻] induces Ca²⁺ mobilization probably through Ca²⁺ influx, followed by the release of NO and PGE₂, thereby modulating various cellular functions.     

Long-Acting β<Subscript>2</Subscript>-Agonists

Salmeterol and formoterol are both long-acting β₂-adrenoceptor agonists (β₂-agonists). They both provide excellent bronchodilating and bronchoprotective effects in patients with asthma but their are some differences between these two long-acting β₂-agonists in vitro and in vivo. Formoterol has a greater potency and intrinsic activity than salmeterol, which can become especially apparent at higher doses than that clinically recommended, and in contracted bronchi. Long-term use of long-acting β₂-agonists can induce tolerance, which can be partially reversed with corticosteroids. Long-acting β₂-agonists have some anti-inflammatory effects in vitro, but data in vivo are less convincing. Compared with doubling the dose of inhaled corticosteroids, the addition of inhaled long-acting β₂-agonists to inhaled corticosteroids improves symptom control in patients with asthma and reduces both the exacerbation rate of asthma and hospital admission rate. No enhanced airway responsiveness or loss of perception of dyspnea has been observed with the use of inhaled long-acting β₂-agonists. Monotherapy with long-acting β₂-agonists is not recommended.     

β<Subscript>2</Subscript>-Agonist Eutomers

β₂-adrenoceptor agonists (β₂-agonists) such as albuterol (salbutamol) and terbutaline and their long-acting analogs salmeterol and formoterol are widely used as bronchodilators in the treatment of asthma. They are chiral drugs historically marketed as racemic mixtures of an active (eutomer) and essentially inactive (distomer) stereoisomer. Despite their obvious therapeutic value and widespread use, β₂-agonists have been implicated, somewhat controversially, in causing an increase in asthma mortality and a deterioration of asthma control by a mechanism that remains elusive. Inherent toxicity of the distomers has been widely touted as an explanation and has given rise to pressure for the replacement of the racemates with pure eutomer formulations (the so-called chiral or racemic switch). This has culminated in the recent introduction into clinical practice of the single active stereoisomer of albuterol (levalbuterol) and the promise of other pure β₂-agonist eutomer formulations to follow. This article examines the evidence on which these chiral switches are based.Clinical studies designed to reveal negative effects of β₂-agonists have searched for reductions in lung function, increases in airway responsiveness to bronchoconstrictor mediators and worsening of asthma control. Crossover studies administering the pure stereoisomers and racemate of albuterol have not shown a clear superiority of the pure eutomer formulation over the racemate in terms of either bronchial hyperresponsiveness, tachyphylaxis to bronchoprotective effects or improvements in lung function. Clinical toxicity of β₂-agonist distomers on any aspect of asthmatic lung function has also not been demonstrated in the relatively short-term inhalational studies (single dose or repeated dose studies <1 week) that have been carried out.In animal studies, the administration of β₂-agonist racemates and distomers has been shown to enhance bronchial hyperresponsiveness but only in ovalbumin-sensitized animals where the relevance to humans is questionable.The pharmacokinetics and metabolism of β₂-agonist stereoisomers appear to be essentially similar whether administered as single stereoisomers or as racemates. Levalbuterol may be slightly more potent than an equivalent dose given as racemate, but there is some evidence that it forms a small amount of the distomer in vivo which detracts somewhat from its purported benefits over use of the racemate.Whilst there remains a clear need for studies of longer duration with sensitive clinical endpoints to evaluate the benefits of β₂-agonist eutomers and to investigate distomer toxicity, the chiral switch for β₂-agonists in general, and for albuterol in particular, does not appear to be justified on the basis of the evidence available to date.     

Optical Feedback Training of Inhalation with Autohaler<Superscript>®</Superscript> and Turbuhaler<Superscript>®</Superscript> in COPD Patients

The success of inhalation therapy depends on patients inhalation technique and correct handling of the inhalation device. In this study the effort to train correct handling and optimal inhalation technique of patients using Autohaler^® and Turbuhaler^® was assessed. The Bad-Reichenhall-Aerosol-Therapy-Trial (BREATH) was a prospective, randomized, cross-over trial in 200 patients who were not familiar with either of the test systems. The correct handling of Autohaler^® and Turbuhaler^® was assessed by means of a checklist (observation score). An optimal inhalation maneuver was used evaluated with the computer-based Inhalation Manager (optical feedback, computer score). The Autohaler^® reached 6.58±3.64 (mean ± SD) out of nine points in the observation score and 66.85 ± 29.84% in the computer score before training. After training the scores increased significantly to 8.33 ± 2.08 points and 86 ± 23.40% respectively. The use of the Turbuhaler^® also significantly improved from 4.28 ± 4.24 points and 56.67 ± 42.97% to 7.78 ± 2.74 points and 85.80 ± 27.63%, respectively. The significant improvement of patients inhalation technique after training emphasizes the importance of training in inhalation therapy. In addition, it could be demonstrated that the optical feedback given by the Inhalation Manager was a useful tool for improving the inhalation technique of patients using Autohaler^® and Turbuhaler^®.     

Acute but not chronic graft-versus-host disease is associated with a reduction of circulating CD4<Superscript>+</Superscript>CD25<Superscript>high</Superscript>CD127<Superscript>low/−</Superscript> regulatory T cells

Defects in central and peripheral tolerance are thought to contribute to life-threatening graft-versus-host disease (GvHD), a severe complication following allogeneic stem cell transplantation (SCT). Recent investigations have demonstrated regulatory T cells (Tregs) to suppress allogeneic immune reactions. Therefore, SCT patients with no or critically low numbers of Tregs may have an increased risk of GvHD. To address this hypothesis, we analyzed the recovery of CD4⁺CD25^highCD127^low/− Tregs in the peripheral blood of patients who have never developed GvHD (n = 6), patients who developed acute/chronic GvHD (n = 18), and patients who developed chronic GvHD without an earlier acute manifestation (n = 5) every 30 days for the first 6 months after peripheral blood SCT (PBSCT). The number of Tregs continuously improved in acute/chronic GvHD patients, but always remained lower than Tregs quantified in patients who never developed a GvHD. In contrast, chronic GvHD patients who did not develop acute GvHD earlier displayed significantly increased Treg cell numbers at the timepoint of chronic inflammation. These results indicate that numerically deficient Tregs following PBSCT are associated with the development of acute but not chronic GvHD.     

Evolution of recombinant factor VIII safety: KOGENATE<Superscript>?</Superscript> and Kogenate<Superscript>?</Superscript> FS/Bayer

The use of factor VIII (FVIII) concentrates in the treatment of hemophilia A has raised important safety issues, historically of pathogen transmission and increasingly of inhibitor development to FVIII treatment. While manufacturing processes of current recombinant FVIII products have been shaped entirely around preventing pathogen transmission, the same modifications that afford a greater margin of safety could affect immunogenicity of the product, consequences of which could only be seen through long-term clinical experience. This review summarizes pathogen safety and inhibitor reports from clinical trials, post-marketing surveillance studies, and study reports on KOGENATE^® and its successor, Kogenate^® FS/Bayer. Although KOGENATE and Kogenate FS/Bayer are nearly identical products, subtle manufacturing improvements to address the need for greater margins of safety from a pathogen transmission perspective have also led to a potentially improved immunogenicity profile (15% in previously untreated/minimally treated patients with severe hemophilia A for Kogenate FS/Bayer). Notably, there has been no occurrence of pathogen contamination, and minimal de novo inhibitor formation in previously treated patients throughout the use of both products. Overall, KOGENATE and Kogenate FS/Bayer have a long history of safety in a variety of clinical settings, including treatment of bleeding, surgical management, and prophylaxis therapy.     

Taurodeoxycholate Modulates Apical Cl<Superscript>−</Superscript>/OH<Superscript>−</Superscript> Exchange Activity in Caco2 Cells

Bile acid malabsorption has been shown to be associated with diarrhea in cases such as ileal resection Crohn’s disease of the ileum, and radiation enteritis. The mechanisms of bile acid-induced diarrhea are not fully understood. Although the induction of colonic chloride secretion in response to bile acids has been extensively investigated, to date the direct effect of bile acids on intestinal chloride absorption has not been well defined. Therefore, the current studies were undertaken to investigate the effect of bile acids on the apical Cl⁻/OH⁻ exchange process utilizing Caco2 monolayers as an in vitro cellular model. Cl⁻/OH⁻ exchange activity was measured as DIDS-sensitive pH gradient-driven ³⁶Cl uptake. The results are summarized as follows: (i) short-term exposure (20 min) of Caco2 cells to taurodeoxycholate (TDC; 200 μM) and glycochenodeoxycholate (GCDC; 200 μM) acids significantly inhibited apical Cl⁻/OH⁻ exchange (by ∼60–70%); (ii) the Ca²⁺ chelator BAPTA-AM blocked the inhibition by TDC; (iii) the reduction in Cl⁻/OH⁻ exchange by TDC was reversed by the PKC inhibitor, chelerythrine chloride; (iv) functional and inhibitor studies indicated that TDC induced inhibition of Cl⁻/OH⁻ exchange was mediated via the activation of the PKCβI isoform; (v) the effect of TDC on apical Cl⁻/OH⁻ exchange was completely blocked by the PI3 kinase inhibitor LY294002 (5 μM); and (vi) the PKA inhibitor, RpcAMP, had no effect on TDC induced inhibition of Cl⁻/OH⁻ exchange. In conclusion, our studies provide direct evidence for inhibition of human intestinal apical Cl⁻/OH⁻ exchange activity by bile acids via Ca²⁺-, PI3 kinase-, and PKCβI-dependent pathways in Caco2 cells.     

Intracellular [Na<Superscript>+</Superscript>] modulates synergy between Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger and L-type Ca<Superscript>2+</Superscript> current in cardiac excitation–contraction coupling during action potentials

Excitation–contraction coupling (ECC) in cardiac myocytes involves triggering of Ca²⁺ release from the sarcoplasmic reticulum (SR) by L-type Ca channels, whose activity is strongly influenced by action potential (AP) profile. The contribution of Ca²⁺ entry via the Na⁺/Ca²⁺ exchanger (NCX) to trigger SR Ca²⁺ release during ECC in response to an AP remains uncertain. To isolate the contribution of NCX to SR Ca²⁺ release, independent of effects on SR Ca²⁺ load, Ca²⁺ release was determined by recording Ca²⁺ spikes using confocal microscopy on patch-clamped rat ventricular myocytes with [Ca²⁺]_i fixed at 150 nmol/L. In response to AP clamps, normalized Ca²⁺ spike amplitudes (ΔF/F ₀) increased sigmoidally and doubled as [Na⁺]_i was elevated from 0 to 20 mmol/L with an EC₅₀ of ~10 mmol/L. This [Na⁺]_i-dependence was independent of I _Na as well as SR Ca²⁺ load, which was unchanged under our experimental conditions. However, NCX inhibition using either KB-R7943 or XIP reduced ΔF/F ₀ amplitude in myocytes with 20 mmol/L [Na⁺]_i, but not with 5 mmol/L [Na⁺]_i. SR Ca²⁺ release was complete before the membrane repolarized to −15 mV, indicating Ca²⁺ entry into the dyad (not reduced extrusion) underlies [Na⁺]_i-dependent enhancement of ECC. Because I _Ca,L inhibition with 50 mmol/L Cd²⁺ abolished Ca²⁺ spikes, our results demonstrate that during cardiac APs, NCX enhances SR Ca²⁺ release by synergistically increasing the efficiency of I _Ca,L-mediated ECC.     

Diminution of Circulating CD4<Superscript>+</Superscript>CD25<Superscript>high</Superscript> T Cells in Na?ve Crohn’s Disease

Crohn’s disease is considered to be caused either by an excess of T-cell effector functions and/or by a defective regulatory T-cell compartment. The aim of this study was to assess in Crohn’s disease the frequency of circulating CD4⁺CD25^high T cells that possess regulatory T-cell functions and CD4⁺CD25^low T cells that contain activated T cells. Flow cytometry of peripheral blood was used to assess CD4⁺CD25^high and CD4⁺CD25^low T-cell frequencies in a cohort of 66 patients with Crohn’s disease in comparison to 19 patients with ulcerative colitis and 31 healthy individuals enrolled as controls. The CD4⁺CD25^high T-cell frequency was significantly lowered in naïve Crohn’s disease (P = 0.013) and in ulcerative colitis (P = 0.001). CD4⁺CD25^low T-cell frequency was increased in Crohn’s disease (P = 0.0001) and in ulcerative colitis (P = 0.0002). Both CD4⁺CD25^high and CD4⁺CD25^low T-cell frequencies are altered in naïve Crohn’s disease resulting in an imbalance between both populations and a relative contraction of the CD4⁺CD25^high T-cell population.     

The roles of Transforming growth Factor Type β<Subscript>3</Subscript>(TGF-β<Subscript>3</Subscript>) and mast cells in the pathogenesis of scleroderma

Scleroderma is a connective tissue disorder characterised by excessive accumulation of collagen in the skin and internal organs. The most likely explanation for this process is local activation of collagen synthesis from fibroblasts. Our intention was to elucidate whether TGF-₃ and mast cells play a pathogenic role in abnormal connective tissue formation in scleroderma. In this study, skin biopsies from 20 patients with scleroderma and five from healthy individuals were studied by an indirect immunoperoxidase technique to determine the immunoreactivity of TGF-₃ in the dermis. In addition, skin samples were stained with toluidine blue to count the number of mast cells in scleroderma, and tissues were examined under the electron microscope to evaluate the ultrastructural changes. Increased TGF-₃ immunoreactivities were detected in the dermis in the patient's skin, suggesting the presence of a subpopulation responsible for the increased collagen production. Mast cell counts in the skin of patients with scleroderma were significantly greater (19.2 ± 4.1/unit) than those of normal controls (4.4 ± 1.2/unit). Ultrastructural observations indicated that there is a close relationship between the mast cells and fibroblasts. These results suggest that fibrosis in scleroderma could evolve through the activation of fibroblasts and the regulatory mechanisms that appear to modulate the behavior of these cells with respect to collagen production.AcknowledgementWe are grateful for the support of Professor M. Kaya in our work with the electron microscope and thank Professor E. Erten for supplying tissue samples.