A20 is an early responding negative regulator of Toll-like receptor 5 signalling in intestinal epithelial cells during inflammation.

Several negative regulatory mechanisms control Toll-like receptor (TLR)-mediated inflammatory responses and restore immune system balance, including the zinc-finger protein A20, a negative regulator of TLR signalling that inhibits nuclear factor kappa B (NF-kappaB) activity. In the present study, we investigated TLR-5-mediated A20 expression and its role in intestinal epithelial cells (IECs) during inflammation. HCT-15 and HT-29 cells were stimulated with flagellin, then the expressions of A20, interleukin-1 receptor-associated kinase (IRAK-M) and Tollip were evaluated using RNase protection assay. Furthermore, experimental colitis was induced in tlr4-deficient CH3/HeJ mice by administration of dextran sodium sulphate (DSS), then flagellin was injected anally, and the colonic expression of A20 was examined by real-time polymerase chain reaction (PCR) and immunohistochemistry. To confirm flagellin-induced expression of A20, we employed an organ culture system. The role of A20 in flagellin-induced tolerance induction was evaluated in vitro, using a gene knock-down method targeting A20. A20 expression increased rapidly and peaked at 1 h after flagellin stimulation in cultured IECs, then declined gradually to the basal level. In vivo, anal injection of flagellin induced epithelial expression of A20 in injured colonic tissue, whereas flagellin did not cause a significant increase in A20 expression in non-injured normal tissue, which was also confirmed in vitro using the organ culture system. Gene knock-down using A20 siRNA did not influence tolerance induced by restimulation with flagellin. A20 is an early response negative regulator of TLR-5 signalling in IECs that functions during intestinal inflammation. Our results provide new insights into the negative feedback regulation of TLR-5 signalling that maintains the innate immune system in the gut.

Down-regulation of single immunoglobulin interleukin-1R-related molecule (SIGIRR)/TIR8 expression in intestinal epithelial cells during inflammation.

Single immunoglobulin (Ig) interleukin-1R-related molecule (SIGIRR) is an Ig-like membrane protein critical for negative regulation of Toll-like receptor (TLR)-4-mediated signalling. We investigated SIGIRR expression and its regulation mechanism in intestinal epithelial cells (IECs) during inflammation. Endoscopic biopsy specimens were obtained from active and inactive colonic mucosa of ulcerative colitis (UC) patients, then SIGIRR expression was examined using real-time polymerase chain reaction (PCR) and immunohistochemistry (IH). Mice experimental colitis models were established by administrations of sulphonic acid (TNBS) and dextran sodium sulphate (DSS), and epithelial expression of SIGIRR was examined using real-time PCR, IH and flow cytometry. The effects of lipopolysaccharide (LPS) and tumour necrosis factor (TNF)-α on SIGIRR expression were evaluated in vitro using cultured IECs. To elucidate SIGIRR expression regulation in IECs, binding ability of the transcription factor SP1 at the responsive element of the SIGIRR promoter was examined using gel-shift and chromatin immunoprecipitation (ChIP) assays. In human colonic samples, SIGIRR was expressed mainly in IECs at levels significantly higher in inactive compared to active mucosa. In the mice, SIGIRR colonic expression decreased rapidly after colitis development and returned gradually to basal levels. Experimental colitis-mediated down-regulation of SIGIRR in IECs was also confirmed by IH and flow cytometry results. Further, inflammatory conditions induced by TLR ligands and TNF-α caused significant down-regulation of SIGIRR expression in IECs, which was dependent upon decreased SP1 binding at the responsive element of the SIGIRR promoter. We found that SIGIRR is expressed in IECs and serves as a negative regulator to maintain gut innate immunity, which is down-regulated during inflammation by inhibition of an SP1-mediated pathway.

Evaluation of modified crystal violet chromoendoscopy procedure using new mucosal pit pattern classification for detection of Barrett's dysplastic lesions.

BACKGROUND: Pit pattern diagnosis is important for endoscopic detection of dysplastic Barrett's lesions, though using magnification endoscopy can be difficult and laborious. We investigated the usefulness of a modified crystal violet chromoendoscopy procedure and utilised a new pit pattern classification for diagnosis of dysplastic Barrett's lesions. METHODS: A total of 1,030 patients suspected of having a columnar lined oesophagus were examined, of whom 816 demonstrated a crystal violet-stained columnar lined oesophagus. The early group of patients underwent 0.05% crystal violet chromoendoscopy, while the later group was examined using 0.03% crystal violet with 3.0% acetate. A targeted biopsy of the columnar lined oesophagus was performed using crystal violet staining after making a diagnosis of closed or open type pit pattern with a newly proposed system of classification. The relationship between type of pit pattern and histologically identified dysplastic Barrett's lesions was evaluated. RESULTS: Dysplastic Barrett's lesions were identified in biopsy samples with an open type pit pattern with a sensitivity of 96.0%. Further, Barrett's mucosa with the intestinal predominant mucin phenotype was closely associated with the open type pit pattern (sensitivity 81.9%, specificity 95.6%). CONCLUSIONS: The new pit pattern classification for diagnosis of Barrett's mucosa was found to be useful for identification of cases with dysplastic lesions and possible malignant potential using a crystal violet chromoendoscopic procedure.

Properties of ADP-degrading activity of human placental syncytiotrophoblast brush border membrane vesicles.

The enzymatic properties of ADP (adenosine diphosphate) degradation in human placental syncytiotrophoblast brush border membrane vesicles (BBMV) were explored and the following results were obtained. BBMV had high ADP degrading activity compared to homogenate of placental villi. ADP degrading activity of BBMV; 1.05 +/- 0.05 mumol/mg protein/min placental villi was 21 times higher than that of homogenate of placental villi. Hydrolysis of ADP by BBMV follows Michaelis-Menten saturation kinetics with an apparent Km of 10.9 +/- 0.8 microM and Vmax of 2.10 +/- 0.17 mumol/mg protein/min. The enzyme has a divalent cation requirement. EDTA (2 mM) was found to abolish ADP degrading activity but this could be restored by the addition of either magnesium or calcium ions. Maximum enzyme activity of ADP degradation in BBMV was observed at a pH close to 8.0. The enzyme was insensitive to vanadate, levamisole, oligomycin, ouabain and N-ethylmaleimide (NEM), omeprazole and adenosine (5') pentaphospho (5') adenosine.

Characterization of human placental activity for transport of L-alanine, using brush border (microvillous) membrane vesicles.

To characterize the placental activity for the transport of amino acids, the uptake of L-alanine was investigated by rapid membrane filtration using brush border membrane vesicles separated from the human placenta of early pregnant (12-13 gestational weeks) and late pregnant (37-38 gestational weeks) women. The uptake of L-alanine into the brush border membrane vesicles at early and late stage of gestation showed a pattern of transport dependent on the intra- and extravesicular Na+ concentration gradient (extravesicular Na+ greater than intravesicular Na+). The Na+ concentration gradient-dependent uptake of L-alanine into the brush border membrane vesicles at early and late stage of gestation also showed a dependency on the potential difference of the internal and external membrane. The transport of L-alanine into the brush border membrane vesicles was markedly augmented at late stage of gestation. On the basis of the double reciprocal plotting of the L-alanine concentration and the Na(+)-dependent uptake of L-alanine into the vesicles, Km and Vmax were calculated as parameters of the Na(+)-dependent uptake of L-alanine into the vesicles. In the early stage of gestation Km was 0.78 mM, and in the late stage of gestation was 0.80 mM. In the early stage of gestation Vmax (nmol/mg protein/20 sec) was 0.62, and in the late stage of gestation was 3.53. From the results, it was considered that the placental active transport mechanism of L-alanine is the same in the early and late stages of gestation, and it was shown that its transport activity increases greatly in late stages compared to the early stages of gestation.

Characterization of human placental activity for transport of taurocholate, using brush border (microvillous) membrane vesicles.

The uptake of taurocholate into brush border membrane vesicles prepared from human full term placenta was studied using a rapid filtration technique. The taurocholate uptake into brush border membrane vesicles was sensitive to extravesicular osmolarity, and pre-incubation of the brush border membrane vesicles with the taurocholate increased the uptake of taurocholate into the brush border membrane vesicles. These findings indicate that the uptake of taurocholate by brush border membrane vesicles represents transport into vesicles. The uptake of taurocholate into vesicles was not dependent on Na+ electrochemical gradient (extravesicular > intravesicular). But this uptake was markedly increased when the intravesicular space was rendered electrically more positive by the use of lowly permeant anions or valinomycin-induced K+ diffusion membrane potentials. These findings indicate that the taurocholate transport into brush border membrane vesicles was dependent on membrane potential. The initial rate of taurocholate transport into brush border membrane vesicles exhibited saturation kinetics with respect to the taurocholate concentration, an apparent Km of 67 microM and Vmax of 0.30 nmol/mg protein/20 sec were calculated.

Detection of IL-6 in human milk and its involvement in IgA production.

A large amount of interleukin-6 (IL-6) was found to be contained in human whey. The concentration of IL-6 in colostrum was significantly higher than that in serum or in milk taken 1 month after parturition. Colostrum contained many more mononuclear cells than late milk. In terms of the proportion of monocytes, T cells and B cells, however, there is no difference between colostrum and late milk. There is a significantly positive correlation between the concentration of IL-6 and the number of mononuclear cells in milk. This demonstrates that IL-6 in whey is derived in part from mononuclear cells. Stimulation of human milk mononuclear cells by Staphylococcus aureus Cowan I in the presence of anti-IL-6 antibody markedly decreased the production of IgA. This suggests that IL-6 contained in milk is closely associated with the local production of IgA in the breast.

Interleukin-2 production by human fetal lymphocytes.

IL-2 production by PHA-stimulated human lymphocytes in umbilical blood was studied ontogenetically. Lymphocytes from the 16-week-old fetus produced appreciable amounts of interleukin-2 (IL-2). In 16- to 36-week-old fetuses the IL-2 production rate was found to be significantly higher than that in adults. When signal transduction, which is mediated by high affinity IL-2 receptors (IL-2R), was studied after adding exogenous IL-2 to PHA-blast lymphocytes, the response of the 24-week-old fetus was similar to that of adults. These results indicate that the attainment of IL-2 production and the function of IL-2 receptors are completed at a relatively early stage of fetal development.

IL-2 receptor expression and function on human cord blood mononuclear cells following PHA and anti-CD3 antibody stimulation.

Interleukin-2 receptors (IL-2R) on mononuclear cells from human umbilical cord blood were investigated after stimulation by phytohemagglutinin (PHA) and anti-CD3 antibody. The proportion of cells expressing the Tac antigen (IL-2R alpha, p55) did not differ from that for adult mononuclear cells. However, the levels of high affinity IL-2R (H-IL-2R) and low affinity IL-2R (L-IL-2R) present on the PHA blasts of cord blood cells were shown to be twice and three times, respectively, that of adults by interleukin-2 (IL-2) binding assay. The level of low affinity IL-2R in the cord blood cells was approximately double that of adult cells after activation by anti-CD3 antibody, but no difference was observed in the levels of high affinity IL-2R. Functional investigation of IL-2R revealed that the amount of internalized IL-2 in PHA-stimulated cord blood mononuclear cells was twice that in adult mononuclear cells but there were no differences between them in the time course of internalization and degradation. Although there were significant numbers of H-IL-2R in premature (25 and 28 weeks) infants following PHA stimulation, there were markedly fewer following anti-CD3 stimulation. Prematurity of the T-cell activation system through the CD3 pathway at this stage is therefore indicated.

Production of IL-6 (BSF-2/IFN beta 2) by mononuclear cells in premature and term infants.

The production of interleukin 6 (IL-6) was examined in premature neonates (48 cases, including 3 miscarried fetuses) and fullterm neonates (20 cases). The IL-6 production by mononuclear cells was measured after stimulation with Staphylococcus aureus Cowan Strain I (SAC), phytohemagglutinin (PHA) or lipopolysaccharide (LPS). The production in full-term neonates was similar to that in healthy adults, whereas it was significantly lower in premature neonates without premature rupture of the membrane (PROM). However, in premature infants with PROM a normal level of IL-6 production was observed in mononuclear cells stimulated with SAC, PHA and LPS. Furthermore, there was a positive correlation between the IgM concentration in the cord serum and IL-6 production by LPS-stimulated mononuclear cells.

Primary infection of Japanese infants with adult T-cell leukaemia-associated retrovirus (ATLV): evidence for viral transmission from mothers to children.

Primary infection with adult T-cell leukemia virus (ATLV) was investigated by follow-up studies on 16 ATLV-seropositive mothers and their breastfed infants in an ATLV-endemic area of Japan. Maternal antibody to ATLV decreased in all the infants, and was detectable in only three of 12 infants tested 6 months after birth. Reappearance of the antibody 9-18 months after birth was observed in only four of the 16 infants. The ATLV-bearing cells in peripheral blood were detected in all 16 mothers after delivery. None of the 16 infants showed ATLV-bearing cells in peripheral or cord blood sampled at birth, or 1, 3 or 6 months after birth. However, virus-bearing cells in the blood became detectable 9-18 months after birth in 13 of the 16 infants. Maternal antibody and virus-bearing cells were never detected in a control group of seven infants of ATLV-seronegative mothers. These findings provide evidence for the high incidence of primary ATLV infection during early infancy among infants born to ATLV-seropositive mothers and suggest maternal viral transmission. Furthermore, samples of breast milk from all 12 seropositive mothers examined contained cell-associated ATLV capable of being transmitted to peripheral leucocytes of neonates. This finding suggests that one of the possible maternal transmission routes of ATLV is via breast milk.

Bottle-feeding can prevent transmission of HTLV-I from mothers to their babies.

Breast-feeding is a major factor in the vertical transmission of human T-lymphotropic virus type I (HTLV-I). We studied whether such transmission may be prevented by bottle-feeding. HTLV-I infection was detected by both HTLV-I antigen and antibody tests. Thirty bottle-fed babies were examined 24 months after birth; only one was found to be HTLV-I antigen-positive. This infection rate was lower than that for breast-fed babies in whom HTLV-I antigen was detected in 24 of the 31 24-month-old babies born to HTLV-I positive mothers in a previous study. These results suggest that most vertical transmission of HTLV-I is attributable to breast-feeding and can be prevented by bottle-feeding.

Maxillary osteomyelitis secondary to osteopetrosis.

A 41-year-old Japanese woman complained of a gradually enlarging swelling of her left cheek for seven months. She was diagnosed with osteopetrosis by standard skeletal radiographs, and her cheek swelling was diagnosed as maxillary osteomyelitis secondary to osteopetrosis. She underwent a left partial maxillectomy, and her post-operative course was stable with no complications. A literature review is also presented.

[Fundamental and clinical study on ceftriaxone in the field of obstetrics and gynecology].

It is reported that ceftriaxone (Ro 13-9904, CTRX) has a half-life time of 7 to 8 hours. In the present study, the serum level 18 hours after intravenous injection with 1 g CTRX was as high as 9.3 micrograms/ml while obviously a higher tissue concentration was maintained compared with other drugs. These facts suggest that CTRX is effective against infections and that the dosage and frequency of administration could be reduced. The global evaluation revealed that CTRX was clinically effective in all the 4 cases with infections. As the adverse reaction, light leukopenia was observed only in 1 case out of the present 4 cases and 20 others administered with CTRX.

[Clinical studies on sulbactam/cefoperazone in the field of obstetrics and gynecology].

Sulbactam/cefoperazone (SBT/CPZ), a new developed antibiotic, was clinically studied. The following results were obtained. Total of 5 cases comprising 1 with endometritis, 3 with Bartholin's abscess, 1 with pyometra (due to corpus cancer) were treated with SBT/CPZ at a dose of 1 g twice daily for 4-5 days by intravenous injection or intravenous drip infusion. The clinical response was good in all cases. A case with pyometra did not respond to the therapy in bacteriologically. Side effects and abnormal laboratory findings due to the drug were not noted.

[Pharmacokinetic and clinical studies of cefodizime in the field of obstetrics and gynecology].

Cefodizime (THR-221, CDZM), a new antibiotic, was studied pharmacokinetically and clinically. The results obtained are summarized as follows: 1. Concentrations of CDZM in internal genital tissues were quite high after an intravenous infusion. 2. Clinical effects of the therapy with CDZM using intravenous infusion twice daily were evaluated in 1 patient with endometritis, 2 patients with pyometra, 1 patient with extragenital abscess and 1 patient with BARTHOLIN'S gland abscess. Clinical responses were good in all 5 patients. No side effects nor abnormal laboratory test values due to the drug were noted.

[Acute myelogenous leukemia developing in pregnancy with complete remission--a case report].

A 27-year-old woman was admitted to our hospital complaining of purpuras and legs' edema in the 38th week of pregnancy. On admission, the hemoglobin was 7.8 g/dl, platelets 20,000/microliter and WBC 6,600/microliters with 52% blast cells. Bone marrow aspirate demonstrated 77.2% myeloblasts with prominent Auer rods, consistent with acute myelogenous leukemia. Receiving packed-red-cell and platelet transfusions, she delivered a normal male infant in the 39th week of pregnancy by normal labor. After delivery, she was placed on a combination chemotherapy of BHAC-MMP, subsequently DNR added. Four weeks later, a complete remission was obtained, lasting for almost one year, and her child has grown well without hematological disorder. Microscopic findings of the placenta obtained at delivery revealed no invasion of leukemic cells, but 9% blast cells were present in the placental cord blood. We reviewed 18 cases reported in Japan of acute leukemia in gestational period, that could obtain complete remission and keep the children growing well. Placental transmission of leukemic cells from mother to infant was discussed.

[Experimental and clinical studies on laserthermia in head and neck cancers].

A synthetic ceramic probe allows a wider angle of irradiation and diffusion of low power energy, below 3W, using an interstitial technique for producing local hyperthermia of Nd: YAG laser (Laserthermia). We studied the influences of this laserthermia procedure in experimental animals and patients with head and neck cancer. The histological findings after laserthermia in the rabbit tongues showed no marked tissue damage in the heated area. Biochemical analysis of arachidonic acid cascade metabolites in these tissues revealed an immediate increase of 12-, 15- HETEs and PGE2 and 3 at days after hyperthermia, but at 8 days these metabolites returned to control levels. The effectiveness of laserthermia on human thyroid cancer implanted in nude mice was compared with that in cisplatin treatment and in combined therapy with both. The most predominant effectiveness was found in the animals receiving therapy combining cisplatin and laserthermia. Laserthermia in combination with radio-chemotherapy was applied in 21 cases with head and neck cancer. Good efficacy was obtained in 17 patients, especially in those with oropharyngeal cancer, showing CR in 4 cases and PR in 13 cases.

[A phase II study of Futraful suppository in head and neck cancer].

A phase II study of FT-207 suppository (Futraful Suppository) for head and neck cancer was conducted at the Kagoshima University Hospital Dept. of Otorhinolaryngology and its 6 affiliated hospitals. Forty cases with head and neck cancer were entered in the trial, of which 37 were perfectly evaluable. Partial response (PR) (16.7%) was achieved in one of 6 patients with maxillary cancer, in 2 of 9 cases with oral cancer (22.2%), in one of 7 with laryngeal cancer (14.3%), in 3 of 13 with pharyngeal cancer, while 2 of the same 13 achieved a complete response (CR) (38.5%), for a total response rate of 24.3%. FT-207 suppository was continuously administered as standard, at a dose of 750 mg each twice daily, or 1,500 mg/day. The response rate was dose-dependent; 2 CR cases with a total dose of 105 g for administration over a total period of more than 150 days. Adverse effects appearing in 35.0% of the overall were mainly anorexia, nausea and vomiting; none of them, however, were serious.