Hemoadsorption of high-mobility group box chromosomal protein 1 using a column for large animals.

BACKGROUND: High-mobility group box chromosomal protein 1 (HMGB1) has recently been identified as an important mediator of various kinds of acute and chronic inflammation. A method for efficiently removing HMGB1 from the systemic circulation could be a promising therapy for HMGB1-mediated inflammatory diseases. MATERIALS AND METHODS: In this study, we produced a new adsorbent material by chemically treating polystyrene fiber. We first determined whether the adsorbent material efficiently adsorbed HMGB1 in vitro using a bovine HMGB1 solution and a plasma sample from a swine model of acute liver failure. We then constructed a column by embedding fabric sheets of the newly developed fibers into a cartridge and tested the ability of the column to reduce plasma HMGB1 levels during a 4-hour extracorporeal hemoperfusion in a swine model of acute liver failure. RESULTS: The in vitro adsorption test of the new fiber showed high performance for HMGB1 adsorption (96% adsorption in the bovine HMGB1 solution and 94% in the acute liver failure swine plasma, 2 h incubation at 37°C; p < 0.05 vs. incubation with no adsorbent). In the in vivo study, the ratio of the HMGB1 concentration at the outlet versus the inlet of the column was significantly lower in swine hemoperfused with the newly developed column (53 and 61% at the beginning and end of perfusion, respectively) than in those animals hemoperfused with the control column (94 and 93% at the beginning and end of perfusion, respectively; p < 0.05). Moreover, the normalized plasma level of HMGB1 was significantly lower during perfusion with the new column than with the control column (p < 0.05 at 1, 2, and 3 h after initiation of perfusion). CONCLUSION: These data suggest that the newly developed column has the potential to effectively adsorb HMGB1 during hemoperfusion in swine.

Granulocyte and monocyte adsorption apheresis as an effective treatment for Reiter disease.

Reiter disease (RD) is characterized by a triad of sterile arthritis, urethritis and conjunctivitis. The conditions occur concomitantly or sequentially, and are associated with mucocutaneous features such as circinate balanitis and stomatitis. Arthritis usually occurs in attacks followed by recovery, but it sometimes progresses to permanent damage of the affected joints. Because the symptoms of this disorder are attributable to activated neutrophils, we assessed the efficacy of granulocyte and monocyte adsorption apheresis (GCAP) in a 73-year-old man with RD who had skin rashes on his penis, scrotum and right hand, with severe arthralgia. The patient's skin rash and joint pain responded dramatically to five sessions of GCAP delivered at intervals of 5 days. We present a detailed description of the patient and discuss the mechanisms of GCAP, and suggest that GCAP may be useful for treating RD.

Mitigation of occult lung injury by pneumonectomy via minithoracotomy in mice.

BACKGROUND: The systemic and pulmonary inflammatory response associated with pneumonectomy performed via minithoracotomy versus that after open posterolateral thoracotomy is uncertain. METHODS: Groups consisting of 7 randomly assigned mice underwent a) minithoracotomy (with 5-mm long incisions and sparing of the muscles) alone, b) posterolateral thoracotomy (with 20-mm long incisions) alone, c) pneumonectomy via minithoracotomy, or d) pneumonectomy via posterolateral thoracotomy. The animals' daily food intake, body weight changes and spontaneous activity were monitored for 10 days, and lung water accumulation and vascular hyperpermeability in the remaining right lung were measured at 24 h after surgery. Concentrations of high mobility group box 1 protein (HMGB1), a mediator of inflammation and shock, were measured in the bronchoalveolar lavage fluid. RESULTS: Compared with posterolateral thoracotomy, pneumonectomy via minithoracotomy was associated with significantly less weight loss (p < 0.05), despite a similar daily food intake among the groups. Spontaneous activity after pneumonectomy via minithoracotomy returned earlier than after posterolateral thoracotomy. Pulmonary vascular hyperpermeability and water retention in the residual lung were significantly less prominent after pneumonectomy performed via minithoracotomy than after pneumonectomy via posterolateral thoracotomy (both comparisons p < 0.05). HMGB1 concentrations in the bronchoalveolar lavage fluid collected from the residual lung were significantly lower (p < 0.05) after minithoracotomy than after posterolateral thoracotomy. CONCLUSIONS: Based on postoperative weight loss, spontaneous activity, and the degree of pulmonary capillary injury in the residual lung, pneumonectomy via minithoracotomy was less invasive than posterolateral thoracotomy. The lower increase in HMGB1 associated with minithoracotomy might result in lower pulmonary vascular hyperpermeability and reflect less surgical invasiveness.

Increased plasma levels of high mobility group box 1 in patients with acute liver failure.

BACKGROUND: High-mobility group box 1 (HMGB1) is a monocyte-derived late-acting inflammatory mediator, which is released in conditions such as shock, tissue injury and endotoxin-induced lethality. In this study, we determined the plasma and hepatic tissue levels of HMGB1 in patients with acute liver failure (ALF). PATIENTS AND METHODS: We determined the plasma levels of HMGB1 and aspartate aminotransferase (AST) in 7 healthy volunteers (HVs), 40 patients with liver cirrhosis (LC), 37 patients with chronic hepatitis (CH), 18 patients with severe acute hepatitis (AH), and 14 patients with fulminant hepatitis (FH). The 14 patients with FH were divided into two subgroups depending upon the history of plasma exchange (PE) before their plasma sample collection. The hepatic levels of HMGB1 were measured in tissue samples from 3 patients with FH who underwent living-donor liver transplantation and from 3 healthy living donors. Hepatic tissue samples were also subjected to immunohistochemical examination for HMGB1. RESULTS: The plasma levels of HMGB1 (ng/ml) were higher in patients with liver diseases, especially in FH patients with no history of PE, than in HVs (0.3 ± 0.3 in HVs, 4.0 ± 2.0 in LC, 5.2 ± 2.6 in CH, 8.6 ± 4.8 in severe AH, 7.8 ± 2.7 in FH with a history of PE, and 12.5 ± 2.6 in FH with no history of PE, p < 0.05 in each comparison). There was a strong and statistically significant relationship between the mean plasma HMGB1 level and the logarithm of the mean AST level (R = 0.900, p < 0.05). The hepatic tissue levels of HMGB1 (ng/mg tissue protein) were lower in patients with FH than in healthy donors (539 ± 116 in FH vs. 874 ± 81 in healthy donors, p < 0.05). Immunohistochemical staining for HMGB1 was strong and clear in the nuclei of hepatocytes in liver sections from healthy donors, but little staining in either nuclei or cytoplasm was evident in specimens from patients with FH. CONCLUSION: We confirmed that plasma HMGB1 levels were increased in patients with ALF. Based on a comparison between HMGB1 contents in normal and ALF livers, it is very likely that HMGB1 is released from injured liver tissue.

1,5-Anhydroglucitol attenuates cytokine release and protects mice with type 2 diabetes from inflammatory reactions.

1,5-anhydroglucitol (1,5-AG) decreases in diabetic patients and is used as a marker of glycemic control. Type 2 diabetic patients are susceptibile to lipopolysaccharides (LPS), which stimulate macrophages to release large quantities of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6. This study examines the effects of 1,5-AG on lung inflammation induced by LPS and consequent systemic inflammation to determine whether the decrease of 1,5-AG concentration induces susceptibility to LPS. Before the challenge with LPS (1 mg/kg in vivo and 500 ng/ml in vitro), we pretreated db/db mice and RAW264.7 cells with 1,5-AG at 38.5 mg/kg and 500 microg/ml, respectively. The levels of IL-6, TNF-alpha, macrophage chemoattractant protein (MCP)-1 and IL-1beta in the serum and in the cell supernatants were measured. We also measured macrophage recruitment and the expression of inducible nitric oxide synthase (iNOS) in pulmonary tissues. We found that 1,5-AG attenuated serum cytokine release and protected db/db mice from LPS-induced pulmonary inflammation. In addition, 1,5-AG suppressed cytokine release and iNOS expression by suppressing Akt/NF-kB activity in RAW264.7 cells. These results suggest that 1,5-AG may be a mediator in, as well as marker for diabetes, and 1,5-AG intake may confer tolerance to LPS in patients with type 2 diabetes.

Thrombomodulin is found on endothelium of arteries, veins, capillaries, and lymphatics, and on syncytiotrophoblast of human placenta.

We have used antibodies to human thrombomodulin isolated from placenta to investigate the distribution of this cofactor for protein C activation in human tissues. Thrombomodulin was found on endothelial cells of arteries, veins, capillaries, and lymphatics by immunocytochemical staining using an avidin-biotin peroxidase method. Thrombomodulin was not detected on sinusoidal lining cells of liver or on postcapillary high-endothelial venules of lymph node, although the latter contained another endothelial antigen, von Willebrand factor. Other cells noted to contain thrombomodulin antigen are those of the syncytiotrophoblast in placenta. The thrombomodulin in syncytiotrophoblast was primarily on the plasma membrane surface that forms the maternal blood sinus. Syncytiotrophoblast also stained with antibodies to von Willebrand factor, which implies that these cells have multiple endothelial functions. Thrombomodulin antigen was found in all organs studied, with the notable exception of brain.

The role of water channel aquaporin 3 in the mechanism of TNF-alpha-mediated proinflammatory events: Implication in periodontal inflammation.

Aquaporin 3 (AQP3) is the predominant water channel protein in human keratinocytes and acts as an inflammatory mediator in some lesions. A chronic, inflammatory process of periodontitis is related with a dramatic change of surrounding fluid homeostasis to plasma extravasation. The exact pattern of aquaporin (AQP) water channel expression and its mechanism in periodontal disease is still unknown. We describe herein an up-regulated AQP3 expression in the epithelial lesion with chronic periodontitis and its functional role. The levels of AQP3 expression in inflamed gingival epithelial tissues were significantly higher than those of healthy subjects. Consistent with these results, AQP3 expression (i.e., levels of mRNA and protein) in cultured rat primary gingival epithelial cells and the human gingival epithelial cell line Ca9-22 were strongly increased in response to TNF-alpha treatment through the 55 kDa TNF-alpha receptor (TNFR I). In this context, small interfering RNA- (siRNA)-mediated "aqp-3 gene silencing," which could reduce AQP3 expression by more than 65%, significantly attenuated selected proinflammatory events of ICAM-1 expression induced by TNF-alpha in Ca9-22. A sixfold increase in leukocyte adherence to TNF-alpha-stimulated epithelial cells was demonstrated by an adherence assay (P < 0.001) and pretreatment with AQP3 siRNA and anti-ICAM-1 antibody reduced leukocyte retention by 85% (P < 0.001). Our study indicates for the first time a novel important mode in the regulation of the inflammatory response through TNF-alpha/TNFR I ligation at the site of epithelial lesions by specialized membrane channel AQP3 and ICAM-1 protein, which is closely implicated in the development of periodontitis mechanisms.

Tumor necrosis factor-alpha stimulates gingival epithelial cells to release high mobility-group box 1.

BACKGROUND AND OBJECTIVE: High-mobility-group box 1 functions as a late-phase inflammatory mediator. It can be released extracellularly by macrophages and necrotic cells through lipopolysaccharide and tumor necrosis factor-alpha. The objective of this study was to clarify the source of high-mobility-group box 1 in chronic periodontitis tissues and tumor necrosis factor-alpha-stimulated gingival epithelial cells, and subsequently elucidate its inducible inflammatory pathway. MATERIAL AND METHODS: Chronic periodontitis and healthy gingival sections were stained for high-mobility-group box 1 by immunohistochemistry and immunofluorescence. The amounts of high-mobility-group box 1 released into the gingival crevicular fluid and supernatants from gingival epithelial cells stimulated by tumor necrosis factor-alpha were examined by western blot. The phosphorylation of mitogen-activated protein kinases (MAPKs) in gingival epithelial cells was also examined. RESULTS: High-mobility-group box 1 was detected in the cytoplasm and nucleus of gingival epithelial cells with periodontitis. Western blotting revealed a significant increase in high-mobility-group box 1 expression in the gingival crevicular fluid from periodontitis patients. High-mobility-group box 1 production in gingival epithelial cells was increased following stimulation with tumor necrosis factor-alpha. The molecular dialogue between tumor necrosis factor-alpha and gingival epithelial cells involved modulation of the activities of p38MAPK, Jun N-terminal kinase and p44/42. Interestingly, only phosphorylation of p38MAPK contributed to more than half of the signaling initiated by tumor necrosis factor-alpha-elicited high-mobility-group box 1 release. CONCLUSION: High-mobility-group box 1 is continuously released from the gingival epithelial cells modulated by tumor necrosis factor-alpha. These findings imply that high-mobility-group box 1 expression and possibly p38MAPK constitute important features in periodontitis.

Fe-oxide concretions formed by interacting carbonate and acidic waters on Earth and Mars.

Spherical Fe-oxide concretions on Earth, especially in Utah, USA, have been investigated as an analog of hematite spherules found in Meridiani Planum on Mars to support interpretations of water-rock interactions in early Mars. Although several formation mechanisms have been proposed for the Fe-oxide concretions on Earth, it is still unclear whether these mechanisms are viable because a precise formation process and precursor of the concretions are missing. This paper presents evidence that Fe-oxide concretions in Utah and newly found Fe-oxide concretions in Mongolia had spherical calcite concretions as precursors. Different formation stages of calcite and Fe-oxide concretions observed, both in Utah and Mongolia, indicate that calcite concretions initially formed within eolian sandstone strata and were dissolved by infiltrating Fe-rich acidic waters to form spherical FeO(OH) crusts due to pH buffering. The similarity between these Fe-oxide concretions on Earth and the hematite spherule occurrences in Meridiani Planum, combined with evidence of acid sulfate water influences on Mars, suggest that the hematite spherules also formed from dissolution of preexisting carbonate spherules possibly formed under a dense carbon dioxide early martian atmosphere.

Coagulation factor Va binds to human umbilical vein endothelial cells and accelerates protein C activation.

In vitro the rate of protein C activation by thrombin is significantly accelerated by two distinct cofactors (a) the endothelial cell surface protein, thrombomodulin, and (b) human coagulation Factor Va. We have recently reported that the activity of Factor Va is contained in the 78,000-D light chain. In this study we have investigated the effects of Factor Va and its light chain on the activation of protein C in the presence of cultured endothelial cells. Thrombin-catalyzed protein C activation on human umbilical vein endothelial cells was enhanced by Factor Va. The ability of Factor Va to stimulate protein C activation on these cells was saturated at 50 nM Factor Va and was observed at several protein C concentrations. Isolated Factor Va light chain in concentrations up to 50 nM also accelerated protein C activation on endothelial cells, but higher concentrations inhibited the reaction. The effects of Factor Va or its light chain on protein C activation were also shown on a mouse hemangioma cell line but not on human fibroblasts nor on a human amelanotic melanoma cell line. Protein C activation on endothelial cells was partially inhibited by a goat anti-thrombomodulin antibody and further addition of a polyclonal rabbit anti-Factor V(Va) antibody resulted in additional inhibition. Endothelial cells grown in medium supplemented with human serum, devoid of Factor V coagulant activity, contained cell surface Factor V(Va) (approximately 15,000 molecules/cell) as measured by the binding of a monoclonal IgG directed against Factor V(Va). These cells also bound an additional 6,000-10,000 molecules Factor Va per cell as determined by direct binding studies using 125I-Factor Va. We suggest that thrombomodulin and Factor Va act in concert to regulate protein C activation on the surface of endothelial cells.

Human thrombomodulin is not an efficient inhibitor of the procoagulant activity of thrombin.

The effect of human thrombomodulin isolated from placenta on the procoagulant activity of thrombin was studied and compared to that of rabbit thrombomodulin. The isolated protein was proved to be thrombomodulin because a rabbit antibody against the isolated protein blocked protein C activation by thrombomodulin in solution and also blocked the protein-C-activating cofactor activity of human umbilical vein endothelial cells. The affinity of human thrombomodulin for human thrombin in the presence of fibrinogen is 30 times less than that of rabbit thrombomodulin. This value is based on the measurements of the clotting time of human fibrinogen and thrombin in the presence of increasing amounts of thrombomodulin. Human thrombomodulin was also much less effective compared with rabbit thrombomodulin in inhibiting thrombin-induced human coagulation factor V activation. The ability to inhibit release of [3H]serotonin from washed human platelets was at least 10 times less using human thrombomodulin compared with rabbit thrombomodulin. A partially purified preparation of human lung thrombomodulin was also relatively ineffective in inhibiting thrombin-induced serotonin release from platelets, indicating that the difference between rabbit and human thrombomodulin is one of species rather than of tissue. Thus, while human thrombomodulin is a potent cofactor in protein C activation, it is not an efficient inhibitor of the procoagulant actions of thrombin.

Overgrowth of human synovial cells driven by the human T cell leukemia virus type I tax gene.

One of the salient pathological features of rheumatoid arthritis is synovial cell proliferation with bone erosion. Despite extensive investigation, the factors essential for synovial cell proliferation remain to be identified. Recent studies suggest that human T cell leukemia virus type I (HTLV-I) may play an important role in synovial overgrowth observed in patients with one type of chronic inflammatory synovitis. In order to confirm and extend these observations, we have established synovial cell clones (SCCs) from three HTLV-I carriers who demonstrated synovial overgrowth but were otherwise asymptomatic. HTLV-I proviral DNA randomly integrated into the cellular genome was present in 20-30% of SCCs. The SCCs carrying HTLV-I proviral DNA and expressing the tax gene exhibited high levels of proliferative potential. HTLV-I was found to function as a transcriptional trans-activator in these SCCs. Moreover, transfection of the tax expression plasmid into SCCs resulted in the same phenotype of increased proliferation and cytokine expression as exhibited by HTLV-I provirus-carrying and tax-expressing SCCs. These data suggest that tax plays a critical role not only in leukemogenesis but also in synovial overgrowth in humans.

Biosynthesis of coagulation Factor V by a human hepatocellular carcinoma cell line.

A human hepatocellular carcinoma line, HepG2, was found to secrete coagulation Factor V. Factor V activity in HepG2 culture fluid increased nearly linearly during a 20-h time course (5 ng Factor Va/h per 10(6) cells). Thrombin treatment increased Factor V activity in HepG2 culture medium six- to ninefold, indicating that the medium accumulates a mixture of Factors V and Va. To demonstrate de novo synthesis of Factor V, HepG2 cells were incubated in culture medium containing [35S]methionine. Labeled Factor V was immunoprecipitated from the medium and was shown to co-migrate with purified plasma Factor V upon sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography. When medium was treated with thrombin before immunoprecipitation and fluorography, the 330,000-Mr [35S]methionine-labeled Factor V was converted to Factor Va. Factor Va coagulant activities from HepG2 cells and human plasma were inhibited in parallel by anti-Factor V antibody, indicating that HepG2 and plasma Factor Va have the same intrinsic activity. If normal hepatocytes synthesize Factor V at the same rate as HepG2 cells, then hepatocyte secretion can account for the total Factor V present in plasma. The production of Factor V by cultured human umbilical vein endothelial cells was also examined. Spent culture medium from endothelial cells contained only Factor Va and the amount was less than 1% of the activity found in medium from HepG2 cells under comparable conditions. The amount of Factor V activity in endothelial cell culture fluid did not change with time in culture.

Detection of human T cell lymphotropic virus type I proviral DNA and its gene expression in synovial cells in chronic inflammatory arthropathy.

To investigate the pathogenesis of human T cell lymphotropic virus type I (HTLV-I)-associated chronic inflammatory arthropathy (HAAP), we sought to detect proviral DNA in the articular lesions. For the detection of proviral DNA, we used the polymerase chain reaction (PCR). Proviral DNA was detected not only in the peripheral blood mononuclear cells (PBMCs) and synovial fluid cells (SFCs), but also in the T lymphocyte-depleted cultured synovial cells (CSCs). These findings suggest that the infection by HTLV-I might occur in vivo in non-T cells. Furthermore, we detected HTLV-I tax1/rex1 messenger RNA in fresh synovial tissues and CSCs but not in fresh PBMCs and fresh SFCs using reverse transcription and PCR. Immunohistochemically, the CSCs from HAAP patients were also shown to express the HTLV-I antigens. These data indicate that HTLV-I in the non-T synovial cells can be transcribed and expressed. Moreover, the sequences of pXII regions in the CSCs demonstrated 97.5-99.4% homology to that in MT-2 cells, HTLV-I-infected cell line. This confirmed that the PCR-amplified bands reflect HTLV-I itself. These results suggest that this organ-specific inflammation can be attributed to non-T cell virus infection in articular lesions.

High-mobility group box 1 protein promotes development of microvascular thrombosis in rats.

BACKGROUND: Sepsis is a life-threatening disorder resulting from systemic inflammatory and coagulatory responses to infection. High-mobility group box 1 protein (HMGB1), an abundant intranuclear protein, was recently identified as a potent lethal mediator of sepsis. However, the precise mechanisms by which HMGB1 exerts its lethal effects in sepsis have yet to be confirmed. We recently reported that plasma HMGB1 levels correlated with disseminated intravascular coagulation (DIC) score, indicating that HMGB1 might play an important role in the pathogenesis of DIC. OBJECTIVES: To investigate the mechanisms responsible for the lethal effects of HMGB1, and more specifically, to explore the effects of HMGB1 on the coagulation system. METHODS: Rats were exposed to thrombin with or without HMGB1, and a survival analysis, pathologic analyses and blood tests were conducted. The effects of HMGB1 on the coagulation cascade, anticoagulant pathways and surface expression of procoagulant or anticoagulant molecules were examined in vitro. RESULTS: Compared to thrombin alone, combined administration of thrombin and HMGB1 resulted in excessive fibrin deposition in glomeruli, prolonged plasma clotting times, and increased mortality. In vitro, HMGB1 did not affect clotting times, but inhibited the anticoagulant protein C pathway mediated by the thrombin-thrombomodulin complex, and stimulated tissue factor expression on monocytes. CONCLUSIONS: These findings demonstrate the procoagulant role of HMGB1 in vivo and in vitro. During sepsis, massive accumulation of HMGB1 in the systemic circulation would promote the development of DIC.

Efficacy and safety of recombinant human soluble thrombomodulin (ART-123) in disseminated intravascular coagulation: results of a phase III, randomized, double-blind clinical trial.

BACKGROUND: Soluble thrombomodulin is a promising therapeutic natural anticoagulant that is comparable to antithrombin, tissue factor pathway inhibitor and activated protein C. OBJECTIVES: We conducted a multicenter, double-blind, randomized, parallel-group trial to compare the efficacy and safety of recombinant human soluble thrombomodulin (ART-123) to those of low-dose heparin for the treatment of disseminated intravascular coagulation (DIC) associated with hematologic malignancy or infection. METHODS: DIC patients (n = 234) were assigned to receive ART-123 (0.06 mg kg(-1) for 30 min, once daily) or heparin sodium (8 U kg(-1) h(-1) for 24 h) for 6 days, using a double-dummy method. The primary efficacy endpoint was DIC resolution rate. The secondary endpoints included clinical course of bleeding symptoms and mortality rate at 28 days. RESULTS: DIC was resolved in 66.1% of the ART-123 group, as compared with 49.9% of the heparin group [difference 16.2%; 95% confidence interval (CI) 3.3-29.1]. Patients in the ART-123 group also showed more marked improvement in clinical course of bleeding symptoms (P = 0.0271). The incidence of bleeding-related adverse events up to 7 days after the start of infusion was lower in the ART-123 group than in the heparin group (43.1% vs. 56.5%, P = 0.0487). CONCLUSIONS: When compared with heparin therapy, ART-123 therapy more significantly improves DIC and alleviates bleeding symptoms in DIC patients.

Efficient epitope mapping by bacteriophage lambda surface display.

A bacteriophage lambda surface expression system, lambda foo, was used for epitope mapping of human galectin-3. We constructed random epitope and peptide libraries and compared their efficiencies in the mapping. The galectin-3 cDNA was randomly digested by DNase 1 to make random epitope libraries. The libraries were screened by affinity selection using a microtiter plate coated with monoclonal antibodies. Direct DNA sequencing of the selected clones defined two distinct epitope sites consisting of nine and 11 amino-acid residues. Affinity selection of random peptide libraries recovered a number of sequences that were similar to each other but distinct from the galectin-3 sequence. These results demonstrate that a single affinity selection of epitope libraries with antibodies is able to define an epitope determinant as small as nine residues long and is more efficient in epitope mapping than random peptide libraries.

Expression of multiple UNC-13 proteins in the Caenorhabditis elegans nervous system.

The Caenorhabditis elegans UNC-13 protein and its mammalian homologues are important for normal neurotransmitter release. We have identified a set of transcripts from the unc-13 locus in C. elegans resulting from alternative splicing and apparent alternative promoters. These transcripts encode proteins that are identical in their C-terminal regions but that vary in their N-terminal regions. The most abundant protein form is localized to most or all synapses. We have analyzed the sequence alterations, immunostaining patterns, and behavioral phenotypes of 31 independent unc-13 alleles. Many of these mutations are transcript-specific; their phenotypes suggest that the different UNC-13 forms have different cellular functions. We have also isolated a deletion allele that is predicted to disrupt all UNC-13 protein products; animals homozygous for this null allele are able to complete embryogenesis and hatch, but they die as paralyzed first-stage larvae. Transgenic expression of the entire gene rescues the behavior of mutants fully; transgenic overexpression of one of the transcripts can partially compensate for the genetic loss of another. This finding suggests some degree of functional overlap of the different protein products.

Expression of thrombomodulin in esophageal squamous cell carcinoma and its relationship to lymph node metastasis.

Thrombomodulin (TM) is thrombin receptor that was identified originally on the endothelium and acts as a natural anticoagulant. However, we reported previously that TM was also expressed in the squamous epithelium mainly at the intercellular bridges. In this study, we examined TM expression in the primary lesions of 106 patients with esophageal squamous cell carcinomas and in the lymph node metastatic lesions of 59 patients using immunohistochemical methods. The carcinoma tissues expressed TM mainly at the cell-cell boundaries and in the cytoplasm. When TM expression was compared between the primary and metastatic lesions in the 59 patients who had lymph node metastasis, 41 (69%) showed decreased TM expression, 18 (31%) showed no change, and none (0%) showed an increase in the metastatic lesions. Wilcoxon's signed-rank test indicated that tumor cells that were positive for TM expression were significantly rarer in the metastatic lesions than in the primary tumors (P < 0.0001). This result indicates that the decrease in TM expression is associated with metastasis of the carcinoma cells. This phenomenon is very similar to that of E-cadherin, although the structures of both molecules are quite different. The reduction of TM expression seems to play an important role in the metastatic process of esophageal cancer.

Comparison of Phosphate Binding Capacities of PA21, A Novel Phosphate Binder, with those of other Phosphate Binders in vitro and in vivo.

PURPOSE: The phosphate binding capacity of PA21, a novel phosphate binder, was compared with those of other phosphate binders in vitro and in vivo. METHODS: 1) For in vitro studies, PA21, sevelamer hydrochloride, lanthanum carbonate hydrate, calcium carbonate, and ferric citrate hydrate were incubated with a phosphate solution at 37°C for 2 h. Phosphate binding capacity was assessed at simulated gastrointestinal tract pH levels of 2, 5, and 8 for estimation of clinical effects, and the quantity of phosphate adsorbed by each phosphate binder was determined. 2) For in vivo studies, rats were orally administered various phosphate binders after the oral administration of phosphate solution (100 mg/kg) adjusted to pH 2, 5, or 8, and the effects of PA21 and other phosphate binders on the serum phosphorus level of the rats were investigated. RESULTS: 1) The in vitro studies revealed that PA21 and sevelamer hydrochloride adsorbed phosphate better at all tested pH levels than lanthanum carbonate hydrate, calcium carbonate, and ferric citrate hydrate, and PA21 showed the most potent phosphate binding capacity among the tested compounds. 2) The in vivo studies showed that PA21 dose-dependently inhibited the increase in the serum phosphorus level after the administration of phosphate solution and no difference in the extent of inhibition by PA21 was observed at the different pH levels (in contrast to other phosphate binders). CONCLUSION: These results indicated that PA21 has a phosphate binding capacity over the entire pH range of the GI tract.