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.

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.

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.

Association of a decrease in antithrombin III activity with a perinatal elevation in aspartate aminotransferase in women with twin pregnancies: relevance to the HELLP syndrome.

BACKGROUND/AIMS: Decreased antithrombin III (AT-III) activity and/or thrombocytopenia associated with an elevated serum level of aspartate aminotransferase in late pregnancy can threaten the lives of both the mother and the fetus. We investigated whether antenatal declines in AT-III activity and the platelet count occur in late twin pregnancy and whether reduced AT-III activity and/or thrombocytopenia precedes an increase in the serum level of aspartate aminotransferase. METHODS: The platelet count, AT-III activity, and the serum level of aspartate aminotransferase were determined weekly or biweekly in 237 women with twin pregnancies in a longitudinal and partly prospective study. RESULTS: Both AT-III activity and the platelet count decreased gradually in the last month of pregnancy, irrespective of the presence or absence of clinical signs of pre-eclampsia. A perinatal elevation in aspartate aminotransferase occurred in 36 (15%) of 237 women. The risk of a perinatal elevation in aspartate amino-transferase increased as the antenatal AT-III activity and/or the platelet count decreased. Pre-eclampsia developed in 60 women (25%). The relative risk of a perinatal aspartate aminotransferase elevation (95% confidence interval) for the 60 women with pre-eclampsia, the 60 women with a platelet count < or = the 25th percentile (164 x 10(9)/1), and the 60 women with AT-III activity < or = the 25th percentile (76% of normal) was 1.9 (1.0 to 3.4), 4.1 (2.3 to 7.5), and 5.9 (3.2 to 11.1), respectively, compared with the remaining 177 women. CONCLUSIONS: AT-III activity and platelet count gradually decreased in the last month of twin pregnancies. A perinatal aspartate aminotransferase elevation was preceded by marked decreases in these parameters in women with twin pregnancies. The monitoring of AT-III activity and platelet count in women who exhibit a gradual decline in these parameters may help to avoid the development of severe HELLP syndrome.

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.

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 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.

[The significance of determination of human herpesvirus-6 (HHV-6) antibody during pregnancy].

HHV-6 which was detected in peripheral blood lymphocytes of lymphocytic patients is now said to be an agent causing exanthema subitum. We investigated the titer of HHV-6 antibody in 100 primigravida and 30 patients with spontaneous abortion by means of the indirect immunofluorescence method (IF method). The positive incidence in which the titer of HHV-6 antibody was more than 10 times in 100 primigravida was 82%. That of HHV-6 IgM antibody was 3% in those who had no complication during pregnancy. This supposes that there may be some women who were infected or were reactivated while they were pregnant. The positive incidence in 30 spontaneous abortion patients was 90%. 3 of them had IgM antibody to HHV-6, and HHV-6 antigen was detected on the epitherium of the chorionic villi by the IF method with HHV-6 monoclonal antibody in two of them. This suggests that the infection or reactivation of HHV-6 may be one of the causes of spontaneous abortions.

[The platelet aggregation inhibiting activity in human placenta--study on the ADP degrading activity of human placental villi].

We studied the platelet aggregation inhibiting activity and ADP degrading activity of human placental villi (tissue culture supernatant) and brush border membrane vesicles (BBMV) and obtained the following results. 1. There existed a platelet aggregation inhibiting activity in tissue culture supernatant of villi (S-villi) but not in that of decidua or amnion. The S-villi inhibited the platelet aggregation induced by ADP, but not that induced by collagen, arachidonic acid or ristocetin. And, there was also ADP degrading activity (ADPase activity) in the S-villi. ADP was quickly degraded by S-villi. When ADP was preincubated with S-villi, the platelet aggregation induced by ADP was completely lost. 2. There was very strong platelet aggregation inhibiting activity in placental BBMV. The BBMV almost completely inhibited the platelet aggregation induced by ADP, collagen, arachidonic acid and ristocetin. And there was very strong ADP degrading activity in the placental BBMV. ADP was quickly degraded by BBMV. When ADP was preincubated with BBMV, the platelet aggregation induced by ADP was completely lost. 3. The enzymatic character (heat stability, enzymatic kinetics, Ca++ dependency and pH dependency) of ADP degrading activity in BBMV was very similar to that in S-villi. 4. The ADP degrading activity of both S-villi and solubilized BBMV were fractionated by anion exchange column chromatography and gel filtration column chromatography in similar patterns, and it was shown that ADP degrading substance of both S-villi and solubilized BBMV had a molecular weight of about 60K.

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.

A study on the placental transport mechanism of vitamin K2 (MK-4).

The present study was primarily concerned with in vitro investigation of vitamin K2 (MK-4) transport using human placental villous tissues, with the objective of elucidating the placental transport mechanism of vitamin K2. 1. When vitamin K2 (MK-4) was not administered to gravidae, the observed concentrations of vitamin K2 were 0.32 +/- 0.07 ng/ml in the maternal venous blood, undetectable in umbilical venous blood, and 1.01 +/- 0.37 ng/g wet tissue in the placental villous tissue. 2. When vitamin K2 was administered to gravidae, (20 mg/day x 7 days per os), the corresponding concentrations were 0.89 +/- 0.21 ng/ml in the maternal venous blood, 0.18 +/- 0.06 ng/ml in umbilical venous blood, and 5.38 +/- 1.05 ng/g wet tissue in the placental villous tissue. 3. In vitro studies using placental villous tissue indicated that vitamin K2 transport into villous tissue is not active, but suggested the existence of a highly vitamin K2 specific transport system in the human placenta. On the other hand, the results of comparison studies of transport of vitamin K2 and vitamin K1 into villous tissue indicated that transport activity with respect to the former vitamin was higher. Moreover, comparison of vitamin K2 transport into villous tissue at the end of the first trimester and at full term revealed that transport was somewhat higher during the final stage. In summary, the above results indicated that whereas transport of vitamin K1 into the fetus is not especially pronounced, transport into the placental villous tissue is comparatively good.

A new instrument for use with Nd:YAG in tonsillectomy.

In tonsillectomy, unexpected bleeding can be one of the most troublesome surgical procedures to perform. To resolve some inherent problems, we have developed a new elevator with contact Nd:YAG laser probe for blunt dissection around the tonsillar capsule. Of 63 cases of laser tonsillectomies performed over 5 years, 51 cases were performed using conventional contact Nd:YAG laser probe and a chisel type dissector. Tonsillectomy in the remaining 12 cases was undertaken by the newly developed elevator with contact Nd:YAG laser probe. We compared clinical evaluation between laser and conventional procedures. Additionally, the advantage of laser tonsillectomy is discussed.

Changes in plasma levels of lipid peroxide and vitamin E during pregnancy.

The present study was undertaken in order to determine the changes in blood levels of lipid peroxide and vitamin E during pregnancy. 1. The man plasma level of lipid peroxide was 0.89 +/- 0.13 nmole/ml for non-pregnant women, while in pregnant women it was increased as the progressed pregnancy reaching 2.71 +/- 0.29 nmole/ml at full term. 2. The mean plasma, red cell and platelet levels of vitamin E were 6.6 +/- 0.9 micrograms/ml, 3.55 +/- 0.38 micrograms/ml packed cell and 99 +/- 25 micrograms/g protein for non-pregnant women, while in pregnant women the levels changed as the progressed pregnancy becoming 15.5 +/- 0.9 micrograms/ml, 2.43 +/- 0.24 micrograms/ml packed cell, and 244 +/- 19 micrograms/g protein at full term. It was shown in this study that during pregnancy the plasma level of lipid peroxide increased while the levels of vitamin E which inhibited the formation of lipid peroxide, increased in plasma and platelets, but decreased in red cells.

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.