Probable implication of mutations of the X open reading frame in the onset of fulminant hepatitis B

A pathogenic role of precore-defective mutation in the onset of fulminant hepatitis B has been suggested. However, precore-defective mutants do not always cause fulminant hepatitis B and are not always isolated from affected patients. These findings strongly suggest the presence of some additional important mutations outside the precore region in fulminant hepatitis. In the present investigation an attempt was made to sequence the X open reading frame of hepatitis B virus DNA isolated from seven patients with fulminant hepatitis B and five patients with acute hepatitis B. The latter were used as controls. Since the X open reading frame encodes the X protein and contains the core promoter/enhancer II complex, some critical mutations may enhance or disrupt the replication and expression of hepatitis B virus DNA leading to fulminant hepatitis. A C-to-T substitution was found at nucleotide (nt) 1655, an A-to-T substitution at nt 1764 and a G-to-A substitution at nt 1766 in 4, 5 and 5 patients, respectively, out of the seven with fulminant hepatitis. These substitutions were not recognized in the patients with acute hepatitis. These mutations might change the function of the X protein and core promoter/enhancer II complex. It is suggested, therefore, that these mutations, as well as the precore-defective mutation, may play an important role in the pathogenesis of fulminant hepatitis. © Wiley-Liss, Inc.     

Possible susceptibility of the HLA-DPB1*0402 and HLA-DPB1*04 alleles to unexplained recurrent abortion: analysis by means of polymerase chain reaction-restricted fragment length polymorphism method

PROBLEM: To clarify whether HLA-DP antigens are associated with patient population of unexplained recurrent abortion. METHOD OF STUDY: The frequency of HLA-DPB1 alleles in patients with unexplained recurrent abortion, and the compatibility of HLA-DPB1 alleles between patient couples, were studied using a polymerase chain reaction (PCR)-restricted fragment length polymorphism (RFLP) method. Thirty patients who had a history of unexplained primary recurrent abortion, and their husbands, were typed for HLA-DPB1 genotype. Two hundred and ninety-nine base pair fragments from the second exon of HLA-DPB1 genes were selectively amplified using the PCR-primers. After amplification, the DNAs were digested with restriction endonucleases, and subjected to electrophoresis in a 12% polyacrilamide gel to determine HLA-DPB1 genotype. RESULTS: The frequency of HLA-DPB1*0402 and DPB1*04 alleles in the patient group (n = 30) was significantly increased, as compared to that in the normal fertile women (n = 30). The frequency of HLA-DPB1*04 allele in the patient group was significantly increased, as compared to that in the general population (n = 112). No significant compatibility of HLA-DPB1 alleles could be observed between patient couples and normal fertile couples. CONCLUSION: These findings suggest a possible new class II association with patient population of unexplained recurrent abortion.     

An enzyme-linked immunosorbent assay for thyroxine in dried blood spotted on filter paper

We have developed a rapid and sensitive enzyme-linked immunosorbent assay (ELISA) for thyroxine (T4) in dried blood samples spotted on filter paper. The assay is carried out on microtiter plates without extraction or centrifugation steps. The detection limit of the assay is 5 pg/disc/well, equivalent to 1.25 micrograms/1 of whole blood or 2.5 micrograms/1 of serum. Intra- and inter-assay coefficients of variation for various T4 concentrations are 2.4-9.0% and 5.9-17.5% respectively. Correlation between the proposed ELISA method and the RIA is good (r = 0.900, n = 62, y(RIA) = 0.99x(ELISA) + 9.90). The ELISA method is useful for mass-screening of neonatal congenital hypothyroidism using dried blood samples on filter paper, is very simple and one person can assay more than 300 samples per day.     

Sleep and waking states in infancy: normative studies

Twelve-hour polygraphic recordings were obtained in 20 normal healthy term infants at 1 week of age, at monthly intervals up to 4 months, and at 6 months of age. Each minute of these recordings was coded into active sleep (AS), quiet sleep (QS), wakefulness (AW), or indeterminate (IN) based on polygraphic and behavioral variables. For each state, a dozen variables were computed with the help of a laboratory computer. Together these variables describe trends in the development of sleep and wakefulness in the laboratory: an increase in QS and a concomitant decrease in AS, an increase in sustained episodes of these states, and continuous sleep onset in AS throughout this time span. Considerable variability appears to characterize immature sleep patterns, but a reduction in variability was noted between 3 and 4 months of age. The number of sustained sleep-state episodes and the percentage of AS and IN proved to be stable characteristics of individual infants. The large variability among and within infants sheds doubt on the usefulness of polygraphic monitoring of sleep states for early detection of abnormalities.     

Trehalose 6,6'-dimycolate (cord factor) of Mycobacterium tuberculosis induces foreign-body- and hypersensitivity-type granulomas in mice

Granulomatous inflammation is characterized morphologically by a compact organized collection of macrophages and their derivatives. It is classified as either a hypersensitivity type or a foreign-body type. Lipid components of the Mycobacterium tuberculosis cell wall participate in the pathogenesis of infection. Strains of M. tuberculosis have cord factor (trehalose 6,6'-dimycolate [TDM]) on their surface. To clarify host responses to TDM, including immunogenicity and pathogenicity, we have analyzed the footpad reaction, histopathology, and cytokine profiles of experimental granulomatous lesions in immunized and unimmunized mice challenged with TDM. In the present study, we have demonstrated for the first time that TDM can induce both foreign-body-type (nonimmune) and hypersensitivity-type (immune) granulomas by acting as a nonspecific irritant and T-cell-dependent antigen. Immunized mice challenged with TDM developed more severe lesions than unimmunized mice. At the active lesion, we found monocyte chemotactic, proinflammatory, and immunoregulatory cytokines. The level was enhanced in immunized mice challenged with TDM. This result implies that both nonimmune and immune mechanisms participate in granulomatous inflammation induced by mycobacterial infection. Taken together with a previous report, this study shows that TDM is a pleiotropic molecule against the host and plays an important role in the pathogenesis of tuberculosis.     

Complete Nucleotide Sequence and Phylogenetic Analyses of Hepatitis B Virus Isolated from Two Pileated Gibbons

Virus-like particles (VLPs, named HmTV1-17), about 40nm in diameter were found in the violet root rot fungus Helicobasidium mompa Tanaka strain No. 17, which had been isolated from an apple tree. Purified preparations of HmTV1-17 contained two species of double-stranded RNA (dsRNA), designated 17L and 17S. cDNAs were constructed from HmTV1-17 genomic dsRNAs purified using CF-11 cellulose column chromatography. The sequences of 17L and 17S cDNA comprised 5207 and 2096bp, respectively. Although 17S has no large open reading flame (ORF) on either strand, 17L has two large overlapping ORFs. The 5 located ORF1 encodes the coat protein (CP, 788 amino acids), whereas the gene product of ORF2, which is in the –1 frame relative to ORF1, shows the typical features of a RNA dependent RNA polymerase (RDRP, 845 amino acids). Phylogenetic analysis based on RDRP showed that HmTV1-17 is closely related to Sphaeropsis sapinea SsRV1, a member of the genus Totivirus from filamentous fungus S. sapinea.     

Duodenal wall cysts may be derived from a ductal component of ectopic pancreatic tissue

AIMS: To clarify the mechanism of origin of duodenal wall cysts in patients with chronic pancreatitis, developing into duodenal stenosis. METHODS AND RESULTS: Specimens from 12 pancreatoduodenectomized patients with chronic pancreatitis and 51 controls were studied histopathologically and immunohistochemically. Variously shaped cystic lesions, averaging about 15 mm in diameter, were found in the duodenum in six of the 12 patients with chronic pancreatitis, but were not observed in the controls. Each case had an average of two cysts, which were located mainly in the muscularis propria of the duodenum with or without submucosal or extraduodenal-peripancreatic extensions. The inner part of the cyst wall consisted of a moderate rim of granulation tissue, with both myofibroblasts and smooth muscle proliferation in the tissue surrounding the cyst and the submucosal layer of the duodenum, occasionally accompanied by an epithelial lining. A ductal structure in the muscularis propria of the duodenum, possibly a ductal component of ectopic pancreatic tissue, was found in five of the six cases. Some of these structures showed cystic changes. Three of the six patients had accompanying duodenal stenosis. CONCLUSIONS: Duodenal wall cysts occur mainly in the muscularis propria of the duodenum associated with both myofibroblasts and smooth muscle proliferation, and may result in duodenal stenosis. These cysts may be derived from a ductal component of ectopic pancreatic tissue.     

Analysis of intestinal HLA-DR bound peptides and dysregulated immune responses to enteric flora in the pathogenesis of inflammatory bowel disease

Isolation of antigenic peptides from the MHC-groove has contributed to the understanding of T cell responses. However, these MHC-associated peptides have been isolated from various murine and human cell lines. The specific antigen responsible for the pathogenesis of inflammatory bowel disease is unknown. We examined antigenic peptides bound to the class II major histocompatibility complex (MHC) groove in human intestine by ion-trap tandem mass spectrometry equipped with online reverse-phase high performance liquid chromatography. We detected 55 parent proteins from 4 controls, 9 patients with ulcerative colitis, and 9 patients with Crohn's disease. The calculated molecular masses (m/z) of these peptides ranged from 874.4 to 2727.4, representing 10-26 amino acid residues. Fifty-one of these 55 parent proteins were exogenous proteins. Escherichia coli-, Saccharomyces cerevisiae-, and Caenorhabditis elegans-derived peptides were found frequently in patients with inflammatory bowel disease. The present results suggest that in vivo antigen processing by antigen-presenting cells and T lymphocytes in human intestine participate with exogenous antigen presentation. Increased immune responses against E. coli, S. cerevisiae and C. elegans found in patients with inflammatory bowel may participate as dysregulated immune responses to enteric flora in the pathogenesis of inflammatory bowel disease.