Medium-chain fatty acids stimulate interleukin-8 production in Caco-2 cells with different mechanisms from long-chain fatty acids.

BACKGROUND AND AIM: It has been suggested that dietary fat exacerbates intestinal inflammation. We investigated the effect of fatty acids on interleukin (IL)-8 production in a human intestinal epithelial cell line (Caco-2). METHODS: The cells were cultured as monolayers on microporous membranes in culture inserts. Oleic acid (OA), capric acid (CA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were applied to the apical compartment of Caco-2 cell monolayers. The concentration of IL-8 in the basolateral medium was measured by using enzyme-linked immunosorbent assay, and the expression of IL-8 mRNA was measured by using competitive reverse transcription--polymerase chain reaction. Protein kinase C inhibitors (GF109203X and calphostin C) and H-7 (a protein kinase inhibitor) were used to study the mechanisms by which IL-8 production is stimulated. RESULTS: Both OA and CA enhanced IL-8 production (approximately fivefold), whereas DHA and EPA did not. Both OA and CA also enhanced IL-1-induced IL-8 production. The onset of OA-induced IL-8 production was delayed compared with that of CA-induced IL-8 production. Both OA and CA enhanced IL-8 mRNA expression (approximately fivefold) after 6 and 3 h, respectively. The protein kinase inhibitor (H-7) reduced both OA- and CA-induced IL-8 production by 88.0 and 85.9%, respectively. The protein kinase C inhibitors (GF109203X and calphostin C) reduced OA-induced IL-8 production by 29.3 and 54.5%, respectively, but showed no effect on CA-induced IL-8 production. CONCLUSIONS: These findings suggest that not only OA but also CA stimulates IL-8 production in intestinal epithelial cells, and the mechanisms of action differ between OA and CA.

Identification of a new point mutation in the human xanthine dehydrogenase gene responsible for a case of classical type I xanthinuria.

A 60-year-old Japanese man was diagnosed as having hypouricemia at an annual health check-up. The routine laboratory data was not remarkable except that the patient's hypouricemia and plasma levels of xanthine and hypoxanthine were much higher than those of normal subjects. Furthermore, the patient's daily urinary excretion of xanthine and hypoxanthine was markedly increased compared with reference values. The xanthine dehyrogenase activity of the duodenal mucosa was below the limits of detection. Nevertheless, allopurinol was metabolized to oxypurinol in vivo. Based on these findings, a subtype of classical xanthinuria (type I) was diagnosed. The xanthine dehyrogenase protein was detected by Western blotting analysis. Sequencing of the cDNA of the xanthine dehyrogenase obtained from the duodenal mucosa revealed that a point mutation of C to T had occurred in nucleotide 445. This changed codon 149 from CGC (Arg) to TGC (Cys), a finding that has not been previously reported in patients with classical xanthinuria type I.

Comparison of tests for fecal lactoferrin and fecal occult blood for colorectal diseases: a prospective pilot study.

OBJECTIVE: This prospective pilot study was conducted to compare the usefulness of measuring fecal lactoferrin (Lf) to that of fecal occult blood (FOB) test for detection of colorectal diseases. PATIENTS AND METHODS: The subjects were 351 patients who underwent colonoscopy. A fecal sample was obtained on the day before colonoscopy. Fecal Lf was measured by enzyme-linked immunosorbent assay. The FOB test was performed by combined assay (latex agglutination) of hemoglobin and transferrin. RESULTS: The specificities of the fecal Lf and FOB tests were the same (88.7%). For patients with colorectal cancer (13), colorectal polyp (69), ulcerative colitis (18), Crohn's disease (13), non-specific colitis (8), internal hemorrhoids (60), colon diverticulum (27), and miscellaneous diseases of the colon (10), the rates of positivity for fecal Lf were 7/13, 14/69, 12/18, 7/13, 4/8, 22/60, 8/27, and 6/10, respectively. The corresponding rates for FOB were 8/13, 12/69, 11/18, 4/13, 4/8, 9/60, 2/27, and 1/10. For patients with internal hemorrhoids, the rate of positivity for fecal Lf was significantly higher than that for FOB. In other disease groups, there was no significant difference in the rate of positivity between fecal Lf and FOB. CONCLUSION: These findings suggest that measurement of fecal Lf is as useful as FOB in detecting colorectal diseases.

Antineutrophil cytoplasmic antibodies in Japanese patients with inflammatory bowel disease: prevalence and recognition of putative antigens.

OBJECTIVE: Our aim was to investigate the prevalence of antineutrophil cytoplasmic antibodies (ANCA) in Japanese patients with ulcerative colitis (UC) and Crohn's disease (CD), and the putative antigens recognized by perinuclear staining pattern ANCA (p-ANCA)-positive sera. METHODS: Sera from UC (n = 52) and CD (n = 43) patients, and from healthy controls (n = 74) were studied. The indirect immunofluorescence (IIF) method was used for the detection of ANCA and its binding pattern. p-ANCA-positive sera were studied further for putative antigens. ELISAs using lactoferrin (Lf), myeloperoxidase (MPO), and cathepsin G (Cat G) as antigens were performed. RESULTS: ANCA was positive in 40 of the 52 (76.9%) UC (p-ANCA in 33) and in 32 of the 43 (74.4%) CD (p-ANCA in 31) patients. UC and CD patients showed significantly higher titers of p-ANCA than controls; however, no significant difference was observed between UC and CD. In UC, 23, 17, and nine of the 33 patients with p-ANCA-positive sera showed reactivity with Lf, MPO, and Cat-G, respectively. In CD, 21, 20, and 11 of the 31 patients with p-ANCA-positive sera showed reactivity with Lf, MPO, and Cat-G, respectively. Fourteen of the UC and six of the CD patients showed reactivity with two different antigens, and seven of the UC and 11 of the CD patients showed reactivity with all three antigens. The presence of anti-Lf and anti-MPO antibodies was further confirmed by Western blotting. CONCLUSIONS: ANCA is useful in distinguishing patients with IBD from normal subjects but is not sufficient for the differential diagnosis of CD and UC. p-ANCA reactivity might be derived from the recognition of heterogeneous neutrophil-associated antigens.

Congenital malformations due to antiepileptic drugs.

To identify the major risk factors for the increased incidence of congenital malformations in offspring of mothers being treated for epilepsy with antiepileptic drugs (AEDs) during pregnancy and, to determine the relative teratogenic risk of AEDs, we prospectively analyzed 983 offspring born in Japan, Italy, and Canada. The incidence of congenital malformations in offspring without drug exposure was 3.1%, versus an incidence with drug exposure of 9.0%. The highest incidence in offspring exposed to a single AED occurred with primidone (PRM; 14.3%), which was followed by valproate (VPA; 11.1%), phenytoin (PHT; 9.1%), carbamazepine (CBZ; 5.7%), and phenobarbital (PB; 5.1%). The VPA dose and level positively correlated with the incidence of malformations. This study first determined a cut-off value of VPA dose and level at 1000 mg/day and 70 microg/ml, respectively, to avoid the occurrence of malformations. The incidence of malformations increases as the number of drugs increases, and as the total daily dose increases. Specific combinations of AEDs such as VPA + CBZ and PHT + PRM + PB produced a higher incidence of congenital malformations. The incidence of malformations was not associated with any background factors studied except for the presence of malformations in siblings. These results indicate that the increased incidence of congenital malformations was caused primarily by AEDs, suggesting that malformations can be prevented by improvements in drug regimen, and by avoiding polypharmacy and high levels of VPA (more than 70 microg/ml) in the treatment of epileptic women of childbearimg age.

Thin cement mantle and osteolysis with a precoated stem.

The radiographic and clinical results of 55 hips (52 patients) implanted with the Harris precoat hip prosthesis using second generation cementing technique were investigated in this study. All metal backed sockets were fixed with cement. The preoperative diagnoses were osteoarthritis secondary to dysplastic hips in 42 patients, primary osteoarthritis in six, rapidly destructive coxarthrosis in five, and osteonecrosis of the femoral head in two. The average age of patients at the time of surgery was 67 years. The average duration of followup was 8 years. The rates of aseptic loosening and osteolysis in the femoral side were 5.5% (three hips) and 12.7% (seven hips), respectively. Three sockets were loosened, and no osteolysis was detected in the acetabular side. The mantle of cement in the femur was graded using the criteria described by Mulroy et al. Twenty-seven of 55 (49%) hips had regions with a thin cement mantle less than 1 mm in thickness or a defect in the cement mantle. Osteolysis was detected in seven of the 27 (26%) hips. Locations of osteolysis were coincidental with those of thin cement mantle or defect with the exception of one hip. This study clearly shows that a thin cement mantle less than 1 mm in thickness and a defect in the cement mantle lead to osteolysis.

Bile acids inhibit tumour necrosis factor alpha-induced interleukin-8 production in human colon epithelial cells.

To clarify the regulatory mechanism of the production of various inflammatory mediators by intestinal epithelial cells, the effect of bile acids (tauroursodeoxycholate, TUDC; taurochenodeoxycholate, TCDC; and taurocholate, TC) on the cytokine-induced production of interleukin (IL)-8 in a human colon epithelial cell line (HT-29) was examined. HT-29 cells were incubated for 24 h in a culture medium containing tumour necrosis factor alpha (TNF alpha; 1 ng/mL) and/or interleukin (IL)-1beta (1 ng/mL) in the presence or absence of bile acids. The IL-8 concentration in the medium was measured by an enzyme-linked immunosorbent assay. The binding assay of TNF alpha was performed using [125I]-TNF alpha (100 pmol/L). Interleukin-8 production during incubation with TNF alpha was markedly reduced in the presence of 0.5 and 1 mmol/LTUDC, 0.5 and 1 mmol/LTCDC and 0.5 and 1 mmol/LTC, by 56, 85, 86, 91, 37 and 70%, respectively. The IL-8 production during incubation with IL-1beta was not significantly reduced in the presence of these bile acids. The specific binding of TNF alpha to cells was inhibited 33, 47, and 14% by 1 mmol/LTUDC, TCDC and TC, respectively. These findings suggest that bile acids inhibit TNF alpha-induced IL-8 production by the colonic cells. The suppression may be partly due to inhibition of TNF alpha binding to the cells by bile acids.

DNA ploidy, c-myc oncoprotein p62 and v-H-ras oncoprotein p21 in colorectal carcinoma.

DNA ploidy and expression of the c-myc oncoprotein p62 and the v-H-ras oncoprotein p21 were examined in 54 colorectal carcinomas. DNA ploidy, determined by DNA flow cytometry, was diploid in 19 samples and aneuploid in 35. Expression of the p62 oncoprotein, determined by immunohistochemical staining, was intensely positive in 18 samples while that of the p21 oncoprotein, also determined by immunohistochemical staining, was positive in 29. There was no correlation between DNA ploidy and expression of the p62 oncoprotein, and DNA ploidy did not correlate with expression of the p21 oncoprotein. There was, however, a close correlation between expression of the p62 oncoprotein and that of the p21 oncoprotein being P < 0.01 according to Peason's chi-square test.

Teratogenicity of antiepileptic drugs: analysis of possible risk factors.

To determine the primary factors responsible for the increased incidence of malformation in the off-spring of antiepileptic drug (AED)-treated epileptic mothers, special attention was paid to drug combinations in a prospective study of 172 deliveries. Variables used for analysis were eight antiepileptic drugs (AEDs) and total daily dosages (drug score), and seven background factors consisting of maternal age at delivery, gravida, outcome of previous pregnancy, etiology and type of epilepsy, occurrence of seizures in the first trimester of pregnancy, and seizure frequency during pregnancy. The overall rate of malformation was 14.0%. Thirty-one patients were administered a single drug, and the rate of malformation was 6.5%. The remaining 141 patients were treated with multiple AEDs, and the rate of malformation was 15.6%. The drug score of the latter group was significantly higher than the former (p = 0.01). There was no definite dose-dependent increase in the incidence of malformations associated with any individual AEDs. There was no relationship between the type of defect and individual AEDs. Wilcoxon rank-sum test revealed significant association between the drug score, valproate (VPA), and congenital malformation. Carbamazepine (CBZ) also reached an almost significant level. Furthermore, VPA polypharmacy produced the highest incidence of malformation, higher than that produced by any other AED or drug combination. There was no significant association between the presence of malformations and the other putative risk factors. These results suggest that high dose of AEDs reflecting polypharmacy, VPA polypharmacy in particular, are primary factors responsible for the increased incidence of congenital malformation in the offspring of treated epileptic mothers.

Metabolism of non-protein energy-substrates in septic rats receiving parenteral nutrition.

The influences of sepsis on the metabolism of fatty acid and glucose in rats receiving parenteral nutrition were investigated. The caecum, with its blood supply, was ligated in 10 rats to produce peritonitis and sepsis (septic rats). Twelve rats (control rats) did not undergo this procedure. Five septic rats and six control rats received glucose as a sole nonprotein calorie (septic-glucose rats), and the remaining five septic rats (septic-lipid rats) and six control rats received the same parenteral solution for the first 44 hours, but 25% of the nonprotein calorie was replaced by 10% lipid emulsion for the last 24 hours. At the termination of the parenteral nutrition, 14C-linoleic acid or 14C-glucose was injected as a bolus in the tail vein, and their degradations to 14CO2 and incorporations into the endogenous fat were compared among the three groups. It was demonstrated that the sepsis accelerated the oxidation of fatty acid but did not affect that of glucose. Hepatic lipogenesis with both fatty acid and glucose was accelerated by an infusion of glucose under a septic condition, while it was inhibited by an infusion of lipid emulsion.

An immunohistochemical study on the river lamprey retina.

The localization of structures immunoreactive to various polypeptides and proteins in the retina of the river lamprey (Lampetra japonica) was investigated by means of an indirect immunohistofluorescence method. In the majority of frozen sections, a subpopulation of amacrine cells showed the immunoreactivity (IR) to one of the examined antisera against corticotropin-releasing factor, glucagon, neuropeptide Y, somatostatin, visinin and 5-hydroxytryptamine, respectively. A few fibers in the inner plexiform layer were immunoreactive to cholecystokinin or substance P antiserum. The visinin-like IR was also found in two types of bipolar cells. The IR was positive in Müller cells against glutamine synthetase but not to glial fibrillary acidic protein. In some flat-mounted preparations, glucagon- and serotonin-like reactive amacrine cells and visinin-like immunoreactive bipolar cells (distally located) could be observed. The results obtained suggest that in the lamprey retina the cytoplasmic property of some cells is similar to, but that of others is different from, vertebrate retinal cells.

[Adenosquamous carcinoma of the stomach].

The appearance of adenosquamous structures in cancer of the stomach was studied in 385 resected specimens. Such structures were observed in 9.1% of the specimens. However, they were not found in cases in which the invasion was limited to the mucosa. The frequency of the appearance increased in correlation with the extent of the cancerous invasion. The type of adenocarcinoma that coexisted with the squamous changes was the intestinal type of Lauren in 68% of the specimens. This indicates that the squamous structures, seen in gastric cancer result from secondary metaplasia of the intestinal type in this condition.

Nitrogen-sparing effect of lipid emulsion in septic dogs.

Effects of intravenously administered lipid emulsion on the nitrogen balance in dogs with intraabdominal infection were investigated. The nitrogen balance in dogs supported by parenteral nutrition (PN) with glucose alone was superior to that in dogs supported by PN with glucose and lipid emulsion, in the absence of intraabdominal infection. On the other hand, the nitrogen balance in dogs supported by PN with glucose and lipid emulsion was superior to that with glucose alone, in the presence of intraabdominal infection. Dogs with intraabdominal infection had an insulin-resistance inability to effectively utilize glucose.

Effects of hemodynamic changes induced by hyper- or hypothermia on intravenous lipid clearance rate and lipoprotein lipase activity in dogs.

Hyper- and hypothermia was induced in dogs by peritoneal perfusion with warm or cold Lactate-Ringer's solution, the objective being to alter cardiac output. Changes in cardiac index, intravenous lipid clearance rate (K-value), and lipoprotein lipase (LPL) activity, concomitantly with changes of the temperature of the mixed venous blood were investigated. The cardiac index increased significantly with the hyperthermia and there was a close correlation between the cardiac index and the K-value. The LPL activity did not change significantly with changes in blood temperature, and the correlation between the LPL activity and the K-value was not significant. The hemodynamics has to be considered when attempting to discuss the intravenous lipid clearance rate.

Clearance rate of intravenously administered lipid in postoperative patients.

Elimination rate (K) of intravenously given triglyceride was studied in seventeen patients who underwent elective abdominal operations, and levels of adrenaline, noradrenaline, insulin and blood sugar were simultaneously measured, as stress-indices. All these indices, except for the insulin/blood sugar ratio, increased post-operatively. The postoperative K value increased significantly in comparison with the preoperative value. There was no significant correlation between K value and any of the stress-indices. These results suggest that the initial catabolism of the lipid emulsion triglyceride is enhanced by surgical stress.

In vitro induction of HLA-restricted cytotoxic T lymphocytes against autologous Epstein-Barr Virus transformed B lymphoblastoid cell line.

Cytotoxic T lymphocytes (CTL) against autologous EBV-transformed B lymphoblastoid cell line (LCL) were induced in vitro by culturing peripheral blood lymphocytes (PBL) of healthy donors together with mitomycin C-treated autologous LCL for 6 days. The cytotoxic cells developed only from the E-rosette-positive fraction but not from the negative fraction of PBL. These CTL killed autologous LCL but not PWM-stimulated autologous PBL. In addition, the CTL killed allogeneic LCL when at least 1 of the HLA-A antigens was identical with that of the LCL of CTL donor. However, identity of HLA-B and HLA-C antigens was not enough for a significant killing of allogeneic LCL. The specificity of the CTL was also confirmed by a cold target inhibition test. These results indicated that the CTL induced specifically recognized EBV-transformed cells with HLA restriction.

Regulation of the in vitro secondary cell-mediated cytotoxic response against syngeneic FBL-3 leukemia by macrophages.

Depletion of macrophages from immune spleen cells by treatment with carbonyl iron and magnet or by in vivo treatment with carrageenan enhanced the in vitro secondary cell-mediated cytotoxic response against a syngeneic Friend virus-induced leukemia, FBL-3 cells of C57BL/6 mice. However, further depletion of macrophages by passing the carbonyl iron-treated immune spleen cells through a nylon wool column abrogated the cytotoxic response. The addition of splenic macrophage-enriched preparations from either FBL-3-immune or normal mice suppressed the cytotoxic response of immune spleen cells treated with carbonyl iron and magnet. This suppressive effect of splenic macrophages presented a marked contrast with the enhancing effect of normal peritoneal macrophages on the same cell-mediated cytotoxic response, indicating regulation of the generation of killer T cells against a syngeneic tumor by functionally distinct macrophages. The suppressed cell-mediated cytotoxic response against FBL-3 cells by immune spleen cells was augmented by the addition of indomethacin to the culture medium, and this augmentation with indomethacin was greatly decreased by depletion of phagocytic cells from the immune spleen by treatment with carbonyl iron and magnet. The mechanisms of regulation of the cell-mediated cytotoxic response with soluble factors released from macrophages are discussed.

LPS-or 8Br-cyclic AMP-induced production of T cell-activating factor(s) in macrophage tumor cell line J774.1.

The macrophage tumor cell line J774.1 replaced the function of normal macrophages in the induction of polyclonal killer T cells with 2-mercaptoethanol. J774.1 does not normally release soluble factor(s) which we have shown to be responsible for the differentiation of T cells to killer T cells. However, stimulation of J774.1 with LPS induced soluble factor(s) for T cell activation. An optimum concentration of LPS for the production of soluble factor(s) was 1 to 10 microgram/ml, which completely inhibited growth of the tumor cells. The production of soluble factor(s) was observed within 6 hr after LPS stimulation and reached its maximum level at 24 hr. Incubation of the cell line with 8Br-cyclic AMP and theophylline induced soluble factor(s), suggesting that LPS stimulation induced an increase in intracellular cyclic AMP which leads to the synthesis of soluble factor(s).