Heat-killed Lactobacillus helveticus improves mood states: a randomised, double-blind, placebo-controlled study.

We investigated the effects of heat-killed Lactobacillus helveticus MCC1848 on daily mood states in healthy young adults. Participants (n=58) were randomised to receive heat-killed L. helveticus MCC1848 powder or placebo powder for 4 weeks. During the study period, adverse events were recorded in the participant diary. Mood states were assessed before and 2 and 4 weeks after initiation of the intervention. The primary outcomes were the shortened version of the Profile of Mood States 2 (POMS 2) scores. Secondary outcomes included other mood state (State-Trait Anxiety Inventory (STAI); visual analogue scale (VAS)), quality of life (acute form of the SF-36v2), sleep (Athens Insomnia Scale (AIS)) and fatigue (Chalder Fatigue Scale (CFS)) scores. Four weeks of heat-killed L. helveticus MCC1848 intake, compared to placebo, significantly improved the shortened version of the POMS 2 'friendliness' and the VAS 'relaxed' scores, which are two indicators of positive mood states. On the other hand, heat-killed L. helveticus MCC1848 intake had no significant effects on negative mood state items (e.g. anger, nervousness, confusion) assessed by the shortened version of the POMS 2, STAI and VAS. AIS and CFS scores also showed no significant differences. No adverse effects were observed with 4 weeks of heat-killed L. helveticus MCC1848 intake. These results suggest that daily consumption of heat-killed L. helveticus MCC1848 is safe and has the potential to improve positive mood states. UMIN Clinical Trial Registry: UMIN000043697.

Pharmacogenomics of gemcitabine: can genetic studies lead to tailor-made therapy?

Gemcitabine is a deoxycytidine analogue that has a broad spectrum of antitumour activity in many solid tumours including pancreatic cancer. We have recently carried out a pharmacogenomic study in cancer patients treated with gemcitabine, and found that one genetic polymorphism of an enzyme involved in gemcitabine metabolism can cause interindividual variations in the pharmacokinetics and toxicity of this agent. In this paper, we review recent genetic studies of gemcitabine, and discuss the possibility of individualised cancer chemotherapy based on a pharmacogenomic approach.

Anti-Helicobacter pylori seropositivity: influence on severity and treatment response in patients with chronic hepatitis C.

We sought to clarify the incidence and role of Helicobacter pylori (H. pylori) seropositivity in patients with hepatitis C virus (HCV) infection and the effect of coinfection on interferon-alpha and ribavirin therapy. The presence of H. pylori was tested using a commercially available enzyme immunoassay in serum samples from 93 patients with chronic hepatitis C. Clinical features, HCV markers and response of HCV to interferon-alpha and ribavirin were compared between H. pylori-positive and H. pylori-negative patients. Anti-H. pylori antibody was detected in 45 (48%) of the 93 patients, whose median HCV-RNA level (495 vs 760 kIU/mL; P = 0.013) and platelet count (128 vs 158 x 10(3)/microL; P = 0.009) were significantly lower than in patients with HCV infection alone. Anti-H. pylori antibody levels were found to be significantly correlated with fibrosis score (P = 0.0083, r = 0.33) but inversely related to platelet count (P = 0.0037, r = -0.34). The sustained response rate for HCV clearance following interferon-alpha and ribavirin treatment did not differ between patients with and without anti-H. pylori seropositivity. The presence of H. pylori [odds ratio (OR) 8.61; 95% confidence interval (CI) 1.59-46.70] and fibrosis score (OR 30.13; 95% CI 5.44-166.78) were found by multivariate analysis to be associated with the decrease of platelet count during therapy. Coexistent H. pylori infection does not demonstrably influence the clinical course of chronic hepatitis C. A possible connection between H. pylori coinfection and thrombocytopenia was found during the treatment course, suggesting that preemptive eradication of H. pylori may facilitate completion of treatment and increased sustained virological response.

Inhibition of NADPH oxidase 4 activates apoptosis via the AKT/apoptosis signal-regulating kinase 1 pathway in pancreatic cancer PANC-1 cells.

Pancreatic adenocarcinoma is an aggressive human malignancy and is characterized by resistance to apoptosis. Recently, NADPH oxidase (Nox) 4-mediated generation of intracellular reactive oxygen species (ROS) was proposed to confer antiapoptotic activity and thus a growth advantage to pancreatic cancer cells. The signaling mechanism by which Nox4 transmits cell survival signals remains unclear. Here, we show that both a flavoprotein inhibitor, diphenylene iodonium (DPI), and small interfering RNAs designed to target Nox4 mRNA (siNox4RNAs) inhibited superoxide production in PANC-1 pancreatic cancer cells, and depletion of ROS by DPI or siNox4RNAs induced apoptosis. Parallely, DPI treatment and siNox4RNA transfection blocked activation of the cell survival kinase AKT by attenuating phosphorylation of AKT. Furthermore, AKT phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) on Ser-83 was reduced by DPI and siNox4RNAs. When ASK1Ser83Ala (an AKT phosphorylation-defective ASK1 mutant) was introduced into PANC-1 cells, this mutant alone induced apoptosis. But, addition of DPI or co-transfection of siNox4RNA had no additive effect, indicating that the mutant can substitute for these reagents in apoptosis induction. Taken together, these findings suggest that ROS generated by Nox4, at least in part, transmit cell survival signals through the AKT-ASK1 pathway in pancreatic cancer cells and their depletion leads to apoptosis.

Laceration of gastric mucosa associated with dialysis-related amyloidosis.

We report a 72-year-old female on long-term hemodialysis, who was admitted to the hospital because of hematemesis. On emergency laparotomy, pylorogastrectomy was performed. The resected specimen showed a giant hematoma and traversing fissure along the lesser curvature of the body of the stomach. Histologically, the specimen showed wide hematoma formation and amyloid deposits in the submucosal layer, especially in the wall of blood vessels. These deposits reacted positively to antihuman beta2-microglobulin antibody. The post-operative course was favorable, and the patient was discharged on the 35th hospital day. In this case, the laceration site on the gastric mucosa was almost intact and did not demonstrate ischemic change, suggesting that the giant hematoma was caused by submucosal vessel rupture, which led to the gastric mucosa laceration. To our knowledge, this is the first case of gastric mucosa laceration associated with dialysis-related amyloidosis.

Age-specific antibody to hepatitis E virus has remained constant during the past 20 years in Japan.

We investigated the presence of antibodies to hepatitis E virus (anti-HEV) and hepatitis A virus (anti-HAV) by enzyme immunoassays in sera from 1015 individuals collected in 1974, 1984 and 1994. Age-specific profiles of anti-HEV remained unchanged with a peak at 40-49 years, while those of anti-HAV started to increase in individuals aged 20-29 years in 1974, 30-39 years in 1984 and 40-49 years in 1994. These results suggest that a silent HEV infection has been taking place in the last 20 years or so in Japan, while HAV infection has been terminated at least since 1974.

Significance of chemokine receptor expression in aggressive NK cell leukemia.

Natural killer (NK) cell-type lymphoproliferative diseases of granular lymphocytes can be subdivided into aggressive NK cell leukemia (ANKL) and chronic NK cell lymphocytosis (CNKL). One reason for the poor outcome in ANKL is leukemic infiltration into multiple organs. The mechanisms of cell trafficking associated with the chemokine system have been investigated in NK cells. To clarify the mechanism of systemic migration of leukemic NK cells, we enrolled nine ANKL and six CNKL cases, and analyzed the expression profiles and functions of chemokine receptors by flowcytometry and chemotaxis assay. CXCR1 was detected on NK cells in all groups, and CCR5 was positive in all ANKL cells. Proliferating NK cells were simultaneously positive for CXCR1 and CCR5 in all ANKL patients examined, and NK cells with this phenotype did not expand in CNKL patients or healthy donors. ANKL cells showed enhanced chemotaxis toward the ligands of these receptors. These results indicated that the chemokine system might play an important role in the pathophysiology of ANKL and that chemokine receptor profiling might be a novel tool for discriminating ANKL cells from benign NK cells.

The efficacy and safety of thymosin alpha-1 in Japanese patients with chronic hepatitis B; results from a randomized clinical trial.

Thymalfasin (thymosin alpha-1; Talpha1) is a 28-amino acid polypeptide that has shown efficacy in the treatment of chronic hepatitis B virus (HBV) infection. The objective of this study was to evaluate the long-term, dose-related efficacy and safety of Talpha1 treatment in chronic hepatitis B patients with positive HBV-DNA and abnormally high alanine aminotransferase (ALT) levels. A total of 316 patients were randomized to receive either 0.8 or 1.6 mg of Talpha1 monotherapy for 24 weeks. At the end of the 72-week observation period (12 months after cessation of therapy), 36.4% of patients in the 1.6-mg treatment group achieved normalization of ALT, 30% achieved clearance of HBV-DNA by branched DNA vs 15% by transcription-mediated amplification, and 22.8% achieved clearance of HBe-antigen. Patients in the 0.8-mg treatment group achieved similar efficacy rates, although patients with advanced fibrosis demonstrated a significantly better response rate when treated with 1.6 mg of Talpha1 monotherapy vs 0.8 mg (as determined by intragroup analysis; patients were not stratified by liver biopsy). All adverse drug reactions were mild and most involved the fluctuation of liver enzymes, which was most likely related to the positive immune effects caused by the response to Talpha1 treatment. Adverse event incidence was similar in the 1.6- and 0.8-mg treatment groups. In conclusion, Talpha1 at doses of 0.8 and 1.6 mg exhibits long-term efficacy against hepatitis B with a good safety profile.

Characterization of hypervariable region in hepatitis C virus envelope protein during acute and chronic infection.

Hepatitis C virus (HCV) causes persistent infection in most patients. To clarify the mechanisms underlying establishment of this persistent infection, nucleotide sequences of the E1/E2 region were characterized in 5 patients with acute and chronic HCV infection. We used direct DNA sequencing methods to identify the major sequence of HCV in each patient. Each HCV genome displayed a high frequency of nucleotide sequence variation in the hypervariable region (HVR) of E2. However, patient-specific conserved nucleotide sequences were identified in the E1/E2 region during the course of infection and conserved the higher-order protein structure. In the acute phase HCV infection, amino acid substitution in HVR-1 as the monthly rate of amino acids substitution per site (%) between each point exceeded 10.2%. In the chronic phase HCV infection, a significantly lower rate of amino acid substitution was observed in patients. The host immune responses to HVR-1 of each HCV isolates from all clinical courses were characterized using synthetic peptides and ELISA. One chronic patient serum (genotype 1b) did not react at all to its own HVR-1 peptides, however another patient (genotype 2b) reacted to all clinical course. These results indicated that HVR-1 might not always exhibit neutralizing epitopes of HCV infection. The sequence variation in HVR-1 may instead indicate the existence of various clones in acute phase infection and the adaption of these clones is thought to have caused persistent and chronic infection in each patient.

Interferon treatment improves survival in chronic hepatitis C patients showing biochemical as well as virological responses by preventing liver-related death.

Interferon therapy for chronic hepatitis C reduces the risk of hepatocellular carcinoma, especially among virological and biochemical responders. However, little is known about the effect of interferon therapy on mortality. We studied the long-term effect of interferon therapy on mortality in patients with chronic hepatitis C. For this retrospective cohort study, 2954 patients with chronic hepatitis C were recruited, of whom 2698 received interferon therapy and 256 did not. The effect of interferon therapy on survival was assessed by standardized mortality ratio (SMR) based on published mortality data for the general Japanese population and by risk ratio calculated by proportional hazard regression. Over 6.0 +/- 2.2 years follow-up, death from liver-related diseases was observed in 69 (68%) of 101 deaths among interferon-treated patients and in 42 (81%) of 52 deaths among untreated patients. Compared with the general population, overall mortality was high among untreated patients (SMR: 2.7; 95% CI: 2.0-3.6) but not among interferon-treated patients (SMR: 0.9; 95% CI: 0.7-1.1). Liver-related mortality was extremely high among untreated patients (SMR: 22.2; 95% CI: 16.0-30.0) and less among interferon-treated patients (SMR: 5.5; 95% CI: 4.3-6.9). The risk of death from all causes was lower for interferon-treated than untreated patients (risk ratio: 0.47; 95% CI: 0.261-0.836; P = 0.01). The risk of death from liver-related diseases was significantly lower for sustained virological responders (risk ratio: 0.04; 95% CI: 0.005-0.301; P = 0.002) compared with untreated patients, but not for nonsustained virological responders. Sustained biochemical responders (risk ratio: 0.03; 95% CI: 0.004-0.230; P < 0.001) and transient biochemical responders (risk ratio: 0.18; 95% CI: 0.063-0.532; P = 0.002) showed a significantly reduced risk of death from liver-related death, whereas biochemical nonresponders did not. Hence interferon treatment improved survival in chronic hepatitis C patients showing a biochemical as well as a virological response by preventing liver-related deaths.

Younger onset myeloperoxidase-specific antineutrophil cytoplasmic antibody- (MPO-ANCA) related glomerulonephritis accompanied with nephrotic syndrome.

It is known that nephrotic syndrome rarely accompanies myeloperoxidase-specific antineutrophil cytoplasmic antibody- (MPO-ANCA) related glomerulonephritis. We present a case of younger onset MPO-ANCA-related glomerulonephritis accompanied with nephrotic syndrome in a female patient. It was diagnosed through the renal biopsy and the detection of a high titer of MPO-ANCA and steroid therapy (intravenous steroid pulse therapy and oral administration), anticoagulant therapy and antiplatelet therapy were initiated. Since her nephrotic syndrome persisted in spite of the decrease of MPO-ANCA, we conducted a second renal biopsy. We found active necrotizing crescentic glomerulonephritis with a small deposition of immunoglobulin and fibrinogen on the glomeruli. To suppress her disease activity, we administered second steroid-pulse therapy and MPO-ANCA titer disappeared. However, as her nephrotic syndrome, which was accompanied by severe hyperlipidemia, persisted, we tried to treat her using low-density lipoprotein (LDL) apheresis. It was effective temporarily, but she finally fell into end-stage renal failure. We discuss here the possibility of double nephropathy by considering her clinical and renal pathologic features.

Clinical evaluation of a new enzyme immunoassay for hepatitis B virus core-related antigen; a marker distinct from viral DNA for monitoring lamivudine treatment.

We aimed to assess the clinical performance of a newly developed chemiluminescence enzyme immunoassay (CLEIA) for the detection of hepatitis B virus (HBV) core-related antigen (HBcrAg) in patients with chronic HBV infection. A total of 82 patients with chronic HBV infection and 167 HBV-negative controls were studied. HBcrAg was measured by CLEIA with monoclonal antibodies to hepatitis B e antigen (HBeAg) and hepatitis B core antigen (HBcAg), and HBV DNA was measured by transcription-mediated amplification assay (TMA) and in-house real-time detection polymerase chain reaction (RTD-PCR). The HBcrAg assay detected viremia in 189 of 216 samples (88%) collected from 72 patients whilst the TMA assay detected viremia in 178 of the 216 samples (82%) (P = 0.019). The HBcrAg concentration correlated linearly with the HBV DNA concentration (P < 0.001) over a range which varied 100 000-fold. The accuracy in the measurement of the patients' HBV load obtained using the HBcrAg assay was not affected by the absence of hepatitis B e antigen from the serum or the presence of precore mutations in the HBV genome. In patients without anti-viral drugs, changes in their serum HBcrAg concentration over time corresponded to their HBV DNA concentration. In six additional patients who were later treated with lamivudine, HBV DNA concentration declined more rapidly than their HBcrAg concentration. Three months after treatment commenced, the ratio of HBcrAg: HBV DNA had increased in all six patients (P = 0.031). The HBcrAg assay is a sensitive and useful test for the assessment of a patient's HBV load. When monitoring the anti-viral effect of lamivudine, HBcrAg provides a viral marker which is independent of HBV DNA.

Long-term follow-up of hepatitis C virus infection: HLA class II loci influences the natural history of the disease.

Hepatitis C virus (HCV) causes various grades of chronic liver disease, ranging from an asymptomatic state to cirrhosis. To assess genetic factors of disease severity, we selected two HCV patient groups according to the following stringent criteria: (i) asymptomatic carrier state (ASC) defined by HCV infection for more than 20 years, normal alanine aminotransferase levels for the past 5 years as well as normal liver histology and/or shape and (ii) liver cirrhosis (LC) as diagnosed by clinical symptoms, liver biopsy and/or ultrasonography. A total of 103 chronically infected Japanese HCV patients (43 ASC and 60 LC) were analyzed. HLA class I and II alleles were established using low resolution DNA typing. HLA-DRB1 and DQB1 genotypes were inferred upon polymerase chain reaction-restriction fragment length polymorphism analysis. Two hundred and one anti-HCV-negative ethnically matched controls were included. The frequencies of DRB1*12 (*1201 and *1202), DQB1*0301 and DRB3*03 alleles were higher in patients with ASC than in those with LC (odds ratio (OR) 11.23, OR 4.25, and OR 3.22, respectively). The frequency of DQB1*0503 were lower in ASC patients compared to LC patients (OR 0.05). No significant differences between groups were observed for age, sex, source of infection, HCV genotype or viral loads. Our findings establish that certain HLA class II alleles strongly influence disease progression following HCV infection.

RNA interference may be more potent than antisense RNA in human cancer cell lines.

1. RNA interference (RNAi) is a newly discovered cellular pathway for the silencing of sequence-specific genes at the mRNA level by the introduction of the cognate double-stranded (ds) RNA. Because antisense (AS) mechanisms have similar effects, we compared these two effects in human cancer cell lines, considering a possible application of RNAi for cancer therapy. 2. We tested RNAi effects by transfecting human hepatoma and pancreatic cancer cell lines with AS and sense (S) RNA expression plasmids corresponding to the exogenous luciferase gene or the endogenous c-raf gene in the form of complexes with a cationic lipopolyamine or a tumour-targeting peptide vector we developed. In addition, we compared the effects of small interfering RNA and AS oligoDNA complexed with the peptide vector. 3. From the viewpoint of AS actions, the effect of the AS RNA may be cancelled by the S RNA, although, interestingly, we found that the combination of the AS and S RNA expression plasmids was more effective than the AS RNA expression plasmids alone in reducing target gene expression, whereas the S RNA expression plasmids had no effects. The combination of the luciferase AS and S RNA had no effects on the expression of either the beta-galactosidase gene or the c-raf gene. In the presence of 2-aminopurine (an inhibitor of dsRNA-activated protein kinase), the inhibitory effect of the combination of AS and S RNA on gene expression did not change in the case of the endogenous c-raf gene, but was reduced in the case of the exogenous luciferase gene. The effect of 22 nucleotide RNA duplexes corresponding to the luciferase gene was by one order stronger than that of the phosphorothioate AS DNA. 4. Thus, it is suggested that RNAi may be more potent than AS RNA in reducing target gene expression in human cancer cell lines, regardless of the length of dsRNA. With further studies on the RNAi phenomenon in cancer cells, RNAi could provide a novel approach for cancer gene therapy.

A case of primary low grade mucosa associated lymphoid tissue (MALT) lymphoma of the oesophagus.

We report a very rare case of primary low grade mucosa associated lymphoid tissue (MALT) lymphoma of the oesophagus. An 83 year old woman was referred to our hospital in June 1999 for further examination and treatment of oesophageal tumour. Although a physical examination and laboratory data showed no significant abnormalities, endoscopic observation revealed two slightly elevated submucosal tumour-like lesions of the oesophagus. Tissue specimens were obtained by endoscopic mucosal resection of the oesophagus using a cap fitted panendoscope. The lesions were composed of diffuse small atypical lymphoid cells--that is, centrocyte-like cells--which were stained with CD20, L26, BCL-2, and kappa, but not with CD3, CD5, CD10, or cyclin D1. Monoclonality was detected by polymerase chain reaction analysis using the primer for CDR-3 of immunoglobulin H and diagnosed as low grade MALT lymphoma of the oesophagus. The tumours were considered to be completely resected and therefore additional treatment was not administered. The patient is alive and well 22 months after treatment and diagnosis.

Association between severity of type A hepatitis and nucleotide variations in the 5' non-translated region of hepatitis A virus RNA: strains from fulminant hepatitis have fewer nucleotide substitutions.

BACKGROUND: Type A hepatitis is still a considerable problem in both underdeveloped and developed countries. Why some patients progress to fulminant type A hepatitis and others do not is unclear. AIMS: To determine if nucleotide differences in the genome of hepatitis A virus (HAV) are responsible for the range of clinical severities, we analysed the 5' non-translated region (5'NTR) of the HAV genome, which has an internal ribosomal entry site and is important for cap independent translation of the viral message. METHODS: Serum samples from 84 Japanese patients with sporadic type A hepatitis from five distant regions of Japan, comprising 12 patients with fulminant hepatitis (FH), 13 with severe acute hepatitis (AHs), and 59 with acute hepatitis (AH), were examined for HAV RNA. The fragment between nucleotides 75 and 638 of the 5'NTR was amplified by reverse transcription-polymerase chain reaction, and the nucleotide sequence was determined by direct sequencing. RESULTS: Comparison of sequences of the 5'NTR revealed relatively fewer nucleotide substitutions in FH and AHs patients compared with the considerable sequence variations found in strains of AH. This tendency was most prominent between nucleotides 200 and 500. Strains from FH and AHs cases had fewer nucleotide substitutions (p<0.001) in this region. CONCLUSIONS: Nucleotide variations in the central portion of the 5'NTR of HAV may influence the severity of type A hepatitis.

Amplified UvrA protein can ameliorate the ultraviolet sensitivity of an Escherichia coli recA mutant.

When a recA strain of Escherichia coli was transformed with the multicopy plasmid pSF11 carrying the uvrA gene of E. coli, its extreme ultraviolet (UV) sensitivity was decreased. The sensitivity of the lexA1 (Ind(-)) strain to UV was also decreased by pSF11. The recA cells expressing Neurospora crassa UV damage endonuclease (UVDE), encoding UV-endonuclease, show UV resistance. On the other hand, only partial amelioration of UV sensitivity of the recA strain was observed in the presence of the plasmid pNP10 carrying the uvrB gene. Host cell reactivation of UV-irradiated lambda phage in recA cells with pSF11 was as efficient as that in wild-type cells. Using an antibody to detect cyclobutane pyrimidine dimers, we found that UV-irradiated recA cells removed dimers from their DNA more rapidly if they carried pSF11 than if they carried a vacant control plasmid. Using anti-UvrA antibody, we observed that the expression level of UvrA protein was about 20-fold higher in the recA strain with pSF11 than in the recA strain without pSF11. Our results were consistent with the idea that constitutive level of UvrA protein in the recA cells results in constitutive levels of active UvrABC nuclease which is not enough to operate full nucleotide excision repair (NER), thus leading to extreme UV sensitivity.

Potential tumor-targeting peptide vector of histidylated oligolysine conjugated to a tumor-homing RGD motif.

We have developed a potential tumor-targeting peptide vector (cRGD-hK) that is intended to be systemically and repeatedly administered to patients with advanced solid tumors. The peptide vector of 36 l-amino acid residues, CRGDCF(K[H-]KKK)6, comprises a tumor-homing RGD motif, a DNA-binding oligolysine, and histidyl residues to facilitate the delivery into the cytosol. Using cytomegalovirus-driven luciferase expression plasmids as a reporter, we tested the transfection efficiency of cRGD-hK in hepatoma and pancreatic cancer cell lines. Transfection with the cRGD-hK/plasmid complexes (molar ratio 4000:1) was inhibited by 50 nM bafilomycin A1, an inhibitor of the vacuolar ATPase endosomal proton pump, or 10 microM cycloRGDfV, an integrin alphavbeta3 antagonist, indicating that the three elements of cRGD-hK could function as expected, at least in vitro. In nude mice bearing tumors created by subcutaneous inoculation, luciferase activity in the tumor tissues 48 hours after the injection of the cRGD-hK/plasmid complexes through the tail vein (20 microg plasmids per mouse) was significantly higher than that in the lung, kidney, and spleen, but only slightly higher than that in the liver. Although the latter difference was small, we propose a potential nonviral gene therapy for advanced solid tumors through use of the tumor-targeting peptide vector.