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Transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel that responds to mechanical, thermal, and chemical stimuli in addition to various endogenous ligands, such as arachidonic acid metabolites. The present study aimed to elucidate the expression of TRPV4 in the gastrointestinal tract and the pathogenic roles of TRPV4 in dextran sulphate sodium (DSS)-induced colitis. TRPV4-immunoreactivity was detected in epithelial-like cells of the mouse tongue, esophagus, stomach, ileum, and colon; TRPV4 expression in the tongue was higher than other gastrointestinal tracts. TRPV4 colocalized with a type IV cell marker sonic hedgehog in circumvallate papillae. These findings suggest that TRPV4 contributes to sour taste sensing by regulating type III taste cell differentiation in mice. DSS-induced colitis was significantly attenuated in TRPV4-knockout (TRPV4KO) mice when compared to wild-type mice. DSS treatment upregulated TRPV4 expression in vascular endothelia of colonic mucosa and submucosa. DSS treatment increased vascular permeability, which was abolished in TRPV4KO mice. The activation of TRPV4 decreased VE-cadherin expression in mouse aortic endothelial cells exposed to TNF-α. These findings indicate that the upregulation of TRPV4 in vascular endothelial cells contributes to the progression of colonic inflammation via the activation of vascular permeability. Thus, TRPV4 is an attractive target for the treatment of inflammatory bowel diseases.

The transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel involved in physical sensing in various tissue types. The present study aimed to elucidate the function and expression of TRPV4 in colonic vascular endothelial cells during dextran sulphate sodium (DSS)-induced colitis. The role of TRPV4 in the progression of colonic inflammation was examined in the 2% DSS-induced murine colitis model using immunohistochemical analysis, Western blotting, and Evans blue dye extrusion assay. DSS-induced colitis was significantly attenuated in TRPV4-deficient (TRPV4 KO) mice when compared to wild-type mice. Repeated intrarectal administration of GSK1016790A, a TRPV4 agonist, exacerbated the severity of DSS-induced colitis. Bone marrow transfer experiments demonstrated a dominant role of TRPV4 in non-haematopoietic cells for DSS-induced colitis. DSS treatment upregulated TRPV4 expression in the vascular endothelia of colonic mucosa and submucosa. DSS treatment increased vascular permeability, which was abolished in TRPV4 KO mice. The DSS-induced increase in vascular permeability was further enhanced by intravenous administration of GSK1016790A, which was abrogated by a TRPV4 antagonist RN1734. TRPV4 was co-localized with vascular endothelial (VE)-cadherin, and VE-cadherin expression was decreased by repeated intravenous administration of GSK1016790A during colitis. Furthermore, TRPV4 activation by GSK106790A decreased VE-cadherin expression in mouse aortic endothelial cells exposed to TNF-α. These findings indicate that TRPV4 upregulation in vascular endothelial cells contributes to the progression of colonic inflammation via the activation of vascular permeability. Thus, TRPV4 is an attractive target for the treatment of inflammatory bowel diseases.

Functional thin films containing insulin were prepared using layer-by-layer (LbL) deposition of insulin and negatively- or positively-charged polymers on the surface of solid substrates. LbL films composed of insulin and negatively-charged polymers such as poly(acrylic acid) (PAA), poly(vinylsulfate) (PVS), and dextran sulfate (DS) were prepared through electrostatic affinity between the materials. The insulin/PAA, insulin/PVS, and insulin/DS films were stable in acidic solutions, whereas they decomposed under physiological conditions as a result of a change in the net electric charge of insulin from positive to negative. Interestingly, the insulin-containing LbL films were stable even in the presence of a digestive-enzyme (pepcin) at pH 1.4 (stomach pH). In contrast, LbL films consisting of insulin and positively-charged polymers such as poly(allylamine hydrochloride) (PAH) decomposed in acidic solutions due to the positive charges of insulin generated in acidic media. The insulin-containing LbL films can be prepared not only on the surface of flat substrates, such as quartz slides, but also on the surface of microparticles, such as poly(lactic acid) (PLA) microbeads. Thus, insulin-containing LbL film-coated PLA microbeads can be handled as a powder. In addition, insulin-containing microcapsules were prepared by coating LbL films on the surface of insulin-doped calcium carbonate (CaCO 3 ) microparticles, followed by dissolution of the CaCO 3 core. The release of insulin from the microcapsules was accelerated at pH 7.4, whereas it was suppressed in acidic solutions. These results suggest the potential use of insulin-containing microcapsules in the development of oral formulations of insulin.

We developed a simple separative method for measuring serum amyloid A (SAA) in both high-density-lipoprotein (HDL) and low-density-lipoprotein (LDL) fractions. It was devised using the SAA agglutination method and phosphotungstic acid-Mg2+ precipitation procedure for evaluating HDL-cholesterol (HDL-C). The new method is also able to detect amyloid A (AA) in each fraction with precision. The results of both the present method and the method using SAA agglutination and the dextran sulfate-Mg2+ precipitation procedure showed a strong correlation when used to measure the level of SAA in the LDL fraction of patients (r = 0.997; p < 0.0001). Reference intervals in normal healthy subjects (n=75) ranged from 0.5 to 4.7 microg/ml in the HDL fraction and from 0.1 to 1.9 microg/ml in the LDL fraction. SAA in the LDL fraction of subjects with hyperlipidemia was significantly higher than in normal subjects and subjects with normal lipidemia. SAA in the HDL fraction and total sera of subjects with hyperlipidemia was significantly higher than in normal subjects; however, it was not higher than in patients with normal lipidemia. The present methods for detecting SAA, especially in the LDL fraction, might benefit from analyzing patho-physiological events in various lipid disorders.

Inhibitors of enzymatic amplification in serum may cause false-negative results for direct detection of hepatitis C virus (HCV) by polymerase chain reaction (PCR). This study was undertaken to demonstrate inhibitory effects of the therapeutic reagents on the PCR detection of HCV and to evaluate the efficacy of their elimination by RNA extraction methods. RNA was extracted using a manual method based on organic extraction and precipitation of RNA (SepaGene RV-R, Sanko Junyaku) or an automated system based on specific capture of HCV-RNA with probes and magnetic bead/fluid (B/F) separation (Roche Molecular Systems). When RNA was extracted by SepaGene RV-R from serum containing 10(5) copies per ml of HCV and amplified for HCV-RNA in the presence of hemoglobin, bilirubin, or heparin by Amplicor HCV (Roche Molecular Systems), results tended to be negative. In addition to these, two therapeutic reagents, ATP and dextran sulfate sodium were also found to have inhibitory effects. When ATP at concentrations up to 5 mM was added to the sera and RNA was extracted with SepaGene RV-R, there were no inhibitory effect on the detection of either HCV-RNA or the internal control. In contrast, when dextran sulfate sodium up to 1 mM was added to the sera, there was a dose dependent inhibitory effect on detection of both HCV-RNA and the internal control. When HCV-RNA was extracted by the automated system, the inhibitory effect of dextran sulfate sodium was successfully eliminated. In conclusion, dextran sulfate sodium and ATP were newly identified as inhibitors that may be present in serum, and the efficacy of eliminating these substances varied among RNA extraction methods.

We investigated the quantitative changes in colonic mucins of rats with colitis. Male Wistar rats were treated with dextran sodium sulfate (DSS) or N-ethylmaleimide (NEM) to induce colitis. Both DSS and NEM caused depletion of goblet cells, infiltration of inflammatory cells and erosion at the colonic mucosa around the anus. Though the goblet cells decreased, colonic mucins increased in the distal colon. These phenomena may explain the clinical features of human ulcerative colitis, namely the goblet cell depletion and the mucous stool. The increase of colonic mucins may be a compensatory function of the colon tissue in response to the localized decrease of mucin production.

Most people drugs are hydrophilic molecules with a molecular weight between 300 and 20,000 and such molecules are usually given by parenteral administration. In many cases, enteral administration of these peptides via the gastrointestinal tract is preferred. However, oral administration of peptides and proteins is often limited by their instability in the gastrointestinal environment and/or poor absorption from the gut. To promote the absorption of these drugs, we first discovered unsaturated fatty acids with absorption enhancing activities and less harmful properties to the gastrointestinal membranes in hydrolysates of natural oil. The mechanisms whereby the permeability of drugs was enhanced by the fatty acids are associated with the disorder in the membrane's interior and the interaction of these fatty acids with the polar head group of phospholipid. Furthermore, we suggested that a SH-related substance was involved in the permeability enhancing effect of these fatty acids. Secondly, we developed a lympho-targeting delivery system for bleomycin by the combined effects of an ion-pair complex with dextran sulfate (DS) and an absorption enhancer. We found a very high lymphatic concentration when administered bleomycin-DS together with the absorption enhancer. Its mechanism may be due to a molecular sieving in the blood-lymph barrier in the intestinal tissues. Finally, to improve the intestinal absorption of peptides, we synthesized novel lipophilic derivatives of peptides including TRH (thyrotropin releasing hormone), tetragastrin, enkephalin, calcitonin and insulin by a chemical modification with fatty acids, while maintaining their pharmacological activities. The stability and permeability of these peptides were improved by acylation with some fatty acids having appropriate carbon numbers. Thus, we have established the strategies for improving the delivery of peptide drugs by various approaches. In future, the combination use of these approaches will be expected to develop the delivery systems of these drugs for therapeutic treatment.

In the present study, we investigated the therapeutic effects of 7- or 14-day treatment with BX661A or salazosulfapyridine (SASP) in the DSS-induced UC model in rats. BX661A (10-300 mg/kg, p.o.) dose-dependently decreased the erosion area and the shortening of the large intestine. On the other hand, SASP (30 and 100.mg/kg, p.o.) dose-dependently decreased the erosion area in the treatment for 14 days (on the contrary, % inhibition of erosion area was reduced by the dose of 300 mg/kg), but did not improve the shortening of the large intestine. Secondly, we investigated the therapeutic effects of 5-aminosalicylic acid (5-ASA), 4-aminobenzoyl- beta-alanine (4-ABA) and sulfapyridine (SP) by intrarectal administration on the DSS-induced UC model in rats. 5-ASA significantly decreased the erosion area in the large intestine and improved the length of the large intestine of rats that was shortened by ingesting DSS. On the other hand, 4-ABA and SP improved neither the shortening nor the erosion area of the large intestine. These results suggest that BX661A may be clinically effective and useful in the treatment of patients with ulcerative colitis. Furthermore, it was suggested that 5-ASA may be the active moiety for the therapeutic effects of BX661A and SASP.

To clarify the mechanism of action of immunoglobulin (IgG) in intravenous immunoglobulin therapy for ulcerative colitis (UC) patients, we studied the effect of IgG on the dynamics of immunocompetent cells in the colonic mucosa of experimental colitis induced by dextran sulfate sodium (DSS) in rats. The administration of the same species' IgG suppressed the mucosal infiltration of immunocompetent cells (activated T cells, macrophages and neutrophils), although the different species' IgG didn't. We have already shown that the same species' IgG, suppressed the production of inflammatory cytokines (TNF alpha, IL-1 alpha and IL-8) in the colonic mucosa of experimental colitis induced by DSS. In the present report, we demonstrated the different species' IgG, as well as same species' IgG, suppressed the production of inflammatory cytokines (TNF alpha and IL-1 alpha) from lamina propria mononuclear cells of rat large intestine in vitro. Therefore, it was considered that the suppression of cytokine production was a consequence of the decreased immunocompetent cells in colitis mucosa. Furthermore, we demonstrated that the DSS-treated antigen presenting cells (APCs) activated antigen-specific T cells as a possible mechanism underlying the colitis induced by DSS and the same species' IgG inhibited this activation of T cells.

In order to the reduce the LDL level of Familial hypercholesterolemia (FH), direct LDL removal techniques have been used in several laboratories. In Japan, selective LDL-apheresis by dextran sulfate cellulose column have been developed and is used in treatment of homo- and heterozygous FH patients. In this paper, I reviewed the effectiveness of LDL-apheresis treatment in FH patients with many evidences of regression of atherosclerosis and discussed the potential role of this technique in the future treatment for coronary heart disease. No significant side effect for long term treatment is not experienced.

To clarify the mechanisms of action of immunoglobulin (IgG) in intravenous immunoglobulin therapy for the ulcerative colitis (UC) patients, we studied the therapeutic effect of IgG on experimental rat colitis induced by dextran sulfate sodium and on the production of inflammatory cytokines such as TNF alpha, IL-1 alpha and IL-8. The administration of rat IgG demonstrated to suppress the development of blood stool and the induction of the ulcerative lesion in large intestine. The levels of TNF alpha, IL-1 alpha and IL-8 were increased in blood and mucosa of the large intestine in this model. Rat IgG showed a tendency to decrease the levels of the cytokines in colonic mucosa. This result seems to provide a piece of explanation of how massive administration of IgG shows an improvement in the ulcerative lesion of UC patients.

We have developed a new method that is applicable for a macroscopic and objective evaluation of erosion in the large intestine in the experimental ulcerative colitis model induced by dextran sulfate sodium (DSS) in rats. The large intestine (without cecum) fixed in 10% neutral buffered formalin solution for more than one week was opened and stained with 1% alcian blue solution. The mucosal surface was stained in light and shade-blue. Histopathological examination revealed that the dark blue area on the mucosal surface had no epithelia and that the connective tissues in the lamina propria were stained with alcian blue. Salazosulfapyridine at 15 and 50 mg/kg twice a day inhibited the erosion area (dark blue area) by 29.6% and 50.2%, respectively. Also, prednisolone at 0.5 mg/kg, twice a day inhibited the erosion by 53.3%. Thus, by measuring the dark blue area stained with 1% alcian blue solution, we could estimate macroscopically and objectively the area of erosions. This method seems to be a useful index for assessing the damages produced in the experimental ulcerative colitis model in rats.

Unlabelled: In the severe cases of toxemia of pregnancy, the coagulation-fibrinolysis pattern and the morphological findings of the kidney suggest participation of chronic DIC. Accordingly, it is considered that management of mother and fetus by administration of drugs with anticoagulant and fibrinolytic activities may be useful., Method: For 17 cases of preeclampsia (gestosis index, 9.1 +/- 1.5) daily dose of 3,000-1,500mg DS and 48,000 IU of UK were administered intravenously by one shot or drip (10-30 minutes) twice daily. The following clinical and laboratory examinations were done: Gestosis index (GI), blood pressure (BP), edema, urine volume, urine protein, Ccr (ml/min), platelet counts, PT, PTT, PRT, TT, factors XII, IX, VIII, X, VII, V, II and XIII, Fbg, ELT, eug + SKLA(Std), Plg, SFMC, serum and urine FDP, AT-III, C1-INA, alpha 2-PI, alpha 2-M and alpha 1-AT., Result: In the preeclamptic women, increases in intrinsic coagulating factors, marked decreases in the extrinsic coagulating factors, depletion of ELT, rise in SFMC, marked increases in blood and urine FDP, and decreases in AT-III and alpha 2-PI were observed, suggesting the presence of chronic DIC. After administration of DS and UK, improvement of clinical findings such as a fall of BP, disappearance of edema, reduction in urine protein, and increase in Ccr were observed. In addition, normalization of the intrinsic system, recovery of the extrinsic system, ELT and serum FDP to normal levels, increasing tendency of Plg, AT-III, alpha 2-PI, alpha 2-M, alpha 1-AT, and disappearance of urine FDP were demonstrated at the same time. It is therefore concluded that this treatment for severe preeclampsia seems to be effective.

Heparin and heparinoids are known to produce selective aldosterone deficiency in man and experimental animals. To assess the nature of the hypoaldosteronism caused by heparin and heparinoids, we investigated the production of aldosterone and its precursor steroids in response to angiotensin II (AII), ACTH or potassium in adrenal zona glomerulosa cells from dextran sulfate-treated rats compared with that in the cells from vehicle-treated rats. Dextran sulfate-treated rats had a decrease in plasma aldosterone and a reduction in the width of the zona glomerulosa 4 weeks after the treatment (40 mg/day, intramuscularly). In these rats, PRA and plasma AII tended to be high, and plasma corticosterone was normal. Basal aldosterone production, when corrected to a uniform number of cells per group, was similar in cells from dextran sulfate- and vehicle-treated rats. The cells from dextran sulfate-treated rats had a less sensitive and lower response of aldosterone production to AII; an increase by 4 orders of magnitude in the threshold dose for AII and a decrease in the maximal AII-stimulated level. The maximal AII-stimulated levels, but not the basal levels, of pregnenolone, corticosterone and 18-hydroxycorticosterone production were low in the cells from dextran sulfate-treated rats. ACTH produced a similar stimulatory effect on aldosterone production in the cells from dextran sulfate- and vehicle-treated rats. The cells from dextran sulfate-treated rats had a less sensitive and lower response of aldosterone production to potassium; an increase by one order of magnitude in the threshold dose for potassium and a decrease in the maximum potassium-stimulated level, presumably because of the glomerulosa hyporesponsiveness to AII. These results suggest that long-term treatment with dextran sulfate in rats produces selective impairment of adrenal zona glomerulosa cells, involving the specific receptors and the aldosterone biosynthesis, to AII in addition to a reduction in the glomerulosa width.

The eligibilities for chemotherapy, prognostic factors and effects of chemotherapy were evaluated in pulmonary metastasis from uterine cervical carcinoma. There was no difference in median survival between patients treated with surgery and those with chemotherapy. The mediastinal lymph node involvement and vascular invasion were demonstrated to be important prognostic factors in patients treated with surgery, and it was suggested that systemic chemotherapy should be given to the patients with mediastinal lymph node involvement. The median survivals of patients treated with chemotherapy were 12.3 months in patients with pulmonary metastasis alone and 5 months in those with both pulmonary and other visceral metastasis, respectively. Responders to chemotherapy survived longer than non-responders. Overall response rate was 42.7% (44/103), and the response rate of MDU (Mitomycin C+Dextran sulfate+Urokinase) (62.3%) was higher than other chemotherapeutic regimens, which suggested that appropriate chemotherapy would prolong the survival of patients with pulmonary metastasis from uterine carcinoma.

Highly specific anti-human colorectal carcinoma monoclonal antibody(A7) was developed by fusion of mouse myeloma cells with mouse spleen cells immunized by colon cancer cells. Neocarzinostatin (NCS) was bound to A7 preserving both antibody and drug activities. This conjugate (A7-NCS) was applied for clinical trial. No serious side effects were reported, and half of the eight patients with metastatic liver tumor responded to A7-NCS well. To overcome the variety in the expression of tumor-specific antigen on tumor cells, new types of conjugates were developed. Mitomycin C(MMC) was bound to Dextran sulphate. And this conjugate (M MC-Dex) was bound to A7 with the expectation that MMC would release from Dextran that was delivered to the surface of the cancer cells. This type of conjugate would be effective not only against cancer cells that express antigens detected by A7 but also against any cells around cancer cells detected by A7. The possibility and problems in cancer therapy using immunotoxin are discussed.