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Background: Both immune-mediated thrombotic thrombocytopenic purpura (iTTP) and septic disseminated intravascular coagulation (DIC) are life-threatening disorders developed by platelet-consuming microvascular thrombi and necessitate immediate therapeutic interventions. Although severe deficiencies of plasma haptoglobin in iTTP and factor XIII (FXIII) activity in septic DIC have been reported, few studies have focused on the possibility of using these markers to distinguish between iTTP and septic DIC., Objectives: We investigated whether the plasma levels of haptoglobin and FXIII activity could be helpful for differential diagnosis., Methods: Thirty-five patients with iTTP and 30 with septic DIC were enrolled in the study. Patient characteristics, coagulation, and fibrinolytic markers were collected from the clinical data. Plasma haptoglobin and FXIII activities were measured using chromogenic Enzyme-Linked Immuno Sorbent Assay and an automated instrument, respectively., Results: The median plasma haptoglobin level was 0.39 mg/dL and 54.20 mg/dL in the iTTP and septic DIC groups, respectively. The median plasma FXIII activities were 91.3% and 36.3% in the iTTP and septic DIC groups, respectively. In the receiver operating characteristic curve analysis, the cutoff level of plasma haptoglobin was 2.868 mg/dL and the area under the curve was 0.832. The cutoff level for plasma FXIII activity and the area under the curve were 76.0% and 0.931, respectively. The thrombotic thrombocytopenic purpura (TTP)/DIC index was defined by FXIII activity (percentage) and haptoglobin (milligrams per decilitre). Laboratory TTP was defined as an index ≥60 and laboratory DIC <60. The sensitivity and specificity of the TTP/DIC index were 94.3% and 86.7%, respectively., Conclusion: The TTP/DIC index, composed of plasma levels of haptoglobin and FXIII activity, is useful in differentiating iTTP from septic DIC., (© 2023 The Author(s).)

Monoclonal antibody therapy is a promising option for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and a cocktail of antibodies (REGN-COV) has been administered to infected patients with a favorable outcome. However, it is necessary to continue generating novel sets of monoclonal antibodies with neutralizing activity because viral variants can emerge that show resistance to the currently utilized antibodies. Here, we isolated a new cocktail of antibodies, EV053273 and EV053286, from peripheral blood mononuclear cells derived from convalescent patients infected with wild-type SARS-CoV-2. EV053273 exerted potent antiviral activity against the Wuhan wild-type virus as well as the Alpha and Delta variants in vitro, whereas the antiviral activity of EV053286 was moderate, but it had a wide-range of suppressive activity on the wild-type virus as well as the Alpha, Beta, Delta, Kappa, Omicron BA.1, and BA.2 variants. With the combined use of EV053273 and EV053286, we observed similar inhibitory effects on viral replication as with REGN-COV in vitro. We further assessed their activity in vivo by using a mouse model infected with a recently established viral strain with adopted infectious activity in mice. Independent experiments revealed that the combined use of EV053273 and EV053286 or the single use of each monoclonal antibody efficiently blocked infection in vivo. Together with data showing that these two monoclonal antibodies could neutralize REGN-COV escape variants and the Omicron variant, our findings suggest that the EV053273 and EV053286 monoclonal antibody cocktail is a novel clinically applicable therapeutic candidate for SARS-CoV-2 infection., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Abstract: In this study, the distribution of hygienic indicator bacteria in cattle livers and bile was examined at slaughterhouses. One hundred twenty-seven cattle livers with gallbladders were carefully eviscerated from carcasses at 10 slaughterhouses. Microbiological examination revealed that nine bile samples (7.1% prevalence) and 19 liver parenchyma samples (15.0% prevalence) were positive for Enterobacteriaceae (EB) with means ± standard deviations of 3.68 ± 4.63 log CFU/mL and 1.59 ± 2.47 log CFU/g, respectively; thus, bacterial contamination was apparent even at the postevisceration stage. Subsequently, 70 cattle livers were obtained at the postprocessing and storage stage from 7 of the 10 slaughterhouses. Microbiological analysis revealed significantly higher levels of EB in the liver parenchyma (3.00 ± 3.89 log CFU/g, P = 0.011) than those at the postevisceration stage, suggesting that bacterial dissemination and/or replication occurred in the liver parenchyma during processing and storage. According to 16S rRNA ion semiconductor sequencing analysis of representative samples from 12 cattle, Proteobacteria, Firmicutes, and Actinobacteria were dominant in both the parenchyma and bile in which EB and Escherichia coli were predominant among livers with higher EB levels. These results suggest that bile plays a role as a vehicle for bacterial transmission to the liver parenchyma. This study is the first to evaluate bacterial distribution and community structure in the liver and biliary microecosystem of cattle at slaughter. Our data support the use of EB testing of bile to screen cattle livers contaminated with high levels of fecal indicator bacteria., (Copyright ©, International Association for Food Protection.)

Introduction: The epidemic of coronavirus disease 2019 (COVID-19) rapidly spread worldwide, and the various infection control measures have a significant influence on the spread of many infectious diseases. However, there have been no multicenter studies on how the number of hospitalized children with various infectious diseases changed before and after the outbreak of COVID-19 in Japan., Methods: We conducted a multicenter, prospective survey for hospitalized pediatric patients in 18 hospitals in Hokkaido Prefecture, Japan, from July 2019 to February 2021. We defined July 2019 to February 2020 as pre-COVID-19, and July 2020 to February 2021 as post-COVID-19. We surveyed various infectious diseases by sex and age., Results: In total, 5300 patients were hospitalized during the study period. The number of patients decreased from 4266 in the pre-COVID-19 period to 701 (16.4%) post-COVID-19. Patients with influenza and RSV decreased from 308 to 795 pre-COVID-19 to zero and three (0.4%) post-COVID-19. However, patients with adenovirus (respiratory infection) only decreased to 60.9% (46-28) of pre-COVID levels. Patients with rotavirus, norovirus, and adenovirus gastroenteritis decreased markedly post-COVID-19 to 2.6% (38-1), 27.8% (97-27) and 13.5% (37-5). The number of patients with UTIs was similar across the two periods (109 and 90). KD patients decreased to 31.7% (161-51) post-COVID-19., Conclusions: We suggest that current infection control measures for COVID-19 such as wearing masks, washing hands, and disinfecting hands with alcohol are effective against various infectious diseases. However, these effects vary by disease., (Copyright © 2021 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Abstract: In Japan, strategies for ensuring food safety have been developed without reliable scientific evidence on the relationship between foodborne diseases and food sources. This study aimed to provide information on the proportions of foodborne diseases caused by seven major causative pathogens (Campylobacter spp., Salmonella, enterohemorrhagic Escherichia coli [EHEC], Vibrio parahaemolyticus, Clostridium perfringens, Staphylococcus aureus, and norovirus) attributed to foods and to explore factors affecting changes in these source attribution proportions over time using analysis of outbreak surveillance data. For the calculation of the number of outbreaks attributed to each source, simple-food outbreaks were assigned to the single-food category in question, and complex-food outbreaks were classified under each category proportional to the estimated probability. During 2007 to 2018, 8,730 outbreaks of foodborne diseases caused by seven pathogens were reported, of which 6,690 (76.6%) were of unknown source. We estimated the following source attribution proportions of foodborne diseases: chicken products (80.3%, 95% uncertainty interval [UI] 80.1 to 80.4) for Campylobacter spp.; beef products (50.1%, UI 47.0 to 51.5) and vegetables (42.3%, UI 40.9 to 45.5) for EHEC; eggs (34.6%, UI 27.8 to 41.4) and vegetables (34.4%, UI 27.8 to 40.8) for Salmonella; finfish (50.3%, UI 33.3 to 66.7) and shellfish (49.7%, UI 33.3 to 66.7) for V. parahaemolyticus; grains and beans (57.8%, UI 49.7 to 64.9) for S. aureus; vegetables (63.6%, UI 48.5 to 74.6), chicken products (12.7%, UI 4.6 to 21.5), and beef products (11.1%, UI 8.5 to 13.1) for C. perfringens; and shellfish (75.5%, UI 74.7 to 76.2) for norovirus. In this study, we provide the best available evidence-based information to evaluate the link between foodborne diseases and foods. Our results on source attribution for Campylobacter spp. and EHEC suggest that the strict health regulations for raw beef were reflected in the proportions of these diseases attributed to this food., (Copyright ©, International Association for Food Protection.)

In this study, we examined the prevalence of Shiga toxin-producing Escherichia coli and Salmonella spp. and the distribution of indicator bacteria in 248 samples of game meats (120 venison and 128 wild boar) retailed between November 2015 and March 2016 in Japan. No Salmonella spp. were detected in any of the samples, whereas Shiga toxin-producing Escherichia coli serotype OUT:H25 (stx 2d + , eae - ) was isolated from one deer meat sample, suggesting a possible source for human infection. Plate count assays indicated greater prevalence of coliforms and E. coli in wild boar meat than in venison, whereas their prevalence in processing facilities showed greater variation than in animal species. The 16S rRNA ion semiconductor sequencing analysis of 24 representative samples revealed that the abundances of Acinetobacter and Arthrobacter spp. significantly correlated with the prevalence of E. coli, and quantitative PCR analyses in combination with selective plate count assay verified these correlations. To our knowledge, this is the first report to characterize the diversity of microorganisms of game meats at retail in Japan, together with identification of dominant microbiota. Our data suggest the necessity of bottom-up hygienic assessment in areas of slaughtering and processing facilities to improve microbiological safety.

To reveal the in planta behaviour of caesium (Cs), the stable isotope 133 Cs was administered into 3-year-old Cryptomeria japonica seedlings by the application of 133 CsCl aqueous solution to the bark surface. The administered 133 Cs was quantified by ICP-MS measurements, which showed transportation of 133 Cs in an ascending direction in the stem. Distribution of 133 Cs was visualized using freeze-fixed C. japonica woody stem samples and cryo-time-of-flight secondary ion mass spectrometry/scanning electron microscopy (cryo-TOF-SIMS/SEM) analysis. Cryo-TOF-SIMS/SEM visualization suggested that 133 Cs was rapidly transported radially by ray parenchyma cells followed by axial transportation by pith and axial parenchyma cells. Adsorption experiments using powdered C. japonica wood samples and X-ray absorption fine structure (XAFS) analysis suggested that 133 Cs was in the hydrated state following its deposition into tracheid cell walls., (Copyright © 2017. Published by Elsevier B.V.)

To demonstrate the effect of climatic elements on Campylobacter colonization in broiler chickens reared in Japan, the correlation between Campylobacter isolated from chickens (191 of 236 flocks, 80.9%) between 2008 and 2012 and climatic elements was analyzed by logistic regression. We divided the rearing process into 13 terms of 5 d each (total: 65 d). Terms were numbered backwards, wherein a 0-term lag was considered as the sampling day plus 4 d before sampling; 1-term lag was the 5-d term before the 0-term lag, and so on, until the 12-term lag. We obtained climatic data tracing back from the 0-term to the 12-term lags. For evaluation in each season, we divided chickens reared during periods of rising temperature (spring, summer) and decreasing temperature (autumn, winter). Air temperature showed a positive correlation with Campylobacter colonization from the 0- to 12-term lags in chickens reared during the period of rising temperature (odds ratio [OR], 1.069 to 1.104), and from the 0- to 4- and 6-term lags (OR, 1.079 to 1.105) in chickens reared during the period of decreasing temperature. The strong positive effect of air temperature on Campylobacter colonization, particularly during the period of rising temperature, may be associated with the effect on the Campylobacter environmental sources and/or vectors. A positive correlation was observed between Campylobacter colonization and humidity when chicken houses were empty and new chicks were introduced (from the 9- to 12-term lags) during the period of decreasing temperature (OR, 1.076 to 1.141). Thus, high humidity would be an important factor causing carry-over of Campylobacter infection during the period of decreasing temperature. We also found that solar radiation increased Campylobacter colonization during the period of decreasing temperature, from the 2- to 8-term lags, except for the 4- and 5-term lags, in Japan. The results of this study demonstrate the effects of air temperature, humidity, and solar radiation on Campylobacter colonization in broiler chickens, and are potentially important for developing strategies to reduce the risk of Campylobacter contamination in broiler chickens., (© 2016 Poultry Science Association Inc.)

Incidences of food poisoning traced to nonanimal food products have been increasingly reported. One of these was a recent large outbreak of Shiga toxin-producing Escherichia coli (STEC) O157 infection from the consumption of lightly pickled vegetables, indicating the necessity of imposing hygienic controls during manufacturing. However, little is known about the bacterial contamination levels in these minimally processed vegetables. Here we examined the prevalence of STEC, Salmonella spp., and Listeria monocytogenes in 100 lightly pickled vegetable products manufactured at 55 processing factories. Simultaneously, we also performed quantitative measurements of representative indicator bacteria (total viable counts, coliform counts, and β-glucuronidase-producing E. coli counts). STEC and Salmonella spp. were not detected in any of the samples; L. monocytogenes was detected in 12 samples manufactured at five of the factories. Microbiological surveillance at two factories (two surveys at factory A and three surveys at factory B) between June 2014 and January 2015 determined that the areas predominantly contaminated with L. monocytogenes included the refrigerators and packaging rooms. Genotyping provided further evidence that the contaminants found in these areas were linked to those found in the final products. Taken together, we demonstrated the prevalence of L. monocytogenes in lightly pickled vegetables sold at the retail level. Microbiological surveillance at the manufacturing factories further clarified the sources of the contamination in the retail products. These data indicate the necessity of implementing adequate monitoring programs to minimize health risks attributable to the consumption of these minimally processed vegetables.

Campylobacter jejuni is one of the leading causes of foodborne gastrointestinal illness worldwide. Here we performed ex vivo proteomic analysis of C. jejuni 81-176 in chicken, a main reservoir for human infection. At 0, 1 and 4 weeks post-infection (p.i.) with the GFP-expressing 81-176 strain, inocula were recovered from chicken ceca by cell sorting using flow cytometry. iTRAQ-coupled 2D-LC-MS/MS analyses that detected 55 C. jejuni proteins, among which either 3 (FabG, HydB, CJJ81176&#95;0876) or 7 (MscS, CetB, FlhF, PurH, PglJ, LpxC, Icd) proteins exhibited >1.4-fold-increased expression at 1 or 4 week(s) p.i. compared with those at 0 weeks p.i., respectively. Deletion of the fabG gene clearly decreased the proportion of bacterial unsaturated fatty acids (UFAs) and chicken colonization. The UFA proportion of the parental strain was not altered when grown at 42 °C. These findings suggest that FabG might play a pivotal role in UFA production, linked to bacterial adaptation in the poultry host. To our knowledge, this is the first example of ex vivo C. jejuni proteomics, in which fatty acid metabolism might affect bacterial adaptation to the chicken host., (Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

In March 2012, two patients were transported urgently to the hospital in Tottori Prefecture, Japan, because of symptoms suggestive of botulism. Botulinum neurotoxin type A was detected in the clinical specimens and the food consumed by the two patients (vacuum packed adzuki-batto, a sweet adzuki bean soup containing noodles). We were able to make a prompt diagnosis of food botulism associated with the consumption of adzuki-batto, from which the causative pathogen Clostridium botulinum Ab was cultured., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Salmonella enterica serovar Typhimurium is a major cause of human gastrointestinal illness worldwide. This pathogen can persist in a wide range of environments, making it of great concern to public health. Here, we report that the salmonella pathogenicity island (SPI)-1 effector protein SipB exhibits a membrane topology that confers bacterial osmotolerance. Disruption of the sipB gene or the invG gene (SPI-1 component) significantly reduced the osmotolerance of S. Typhimurium LT2. Biochemical assays showed that NaCl osmolarity increased the membrane topology of SipB, and a neutralising antibody against SipB reduced osmotolerance in the WT strain. The WT strain, but not the sipB mutant, exhibited elevated cyclopropane fatty acid C19:0 during conditions of osmotic stress, correlating with the observed levels of survival and membrane integrity. This result suggests a link between SipB and the altered fatty acid composition induced upon exposure to osmotic stress. Overall, our findings provide the first evidence that the Salmonella virulence translocon SipB affects membrane fluidity and alters bacterial osmotolerance., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In the initial treatment of acute myocardial infarction, it is important to administer oral low-dose acetylsalicylic acid (ASA) within 10 min of arrival at the hospital. However, ASA is supplied as an enteric-coated tablet or buffered tablet to prevent gastric irritation. Although current guidelines recommended that patients should chew their initial dose of ASA, there is little evidence as to whether this is efficacious. Therefore, we aimed to make a direct comparison of the pharmacokinetics and pharmacodynamics of ASA after ingestion of intact and chewed nonenteric-coated buffered ASA tablet (NBA) and enteric-coated ASA tablet (ECA) in a quadruple crossover study in healthy volunteers. Chewing ECA accelerated t(max) of ASA absorption, which became equivalent to that after ingestion of intact or chewed NBA. A significant decrease in serum thromboxane B(2) was observed 20 min after ingestion of chewed ECA, or intact or chewed NBA, and inhibition of platelet aggregation was also observed within 20 min. Thus, chewing of the ECA appears to result in a similar timing of ASA action to that in the case of chewed or unchewed NBA., (Copyright © 2011 Wiley-Liss, Inc.)

With insufficient source separation, construction and demolition (C&D) waste becomes a mixed material that is difficult to recycle. Treatment of mixed C&D waste generates residue that contains gypsum and organic matter and poses a risk of H(2)S formation in landfills. Therefore, removing gypsum and organic matter from the residue is vital. This study investigated the distribution of gypsum and organic matter in a sorting process. Heavy liquid separation was used to determine the density ranges in which gypsum and organic matter were most concentrated. The fine residue that was separated before shredding accounted for 27.9% of the waste mass and contained the greatest quantity of gypsum; therefore, most of the gypsum (52.4%) was distributed in this fraction. When this fine fraction was subjected to heavy liquid separation, 93% of the gypsum was concentrated in the density range of 1.59-2.28, which contained 24% of the total waste mass. Therefore, removing this density range after segregating fine particles should reduce the amount of gypsum sent to landfills. Organic matter tends to float as density increases; nevertheless, separation at 1.0 density could be more efficient., ((c) 2009 Elsevier B.V. All rights reserved.)

During an outbreak of enterohemorrhagic Escherichia coli (EHEC) O157, we showed previously that food isolates were resistant to oxidative stress, while patient isolates were sensitive to it. Because food isolates increased stress-sensitivity after mouse passage, this change most likely occurred during passage through patients. Here we demonstrate that the phenotypic change occurring during mouse passage correlates with the stress response of outer membrane protein W (OmpW) in EHEC O157 strains. Upon induction of oxidative stress, OmpW was highly expressed only in the stress-sensitive MP37 strain, obtained by mouse passage of food strain F2, but not in the F2 strain. Western blotting confirmed that expression of OmpW was induced in the viable but non-culturable (VBNC) state. Deletion of ompW in the MP37 strain increased recovery from dormancy, while overexpression of OmpW in the F2 strain decreased recovery when exposed to oxidative stress, suggesting that high levels of OmpW sensitize the bacteria to stress. DNA alignment revealed that the class I integron (int1I) fragments flanking the ompW gene are oriented in opposite directions between stress-resistant and -sensitive strains. All stress-sensitive strains induced ompW under stress. We propose that the different stress response of OmpW was introduced by genetic alteration during in vivo passage.

Background: Venous thromboembolism (VTE) is a critical complication after hip replacement surgery, so both early diagnosis and prophylaxis are important. The purpose of this study was to clarify the rapid changes of the fibrin monomer complex (FMC) and soluble fibrin (SF) during the perioperative period of hip replacement surgery., Methods: The subjects were 32 patients (7 men, 25 women) who underwent elective hip replacement surgery between November 2004 and January 2006. Their ages ranged between 34 to 82 years (mean 56.8 years). According to their thromboembolic risk, the patients received different prophylaxis: unfractionated heparin (4 patients), danaparoid sodium (14 patients), or mechanical therapy only (14 patients)., Results: FMC and SF became rapidly elevated during the operation and just after surgery but declined to preoperational levels 3 days after surgery; they were higher in lupus anticoagulant (LA)-positive patients. In contrast, FDP and D-dimer had gradually become elevated 3 and 7 days after surgery. According to venous ultrasonography and lung perfusion scintigraphy, VTE occurred in 7 patients overall (21.9%). The incidence of VTE was 7.1% in the danaparoid group, whereas it was 35.7% in the mechanical therapy group. We also found that danaparoid sodium rapidly decreased FMC and SF within 3 days., Conclusions: FMC and SF were rapidly elevated during hip replacement surgery and differentiated in LA-positive and LA-negative patients.

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.

We examined the role of nitric oxide (NO) produced by an inducible isoform of NO synthase (iNOS) using N[6]-(iminoethyl)-lysine (L-NIL), a selective iNOS inhibitor, in the rat model of lipopolysaccharide (LPS)-induced disseminated intravascular coagulation (DIC) and investigated changes in organ function, plasma levels of NOX (metabolites of NO) and endothelin. We induced experimental DIC by the sustained infusion of 30 mg kg(-1) LPS for 4 h via the tail vein. We then investigated the effect of L-NIL (6 mg kg(-1), from - 0.5 to 4 h) on LPS-induced DIC. Blood was withdrawn at 4 and 8 h, and all four groups (LPS with or without L-NIL at 4 and 8 h) consisted of eight rats. Three of the animals in the 8-h LPS group died, and we examined blood samples from five rats in this group. None of the other rats died. The LPS-induced elevation of creatinine, alanine aminotransferase, glomerular fibrin deposition and plasminogen activator inhibitor was significantly suppressed by L-NIL coadministration, although L-NIL did not affect the platelet count, fibrinogen concentration or the level of thrombin-antithrombin complex. Moreover, plasma levels of the D-dimer that reflect the lysis of cross-linked fibrin were significantly increased by L-NIL coadministration in the LPS-induced DIC model. Plasma levels of NOX and endothelin were obviously increased by LPS infusion. However, both levels were significantly suppressed in the LPS + L-NIL group, when compared with the LPS group. Although mean arterial pressure (MAP) was significantly decreased between 2 and 8 h compared with the control in the LPS group, this depression was significantly attenuated in the LPS + L-NIL group. Our results suggest that NO induced by iNOS contributes to hypotension (depressed MAP), the progression of hepatic and renal dysfunction, microthrombus deposition and elevated endothelin levels in the rat model of LPS-induced DIC.

Objective: The objective of the present study was to explore whether prenatal dexamethasone treatment influences mitochondrial maturation in the fetal rat brain., Study Design: Mitochondrial respiration was measured polarographically with homogenates of fetal cerebral cortical tissues on day 16 (with saline solution, n = 8; with dexamethasone, n = 8), day 18 (with saline solution, n = 8; with dexamethasone, n = 8), and day 20 (with saline solution, n = 8; with dexamethasone,n = 8) of gestation. Four doses of dexamethasone (0.1 mg small middle dot kg) or vehicle (saline solution) were given, with an interval of 12 hours, until 12 hours before each measurement., Results: In the vehicle-treated animals, mitochondrial respiratory activity was increased significantly after day 18 of gestation. Dexamethasone-treated animals showed a significant increase in mitochondrial activity at day 16 of gestation compared with vehicle-treated animals., Conclusion: The results indicate that prenatal dexamethasone treatment contributes to the precocious maturation of mitochondrial activity in the fetal rat brain. Because acceleration in cerebral mitochondrial activities is required immediately after birth to maintain high-energy phosphate levels, the precocious maturation may be crucial for the successful outcome of the preterm infant.

A DNA fragment of 163 bp containing 11 GGA repeats formed two-end positioned mononucleosomes as efficiently as that of CTG repeats. However, the rotational positioning of the GGA fragment was weak because clear DNase I cleavage patterns with 10-base periodicity were not seen near the center of the GGA fragment but were detected in the entire region of the CTG fragment. Incubation of the GGA mononucleosomes with the same fragment provided the DNA-DNA complex, which had been shown by using naked DNA fragments. DNase I digestion of the complex exhibited protection in the GGA repeats and in flanking sequences of about 30 bp at both sides, suggesting that both the repeat and flanking regions were involved in the association. Interestingly, histone H1, which enhanced DNA-DNA association on naked DNA, did not affect the complex formation on mononucleosomes. These results imply that GGA microsatellites in genomes could associate with one another at multiple sites and that the association may play a role in functional organization of higher order chromatin architecture., ((c)2002 Elsevier Science.)

Objective: Mitochondfial respiratory activities were measured in neonatal rat brain to compare the influence of transient intrauterine ischemia in the preterm fetus with that in the term fetus and to evaluate the effect of alpha-phenyl-N -tert-butyl-nitrone treatment., Study Design: Intrauterine ischemia was induced by a 30-minute occlusion of the right uterine artery. The control group consisted of term fetuses (20 days old) exposed to normoxia (n = 8) and ischemia (n = 8). For the investigation into maturity effect, preterm fetuses (14 days old) were exposed to normoxia (n = 8) or ischemia (n = 8), and for the alpha-phenyl-N -tert-butyl-nitrone treatment investigation, term fetuses were exposed to ischemia with alpha-phenyl-N -tert-butyl-nitrone (n = 8). All subjects underwent cesarean delivery at 21 days of gestation, and the mitochondrial respiration was measured polarographically 1 hour after delivery., Results: In the control group the neonatal cortical tissue exposed to ischemia showed a significant decrease in mitochondrial activities compared with those in normoxic control animals. In the preterm group the mitochondrial activities of ischemic fetuses were maintained close to normoxic levels. The neonatal mitochondrial deterioration caused by term ischemia was prevented by alpha-phenyl-N -tert-butyl-nitrone., Conclusion: The results indicate that preterm fetuses are more capable than term fetuses of maintaining mitochondrial function under conditions of transient intrauterine ischemia and suggest that oxygen derived free radicals may play a crucial role in the development of neonatal neurologic deficit.

Several studies have previously reported high levels of total tissue factor pathway inhibitor (TFPI) antigen in patients with hypercholesterolemia. The relationship between serum lipid concentrations and total and free-form TFPI antigen in 32 patients with primary type II hypercholesterolemia and 38 age- and gender-matched normolipemic control subjects was studied (Study Group I). Plasma concentrations of total TFPI (tTFPI) antigen, free-form TFPI (fTFPI) antigen, tissue factor antigen, factor VII activity (FVIIc), and prothrombin fragment 1+2 (F1+2) were measured. The median levels of tTFPI, fTFPI, FVIIc, and F1+2 were higher in hyperlipidemic patients compared with those in healthy subjects. The effect of lowering total cholesterol on hypercoagulability in 25 patients with type II hyperlipoproteinemia (Study Group II) were also studied. The median levels of tTFPI, FVIIc, and F1+2 decreased significantly after 6 months of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor therapy in the hypercholesterolemic patients. On the other hand, fTFPI did not change after therapy. Plasma tTFPI was strongly correlated with total cholesterol and low density lipoprotein (LDL)-cholesterol in hyperlipidemic patients. In contrast to the strong correlation between tTFPI and total cholesterol, the correlation between plasma fTFPI and total cholesterol was relatively poor. These results suggest that the activation of the anticoagulant system as well as the activation of the coagulation system may occur in association with hypercholesterolemia. Furthermore, the results of this study may suggest that lowering of total cholesterol in hyperlipidemic patients reduces the thrombin generation in plasma and that down-regulation of LDL does not affect the anticoagulant potency of TFPI in plasma.

Background/aims: Our previous results showed that the percentage of fucosylated species of alpha-fetoprotein (AFP) in total AFP, fucosylation index, was a very useful diagnostic tool to distinguish AFP due to hepatocellular carcinoma from AFP due to non-neoplastic liver diseases. On the other hand, alpha1-6 fucosyl-transferase (alphaFT) catalyzes the addition of fucose from GDP-fucose through an alpha1-6 linkage to the reducing end of N-acetylglucosamine residue of N-linked oligosaccharides of glycoproteins. However, the biological and clinical significance of alphaFT in patients with hepatocellular carcinoma is not fully understood. In the present study, we measured alphaFT activity to elucidate the enzymatic background of fucosylated species of AFP in hepatocellular carcinoma., Methods: Plasma samples from 84 cases of hepatocellular carcinoma, 40 of liver cirrhosis, 40 of chronic hepatitis and 30 of normal controls, and 26 paired samples of hepatocellular carcinoma and surrounding noncancerous tissues were enrolled in the present study. AlphaFT activity was measured by high performance liquid chromatography with a synthesized fluorescence-labeled glycopeptide with an asialoagalactobiantennary sugar chain as a substrate in the presence of GDP-fucose., Results: Plasma alphaFT activities (mean+/-SD, pmol/ml/h) in patients with hepatocellular carcinoma, liver cirrhosis, chronic hepatitis and normal controls were 435+/-271, 490+/-290, 590+/-209 and 380+/-133, respectively. AlphaFT levels in hepatocellular carcinoma and chronic liver diseases were increased compared with that in normal controls. A statistically significant positive correlation was observed between plasma alphaFT activity and fucosylation index of AFP (r=0.34, p= 0.0032) in 60 patients with hepatocellular carcinoma, in which increments of serum AFP were observed. When the tentative cutoff value of fucosylation index was set at 18%, which corresponded to the cutoff value to discriminate between hepatocellular carcinoma and non-neoplastic liver diseases in our previous study, the plasma alphaFT activity in hepatocellular carcinoma patients whose fucosylation index was more than 18% (n=32, 523+/-324 pmol/ml/h) was higher than that in hepatocellular carcinoma patients whose fucosylation index was equal to or less than 18% (n=28, 383+/-229) (p=0.055). An increment of the plasma levels of alphaFT occurred in accordance with an advancement of hepatocellular carcinoma stages. Tissue aFT activity in hepatocellular carcinoma (175+/-178 pmol/mg/h) was higher than those in surrounding noncancerous liver (144+/-134) and in normal liver (79+/-19). The mean alphaFT activities in well-, moderately- and poorly-differentiated hepatocellular carcinoma were 38+/-0.7, 177+/-182 and 219+/-189, respectively, and they tended to increase with dedifferentiation in hepatocellular carcinoma tissues., Conclusions: The present study indicates that alphaFT is responsible for the formation of the fucosylated species of AFP in hepatocellular carcinoma and suggests that the measurement of alphaFT provides a possible aid in the evaluation of the degree of advancement in patients with hepatocellular carcinoma.

The authors discuss a case of pheochromocytoma in a 20-year-old man referred for anemia due to gastrointestinal bleeding. Pheochromocytoma uncommonly presents with gastrointestinal bleeding, and a pathologic diagnosis is rarely made without resection of the tumor. Imaging studies demonstrated a mass on the left kidney with metastases to the liver, and high levels of catecholamines in plasma and urine were suggestive of pheochromocytoma. Endoscopic examination of the gastrointestinal tract demonstrated a mass in the proximal jejunum near the ligament of Treitz and a mass with central ulceration in the upper jejunum; biopsy confirmed the pathologic diagnosis of malignant pheochromocytoma with jejunal invasion and metastases to the liver. Gastrointestinal bleeding is one of the manifestations of pheochromocytoma. This is the first case in which endoscopic biopsy determined a pathologic diagnosis of pheochromocytoma.

Background/aims: Identification of the risk factors of multicentric hepatocarcinogenesis is important for the clinical management of hepatocellular carcinoma. We investigated hyperplastic foci in non-cancerous liver parenchyma, and clarified their pathological features and clinical significance., Methods: Hyperplastic foci were defined as hypercellular areas, which architecturally and cytologically resembled early hepatocellular carcinoma or adenomatous hyperplasia but did not form macroscopically detectable nodules. Surgically resected livers from 155 patients with hepatocellular carcinoma were examined histopathologically and immunohistochemically., Results: Hyperplastic foci were found in 26 of 155 patients (16.8%). All the patients with hyperplastic foci had chronic liver diseases, and the incidence did not differ between those with chronic hepatitis and those with liver cirrhosis. Six of 92 (6.5%) patients with single primary hepatocellular carcinoma nodules, 8 of 42 (19.0%) with two nodules, and 12 of 21 (57.0%) with more than three nodules had hyperplastic foci. The incidence of hyperplastic foci showed a significant positive correlation with the multiplicity of hepatocellular carcinoma nodules. Immunohistochemically, hyperplastic foci were masses of proliferative hepatocytes similar to adenomatous hyperplasia and early hepatocellular carcinoma., Conclusions: Hyperplastic foci reflect the risk of multicentric hepatocarcinogenesis. Our results suggest strongly that hyperplastic foci are precursors of adenomatous hyperplasia or hepatocellular carcinoma.

Background/aims: Fibronectin is a multifunctional glycoprotein and plays important roles in cell-to-cell or cell-to-matrix interaction. The molecular and functional diversity of fibronectin arises from alternative splicing of pre-mRNA at three variable regions, termed ED-A, ED-B and IIICS. Cellular fibronectin with ED-A and ED-B regions has different biological activities from plasma fibronectin lacking these regions. This study was aimed at investigating the type-specific expression of fibronectin in human liver diseases., Methods: Immunohistochemistry with anti-total and anti-cellular fibronectin monoclonal antibodies, in situ hybridization with cDNA probes detecting common and ED-A regions and RT-PCR to amplify each variable region were performed in 35 specimens, including 4 control, 16 chronic hepatitis, 7 liver cirrhosis and 8 hepatocelular carcinoma., Results: In control liver, there were slight deposits of cellular fibronectin [ED-A(+)fibronectin] in portal areas. In chronic hepatitis, it was strongly deposited at the margin of the fibrously enlarged portal areas where new collagen fibers were formed. Cellular fibronectin was evenly and abundantly accumulated in fibrotic septa in liver cirrhosis, and in fibrotic septa and capsules of tumor nodules in hepatocellular carcinoma. In control liver, cellular fibronectin mRNA was localized in a few hepatocytes and non-parenchymal cells around central veins, and was increased in the same cell populations near fibrously enlarged portal areas as hepatic fibrosis progressed. In hepatocellular carcinoma, it was expressed in most hepatoma cells. Fibronectin mRNA with three variable regions was detectable by RT-PCR in control liver as well as in each disease group., Conclusions: The expression of cellular fibronectin was increased in fibrotic human liver and hepatocellular carcinoma. In human liver, both non-parenchymal cells and hepatocytes participated together in cellular fibronectin production. In hepatocellular carcinoma, hepatoma cells were the main producer. Our results indicate that, in human liver, cellular fibronectin may participate in the hepatic fibrogenesis and in the malignant phenotypes of hepatocellular carcinoma.

Diabetes mellitus is associated with disturbances in hemostasis that could contribute to the development of diabetic vascular disease. We investigated the changes in parameters of blood coagulation and the fibrinolytic system and in plasma levels of lipoprotein(a)(Lp(a)) in 124 patients with type II diabetes mellitus and 44 healthy control subjects matched for age and body mass index (BMI) to determine whether hemostatic disturbances may lead to increased cardiovascular mortality. Median levels of fibrinogen (P < 0.0001), thrombin-antithrombin III complex (TAT) (P < 0.005), and plasminogen activator inhibitor-1 (PAI-1) activity (P < 0.05) in plasma were significantly elevated in diabetic patients compared with controls. The median concentration of Lp(a) was significantly higher in diabetic patients than in normal controls (18.2 vs. 12.6 mg/dl. P < 0.0005). Lp(a) levels tended to be elevated in patients with a prolonged history of diabetes. There was no evidence that Lp(a) levels were affected by metabolic control or by type of treatment. Twenty-two diabetics with coronary heart disease (CHD) had significantly higher levels of fibrinogen (P < 0.05), TAT (P < 0.05), and Lp(a) (24.7 vs. 13.7 mg/dl, P < 0.01) than the 51 patients without diabetic angiopathy. Our data indicate that impaired hemostatic balance in diabetes may cause hypercoagulability and may thus contribute to the increased cardiovascular mortality in diabetes.

We investigated how NK cells, extrathymic T cells, and thymus-derived T cells are activated in mice during infection with an intracellular pathogen, Listeria (L.) monocytogenes. Although macrophages and granulocytes are known to be involved in the elimination of this pathogen in an early phase of infection, it was still controversial what type of lymphocytes are induced as effectors in subsequent phases. When mice were ip injected with 1 x 10(3) L. monocytogenes (a sublethal dose), a prominent increase in the number of mononuclear cells in the liver and spleen was induced. Phenotypic analysis revealed that serial induction of lymphocyte subsets, NK cells-->extrathymic T cells-->thymus-derived T cells, occurred in these organs. Extrathymic T cells were estimated to have intermediate CD3 and a high level of IL-2 receptor beta-chain on the surface (i.e., intermediate CD3 cells). These mice became free from infection after 2 weeks. In the case of oral administration, 1 x 10(3) L. monocytogenes increased the number of cells in the liver and the number of intraepithelial and lamina propria lymphocytes in the intestine. Phenotypic analysis also showed a sequential induction of lymphocyte subsets in the liver and the induction of extrathymic T cells in the intestine. Preelimination of intermediate TCR cells and NK cells by in vivo treatment with anti-LFA-1 mAb made mice susceptible to an ip injected sublethal dose of L. monocytogenes. These results reveal a unique order of lymphocyte induction during listerial infection and indicate that extrathymic T cells might be one of the important cells in achieving resistance against L. monocytogenes.

Immunolocalization of a fibronectin-binding proteoglycan (PG-P1) in relation to fibronectin, type IV collagen and laminin, in normal and fibrotic human liver was investigated by light and electron microscopy. HS-42, which is a monoclonal antibody to PG-P1 and is reported to recognize a heparan sulfate proteoglycan named HSPG2/perlecan, was used for this purpose. Light microscopy in the human liver with minimal changes revealed that PG-P1 was present along the hepatic sinusoids as well as fibronectin and type IV collagen, whereas laminin was only weakly detected. In portal areas, PG-P1 was only localized on basement membranes around bile duct systems and blood vessels, as well as laminin and type IV collagen, while fibronectin was scarcely detected in basement membranes. In the fibrotic liver, fibronectin was abundant in necrotic and/or newly fibrosing areas, while PG-P1 was absent in these regions. Using immunoelectron microscopy, PG-P1 was localized in the space of Disse in nearly normal livers and was only detected on basement membranes in portal tracts. In fibrotic livers, PG-P1 in the space of Disse occasionally showed a basement-membrane-like deposition in parallel with the increased light microscopical deposition of laminin in this area, suggesting the positive participation of PG-P1 in the sinusoidal capillarization. Most capillary and sinusoidal endothelial cells, and rarely bile epithelial cells revealed the reaction products of PG-P1 in their rough endoplasmic reticulum and small vesicles. Thus, it was suggested that these cell types are mainly, if not wholly, responsible for PG-P1 production.(ABSTRACT TRUNCATED AT 250 WORDS)

Extrathymic T cells in the hepatic sinusoids and intraepithelial lymphocytes (IEL) in the intestine of mice have both similar and different properties. In this study, both types of extrathymic T cells in mice were further characterized. Lymphocytes obtained from systemic immune organs, including the lamina propria and Peyer's patches, were also compared. Extrathymic T cells in both the liver and the intestine contained a large proportion of gamma delta T cells and expressed the alpha alpha homodimer of CD8. They became more prominent in athymic nude mice and in normal mice with aging, while disappearing in scid mice. Extrathymic T cells in the liver, on the other hand, had TCR of intermediate intensity (i.e., intermediate TCR cells) and IL-2 receptor beta-chains (IL-2R beta) of high intensity, similar to NK cells, whereas IEL had TCR of bright intensity and consisted of cells with both low and high levels of IL-2R beta. Thymus-derived T cells did not express IL-2R beta at all, at least at their resting conditions. Intermediate TCR cells included double-negative CD4-8- cells as well as single-positive cells. In contrast, IEL contained both double-positive (DP) CD4+8+ cells and single-positive cells. More precisely, IEL gamma delta T cells were mainly IL-2R beta + and single-positive (mainly CD8+), while IEL alpha beta T cells were mainly IL-2R beta- and contained both DP CD4+8+ cells and single-positive cells. CD4+ cells were more predominant than CD8+ cells in the liver, while CD8+ cells were overwhelmingly predominant in the intestine. These results suggest that both intermediate TCR cells and IEL are generated as primitive T cells in phylogeny, but later develop along independent pathways at their respective sites.

Chemical structures of the sugar chains of alpha 1-antitrypsin (AAT) from patients with hepatocellular carcinoma (HCC) and from healthy individuals with a different affinity for Lens culinaris agglutinin (LCA) were examined by pyridylamination of their oligosaccharides and stepwise exoglycosidase digestion in combination with reversed-phase and size-fractionation high-performance liquid chromatography. We found that the LCA-reactive species of AAT from patients with HCC carried both the biantennary sugar chain with a fucose residue at the innermost N-acetylglucosamine residue, Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6(Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc-PA, and the biantennary chain without a fucose residue, Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6(Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc-PA, at a ratio of about 1:0.6. The LCA-nonreactive species of AAT contained the biantennary sugar chain Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6(Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc as a major component. These results indicate that a characteristic feature of the carbohydrate chains of AAT from patients with HCC is an increment in fucosylation.

A congenitally dysfunctional form of prothrombin, Prothrombin Himi, shows reduced fibrinogen clotting activity, although it retains full hydrolytic activity toward synthetic substrates. To elucidate the structural abnormality of the variant prothrombin, we first performed genetic analysis of dysprothrombin. Polymerase chain reaction amplification of the exons 8 through 14 of the proband and her family members' prothrombin genes, which code the thrombin moiety, followed by single-strand conformation polymorphism analysis, identified two variant conformers in exon 10 specific to this family. One variant allele detected in the father was inherited by the proband and one of her sisters, and the other detected in the mother was also inherited by them. This result indicates that the proband has two different base pair changes in the gene. Sequencing showed two novel point mutations in the proband's gene. One is a T to C transition at position 8751, resulting in the substitution of threonine for methionine at codon 337 (Thrombin Himi I). The other is a G to A transition at 8904, resulting in the substitution of histidine for arginine at codon 388 (Thrombin Himi II). By sequencing analysis of her parents, it was determined that Thrombin Himi I was inherited from the father and Thrombin Himi II from the mother. These results confirm that Prothrombin Himi is compound heterozygous for two dysfunctional prothrombin molecules.

Biological characteristics of alkaline phosphatase (ALP) from rat intestine were compared among four various fractions (brush border, cytosol, luminal and lymph) to clarify the source and route of ALP appeared in intestinal lymphatics. ALP of each fraction had the same Km values and the same heat stability and all were of intestinal type. However, lymph and cytosol ALP showed similar affinity to three kinds of lectins and were distinct from membrane and luminal ALP. On polyacrylamide gel electrophoresis (PAGE), there seems to be two types of charge isomers of intestinal ALP, namely brush border type and lymph type. On sodium dodecyl sulfate (SDS)-PAGE, the molecular weight of principal band for each form was: 130,000 for brush border type, and 160,000 for lymph type. Fat administration significantly increased the ALP activity in intestinal lumen and intestinal lymph. However, it did not change biological characteristics of ALP including binding ability to lectins and electrophoretic mobility in any of these four fractions. These results suggest that lymphatic ALP may be transported from cytosol of intestinal cells and that fat administration seems to induce quantitative change of intestinal ALP, but not to change its physiological route of transportation into intestinal lymph.

By light and electron microscopic examinations, histologic changes in the liver of mice with graft-versus-host reaction (GVHR) were analyzed. To induce GVHR, C57BL/6 (B6) spleen cells were injected into (B6Xbm1)F1, (B6Xbm12)F1, and (bm1Xbm12)F1 mice. In (B6Xbm12)F1 recipient mice, bile duct changes resembling chronic non-suppurative destructive cholangitis (CNSDC) and a formation of epithelioid granulomas were observed during the course of GVHR. An epithelioid granuloma in the liver of (B6Xbm1)F1 or (bm1.Xbm12)F1 recipients was not detected. By electron microscopy, the bile duct epithelia were seen to be in close contact with infiltrating cells, and marked alterations of their cytoplasm and microvilli were demonstrated; ie, vacuolation of the cytoplasm, deterioration of microvilli, and bleb formation were frequently observed in the liver of class II-disparate hosts. Concerning the basement membrane, no marked changes characteristic of primary biliary cirrhosis (PBC), such as many-layered basement membranes containing osmium positive substance, were detected. Because the major histocompatibility complex (MHC) class II-disparate system was used in our experimental system in the GVHR, the antigen expressed on the bile duct might be a target and be associated with the formation of the initial hepatic lesions in PBC such as CNSDC and epithelioid granuloma formation. Thus, GVHR across the MHC class II antigen is believed to play an important role in the development of PBC.