Influence of age and duration of infection on bacterial load and immune responses to Helicobacter pylori infection in a murine model.

Using a murine model, we previously showed that Helicobacter pylori infects and colonizes offspring via maternal transmission during the nursing period. The aim of this study was to investigate the influence of age and duration of infection on inflammatory and immune responses to H. pylori in infant and adult mice. During the breast-feeding period, the number of bacteria was significantly suppressed in 1-week-old mice infected with H. pylori at an early stage of nursing, compared with adult mice, suggesting that breast-milk induces such low colonization. In addition, these mice had weaker gastric inflammation, especially Th1 cytokine and humoral responses than in mice infected with H. pylori after weaning in spite of elevated levels of Th1 cytokines. Although infant mice showed low inflammatory responses against H. pylori, they produced H. pylori-specific antibodies following vaccination with oral or parenteral adjuvant. Our results suggest the importance of age at the time of primary infection on bacterial load, gastric inflammation and humoral responses in a murine model of H. pylori infection.

Diversity of vacA and cagA genes of Helicobacter pylori in Japanese children.

BACKGROUND: Helicobacter pylori infection is generally acquired in childhood and persists as an asymptomatic infection for decades in most infected individuals. Only a minority develops a clinical outcome even in childhood, such as peptic ulcer. It has been reported that H. pylori infection with the type I strain, which expresses the VacA and CagA antigen, is associated with peptic ulcer. AIM: We examined the diversity of vacA and cagA genes in isolates obtained from Japanese paediatric patients with peptic ulcer or chronic gastritis to investigate the relationship between genetic diversity and clinical outcome. METHODS: The diversity of vacA and cagA genes was investigated by PCR and sequence analysis in 30 isolates obtained from Japanese paediatric patients with peptic ulcer (eight strains) or chronic gastritis (22 strains). RESULTS: All isolates from Japanese children were cagA-positive strains. Twenty-six strains (86.7%) had East Asian type CagA, and 4 (13.3%) had Western type CagA. The predominant vacA genotype was s1c/m1b (22/30, 73.3%). There was no significant association between the diversity of cagA and vacA genes and clinical outcome. All four children infected with Western CagA strain had a history of overseas travel or residence. CONCLUSION: The predominant genotype of H. pylori in Japanese children is East Asian CagA and vacA s1c/m1b genotype, regardless of clinical outcome. Japanese H. pylori strains are homogeneously of the East Asian type; however, Western strains can be introduced into Japan concomitant with host movement from foreign countries in childhood.

Childhood Helicobacter pylori infection in a murine model: maternal transmission and eradication by systemic immunization using bacterial antigen-aluminium hydroxide.

In humans, transmission of Helicobacter pylori is thought to occur largely during childhood. Infected mothers are generally considered to be the main source of the pathogen. However, little is known about when and how often maternal transmission of H. pylori occurs during childhood. In the present study, we examined these issues in an experimental murine model. Pregnant C57BL/6 mice, infected experimentally with H. pylori, delivered and nursed their litters. The stomachs of the infants were isolated and assessed for transmission of H. pylori. We also investigated the effect of systemic immunization using H. pylori antigen-aluminium hydroxide (AlOH) with regard to providing anti-H. pylori immunity and eradicating the maternally transmitted bacteria in infants. Polymerase chain reaction (PCR) was used to examine the presence of transmitted bacteria and their eradication. Maternal transmission of H. pylori varied widely during the nursing period, but almost all litters showed bacterial transmission at 2 weeks postpartum. Systemic immunization with bacterial antigen-AlOH eradicated the bacteria in most litters; this immunization induced a local decrease of Th2 cytokines and a local increase of Th1 cytokines in the gastric tissue, as determined by ELISA. Our results indicate that our H. pylori vaccine provides not only protection, but also eradication of the already transmitted H. pylori.

Highly divergent actin expressed in a Chlamydomonas mutant lacking the conventional actin gene.

The Chlamydomonas mutant ida5 is deficient in the conventional actin gene and its axoneme lacks a subset of inner dynein arms that contain actin as a subunit. However, this mutant retains some other inner dynein arms because a novel protein (NAP) is expressed as a substitute for actin. In this study, we show by sequence analysis that NAP is identical to a putative actin-related protein, the cDNA sequence of which has recently been reported and shown to have 64% amino acid identity with conventional actin. A polyclonal antibody raised against a synthetic polypeptide corresponding to the NH2-terminal sequence of this protein specifically reacted with the spot corresponding to NAP in two-dimensional electrophoresis patterns. NAP apparently can substitute for conventional actin in some, but not all, cellular functions, and therefore can be regarded as a highly divergent actin. This unconventional actin appears to be expressed only when conventional actin is absent.

Recovery of flagellar inner-arm dynein and the fertilization tubule in Chlamydomonas ida5 mutant by transformation with actin genes.

The ida5 mutant of Chlamydomonas, first isolated as a mutant lacking a subset of axonemal inner-arm dyneins, has recently been shown to lack conventional actin owing to a serious mutation in its gene. It lacks inner-arm dyneins probably because actin is an essential subunit for their assembly. In addition, male gametes of ida5 are unable to produce the fertilization tubule, a structure that contains a core of actin filament bundles. To establish that those observed deficiencies are solely attributable to the loss of actin, and to provide a basis for future studies on the actin function in this organism, we examined in this study whether transformation of this mutant with cloned actin genes can rescue the mutant phenotypes. Cotransformation of the double mutant ida5arg2 with the wild-type actin gene and arginino-succinate lyase gene that suppresses the arg2 mutation yielded several transformants that displayed increased motility. All of them were found to have acquired the introduced actin gene in the genome and the product actin in the flagella, and regained the missing inner-arm dyneins and wild-type motility. In addition, most transformants also became able to grow the fertilization tubule when mating reaction was induced. In addition to the wild-type actin gene, we also used a chimeric actin gene in which the N-terminal 12 amino-acid sequence of Chlamydomonas actin was replaced by that of the greatly divergent Tetrahymena actin. Transformants with this gene also resulted in recovery of inner-arm dynein and 70-80% of the wild-type level of motility. These results established that the lack of inner-arm dynein and the fertilization tubule in ida5 are consequences of its loss of conventional actin. Furthermore, they demonstrate that Chlamydomonas offers an excellent experimental system with which to study the structure-function relationship of actin by means of mutant analysis.

A computer simulation study of isometric contraction of latissimus dorsi muscle used for cardiac assistance.

This study was designed to investigate the feasibility of a skeletal muscle pump employing latissimus dorsi muscle (LDM) for cardiac assistance. We developed and used a 2-dimensional mathematical model for LDM to investigate how the size of pneumatic balloons (30, 38, and 45 ml) and the three different locations (proximal, center, and distal) affect the pressure applied to the balloon by LDM. The computer simulation was performed by coding a visco-elastic and nonlinear 2-dimensional program that employed the finite element method (FEM). The muscle specific parameters of LDM were obtained from animal experiment results. The model is based on Hill's characteristic equation and composed of a contractile component and a passive element. The simulation results indicated that the intermediate and largest sized balloon lead to the highest and the lowest power (volume reduction per unit time interval), respectively. On the other hand, when the balloon is inserted in the distal LDM, the power is lower than in the other two positions, regardless of the balloon size. The above results suggest that the optimal size of the balloon should be selected depending on the muscle specific parameters of the actuator, and that the balloon should be inserted either in the proximal portion or center of the actuator.

Chlamydomonas inner-arm dynein mutant, ida5, has a mutation in an actin-encoding gene.

Chlamydomonas flagellar inner-arm dynein consists of seven subspecies (a-g), of which all but f contain actin as subunits. The mutant ida5 and a new strain, ida5-t, lack four subspecies (a, c, d, and e). These mutants were found to have mutations in the conventional actin gene, such that its product is totally lost; ida5 has a single-base deletion that results in a stop codon at a position about two-thirds from the 5' end of the coding region, and ida5-t lacks a large portion of the entire actin gene. Two-dimensional gel electrophoresis patterns of the axonemes and inner-arm subspecies b and g of ida5 lacked the spot of actin (isoelectric point [pI] = approximately 5.3) but had two novel spots with pIs of approximately 5.6 and approximately 5.7 instead. Western blot with different kinds of anti-actin antibodies suggested that the proteins responsible for the two novel spots and conventional actin are different but share some antigenicity. Since Chlamydomonas has been shown to have only a single copy of the conventional actin gene, it is likely that the novel spots in ida5 and ida5-t originated from another gene(s) that codes for a novel actin-like protein(s) (NAP), which has hitherto been undetected in wild-type cells. These mutants retain the two inner-arm subspecies b and g, in addition to f, possibly because NAP can functionally substitute for the actin in these subspecies while they cannot in other subspecies. The net growth rate of ida5 and ida5-t cells did not differ from that of wild type, but the mating efficiency was greatly reduced. This defect was apparently caused by deficient growth of the fertilization tubule. These results suggest that NAP can carry out some, but not all, functions performed by conventional actin in the cytoplasm and raise the possibility that Chlamydomonas can live without ordinary actin.

Effect of hepatocyte volume on energy status in the cirrhotic rat liver.

To evaluate the effect of reduced hepatocyte volume on liver energy status, the relationship between the morphologically quantified hepatocyte volume and biochemical parameters, and the difference in nuclear density between the parenchyma and stroma were assessed in rat livers with thioacetamide-induced cirrhosis. The ratio of hepatocytes to whole liver tissue, defined as the 'hepatocyte area ratio', and the nuclear densities of the parenchyma and stroma were calculated microscopically with an image analysing system. Adenine nucleotide, protein and DNA contents, and the ornithine carbamoyltransferase activity in the liver were assayed. In the cirrhotic group, adenine nucleotide content, protein content and ornithine carbamoyltransferase activity were positively correlated with the hepatocyte area ratio, whereas DNA content was negatively correlated with this ratio. The adenylate energy charge of the cirrhotic liver was maintained at a constant level regardless of the ratio. Adenine nucleotide content, protein content and ornithine carbamoyltransferase activity per fractional 'hepatocyte area ratio' in cirrhotic livers were significantly lower than in control livers. The nuclear density of the stroma was significantly greater than that of the parenchyma. These results suggest that the lowered energy status in the cirrhotic liver is not caused by reduced hepatocyte volume but rather by impaired hepatocyte metabolism. In addition, the morphological measurement with an image analysing system was found to be useful for evaluating the effects of decreased hepatocyte volume on biochemical derangements in cirrhotic tissue.

[Coagulation and fibrinolysis in pregnancy].

We studied on coagulation and fibrinolysis systems during pregnancy by measuring plasma levels of thrombin-antithrombin III complex (TAT), plasmin-alpha 2 plasmin inhibitor complex (PIC), fibrinogen/fibrin degradation products (FDP), plasminogen (PLG) and antithrombin III (AT-III). Ninety seven pregnant, 5 post-delivery and 32 nonpregnant women aged from 20 to 52 years old were included in this study. Plasma concentrations of TAT and PIC in nonpregnant women were 3.38 +/- 1.02 micrograms/l and 0.65 +/- 0.24 micrograms/ml, respectively. TAT gradually increased with the progression of pregnancy and rapidly decreased after the delivery. Whereas PIC and AT-III concentrations did not change significantly during pregnancy. Fibrinogen, PLG and FDP concentrations changed similarly as TAT. Eight pregnants whose plasma PIC concentrations elevated more than 1.0 micrograms/l were further examined. In 6 women out of them (71.5%), FDP concentrations were elevated. In this particular group of subjects, however, they delivered normally without complications such as toxemia. These observations suggest that, at least, a hypercoagulative state progresses with pregnancy, being normalized after the delivery. Although we could not find the relationship between the hypercoagulation and clinical complications such as thrombosis and toxemia of pregnancy, present findings suggest that special caution should be paid on the pregnants whose TAT and FDP levels are elevated.

Comparability of hamster with human faecal unconjugated bile acids in a model of colorectal cancer.

The relationship between experimental colorectal carcinogenesis and bile acids has usually been investigated in the rat, a species with a markedly different bile acid profile from man. In this study, we show that the hamster faecal bile acid profile is similar to that in man. Rectal cancer was induced in hamsters using twice weekly instillations of N-methyl-N-nitro-N-nitrosoguanidine (MNNG) for 4 weeks at doses of 1-8 mg kg-1. The medcian (range) faecal bile acid concentrations of tumour-bearing hamsters (0.52, 0.46-0.84 mumoles g-1 faeces) was reduced compared to controls (1.08, 0.95-1.65, mumoles g-1) and non-tumour bearing MNNG treated hamsters (1.18, 0.64-1.42 mumoles g-1), largely due to a decrease in cholic acid derivatives (all p less than 0.05) at least). This model may be more suitable for studying the relationship between colorectal cancer and bile acids.

[Anticoagulants preventing pseudo-thrombocytopenia].

It is well known that some anticoagulants, especially EDTA (ethylene diamine tetraacetic acid), sometimes lead to pseudo-thrombocytopenia due to the aggregation of thrombocytes. We evaluated appropriate anticoagulants that prevent pseudo-thrombocytopenia without affecting other hematological data. In this study, 10 mg EDTA-2K and 1 mg citric acid were added to 1 ml of blood as anticoagulants. Using these anticoagulants (EC method), an aggregation of thrombocytes was clearly inhibited and the platelet counts remained stable in 10 cases with pseudo-thrombocytopenia. This method did not affect the other blood cell counts, the stainability and the morphology of the cells. Since the addition of citric acid to EDTA could prevent the cell volume change induced by the high concentration of EDTA, the micro-hematocrit values were remained unchanged. Hematological data in 20 cases which did not show any pseudo-thrombocytopenia, correlated significantly between the EDTA method and the EC method, and did not change significantly 24 hours after blood sampling. It is concluded that EDTA and citric acid may be a useful combination of anticoagulants for the prevention of pseudo-thrombocytopenia.

Specific inhibitory effect of dietary eicosapentaenoic acid on N-nitroso-N-methylurea-induced mammary carcinogenesis in female Sprague-Dawley rats.

We have investigated the effects of two qualitatively different types of unsaturated fatty acids on N-nitroso-N-methylurea (NMU)-induced mammary carcinogenesis in female Sprague-Dawley rats. Semipurified diets containing 4.7% eicosapentaenoic acid (EPA) plus 0.3% linoleic acid or 5% linoleic acid were prepared. Animals maintained on these diets were given an i.v. injection of NMU (50 mg/kg body wt) at 50 days of age and killed 20 weeks later. Both tumor incidence and tumor number per rat were significantly lower in the EPA diet group (60.0% and 2.3 +/- 2.5 versus 93.3% and 5.1 +/- 4.5 respectively) for the 5% linoleic acid diet. Furthermore, the average weight of tumor material (total) per rat was significantly lower in the EPA as compared to linoleic acid diet group (2.9 +/- 4.2 g and 11.4 +/- 12.2 g respectively). Analysis of phospholipid fatty acids in the mammary tumors in the EPA diet group showed a higher proportion of C16:0, C18:2, omega-3 fatty acids C20:5 and C22:6 and a lower proportion of C20:4. Furthermore, mammary tumors in rats fed the EPA diet demonstrated significant reduction in prostaglandins. The results thus suggest that inhibition by EPA of NMU-induced mammary carcinogenesis may be mediated via the modulation of lipid metabolism and associated reduction in prostaglandin synthesis.

[A double cancer of the gallbladder and common bile duct associated with an anomalous arrangement of the choledocho-pancreatic ductal junction--a case report and a review of the literature].

Reported is the case of a 65-year-old woman who complained of general fatigue and itching. The cancer of the gallbladder and the common bile duct associated with anomalous arrangement of choledocho-pancreatic ductal junction was diagnosed after examination by ERCP, PTCS, US, and CT. The cancer of the common bile duct had metastasized to the pancreas, the portal vein, and the regional lymph nodes and was unresectable. It is well known that an anomalous arrangement of the choledocho-pancreatic ductal junction is highly associated with a cancer of the common bile duct or the gallbladder. But a case that is associated with double cancers is rare and only 6 cases have been reported in the Japanese literature. In addition to a discussion of this case, the literature is reviewed.

Prognostic significance of serosal invasion and free intraperitoneal cancer cells in gastric cancer.

Survival rates after curative gastrectomy for advanced gastric cancer among 238 patients in whom the cancer was invading the serosa were compared with 283 patients without serosal invasion. Generalized Wilcoxon estimates for 5-year survival rate were 47.1 per cent for patients exhibiting serosal invasion and 75.9 per cent for patients without serosal invasion. The frequency of lymph node metastasis increased proportionately with the extent of serosal invasion: 18.4 per cent in cases of S0; 53.8 per cent in cases of S1; 80.0 per cent in cases of S2; and 91.4 per cent in cases of S3. The higher the aggregate total of S (serosal invasion) and n (lymph node metastasis) factors, the lower the 5-year survival rate. In addition, patients with serosal invasion had a propensity for peritoneal dissemination of cancer cells; the percentage of cases with intraperitoneal free cancer cells increased with the extent of serosal invasion. It is worth noting that when cancer infiltration proceeded to the deeper layers and was accompanied by nodal metastasis, cancerous invasion of the perinodal fatty tissue was frequently evident. Therefore, unfavourable prognosis after curative resection in gastric cancer patients with serosal invasion may be largely dependent on whether or not the cancer has invaded the peritoneal cavity and the perinodal fatty tissue.

Effect of dietary eicosapentaenoic acid on azoxymethane-induced colon carcinogenesis in rats.

The effects of eicosapentaenoic acid (EPA, n-3 polyunsaturated fatty acid) and linoleic acid (n-6 polyunsaturated fatty acid) on azoxymethane-induced colon carcinogenesis in rats were studied. Male Donryu rats were given two types of semipurified diet containing 4.7% EPA plus 0.3% linoleic acid and 5% linoleic acid. The rats were given s.c. injection of azoxymethane (7.4 mg/kg body weight once a week for 11 weeks) and sacrificed 15 weeks after the last injection of azoxymethane. The tumor incidence and tumor yields (tumors per rat) of the colon were significantly lower in rats on the EPA diet compared to those on the linoleic acid diet; i.e., 33%, 0.41 +/- 0.61 and 69%, 1.66 +/- 1.69, respectively. In the analysis of phospholipid fatty acid composition, the colon tumor showed higher levels of arachidonic acid and lower levels of linoleic acid than those in the normal colon mucosa in both diet groups. Despite the increase of arachidonic acid in colon tumor, the EPA diet suppressed the excessive production of prostaglandin E2, which may be accompanied with neoplastic formation, whereas linoleic acid diet caused a marked increase in the tumor content of prostaglandin E2 compared to normal colon mucosa. These results suggest that EPA exerts its inhibitory effect on colon carcinogenesis by modulating lipid metabolism and inhibiting prostaglandin E2 synthesis in tumor cells.

Effects of dietary saturated and unsaturated fatty acids on fecal bile acids and colon carcinogenesis induced by azoxymethane in rats.

To determine whether the kind of dietary fat affects colon carcinogenesis, male Donryu rats were fed a 5% fat diet containing linoleate, an unsaturated fat, or stearate, a saturated fat, in semipurified fat-free chow. The rats were given azoxymethane (7.4 mg/kg body weight) s.c. once a week for 11 weeks and killed 15 weeks after the last injection of the carcinogen. The rats on the unsaturated fat diet had a significantly higher incidence of colon tumors. Fatty acid analysis of cholesterol esters in the liver and examination of the amount of fecal bile acids showed that the unsaturated fat diet increased the level of cholesterol linoleate and arachidonate in the liver and also increased the fecal excretion of bile acids, especially that of lithocholic acid. The colon tumors in rats on the unsaturated fat diet, compared with those in rats on the saturated fat diet, contained a higher level of lysophosphatidylcholine. These results suggest that increased fecal excretion of bile acids due to increased polyunsaturated cholesterol esters in the liver stimulates phospholipase A2 activity of colon initiated cells and enhances colon carcinogenesis in rats on the unsaturated fat diet.