Heterogeneity of hepatitis delta antigen

Hepatitis delta antigen (HDAg) is the only known protein encoded by the hepatitis delta virus (HDV). Two HDAg species of different sizes have been detected in the sera and livers of the infected humans, chimpanzees, and woodchucks, even though only one RNA species was previously identified in most of the HDV strains. To study HDAg heterogeneity, we took advantage of the fact that a single base mutation at nucleotide 1015 (C to U), which results in an amber termination codon in the HDAg open reading frame (ORF), eliminates a unique Ncol restriction enzyme site. We screened various HDV cDNA clones and detected sequence heterogeneity of the HDAg-coding region on the basis of the presence or absence of the Ncol site. Five delta hepatitis patients were examined. In every patient, two types of HDAg-coding sequence were detected at nucleotide 1015: one which contains a C and results in an ORF encoding a delta antigen of 214 amino acids, and the other which possesses a U and results in an amber termination codon and a truncated HDAg species of 195 amino acids. The in vitro translation products of these two ORFs comigrated with the two HDAg species from the patient's plasma on SDS polyacrylamide gels. Polymerase chain reaction (PCR) amplification of the HDV RNA from some patients' sera and subsequent sequencing showed several additional mutations in the HDAg-coding region. These mutations are independent of the C or U nucleotide change at the site of the amber termination codon.(ABSTRACT TRUNCATED AT 250 WORDS)     

Predominance of methicillin-resistant Staphylococcus aureus in the residents and environments of long-term care facilities in Taiwan

Background/purpose

This study investigated the distribution and persistence of multidrug resistant organisms (MDROs) including methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Enterobacteriaceae (CRE), carbapenem-resistant Pseudomonas aeruginosa (CRPA), and multidrug-resistant Acinetobacter baumannii (MDRAB) in six long-term care facilities (LTCFs).

Contribution of histamine and prostanoids to bronchoconstriction provoked by inhaled bradykinin in atopic asthma

Bradykinin, a nonapeptide cleavage product of high molecular weight kininogen, is a potent bronchoconstrictor agonist in asthma; however, its mechanism of action is not known. Since bradykinin has been shown to stimulate mediator release from mast cells and augment the release of prostanoids, we have examined the effect of a selective histamine H1 receptor antagonist, terfenadine and a potent cyclooxygenase inhibitor, flurbiprofen on bronchoconstriction provoked by inhaled bradykinin in asthma. As a bronchial provocation procedure bradykinin challenge was repeatable to within 1 doubling dilution. In nine atopic asthmatic subjects, terfenadine 180 mg, when compared to placebo, increased the geometric mean provocation concentration of inhaled agonist required to reduce FEV1 by 20% of baseline (PC20) from 0.7 to greater than 22.9 mg/ml for histamine (P less than 0.01) and 0.3 to 0.5 mg/ml for bradykinin (P less than 0.01). In a further nine atopic asthmatics, flurbiprofen 150 mg when compared to placebo produced a small but significant protection of the airways against bradykinin, geometric mean PC20 increasing from 0.40 to 0.79 mg/ml (P less than 0.05). We conclude that bradykinin is a potent bronchoconstrictor agonist in asthma, being approximately 9.5 times more potent than histamine in molar terms. Pharmacological intervention with terfenadine and flurbiprofen led to a significant protection of the airways against the constrictor effect of bradykinin but the effect in each case was small. Thus, while histamine and prostanoids may contribute as mediators of bradykinin-induced bronchoconstriction, they are unlikely to account for the majority of the response.     

Expression of cytokeratins in normal and diseased livers and in primary liver carcinomas

Hepatocytes and bile duct epithelium express several types of cytokeratins, the characteristic intermediate-filament proteins of epithelial cells. The cytokeratin antigen expression was studied in normal and diseased livers, intrahepatic cholangiocarcinomas, and hepatocellular carcinomas by immunohistochemical methods using a panel of polyclonal and monoclonal antibodies to cytokeratins. Ten percent formaldehyde solution-fixed, paraffin-embedded sections obtained from ten patients without liver disease, 18 patients without liver disease, 18 patients with different stages of primary biliary cirrhosis, 14 patients with alcoholic hepatitis, ten patients with fatty liver hepatitis secondary to diabetes mellitus or morbid obesity, five patients with hepatocellular carcinomas, and five patients with cholangiocarcinomas were examined. The results suggested that hepatocytes and bile duct epithelium retain their distinct cytokeratin profiles in liver disease, including malignant transformation. Therefore, demonstration of cytokeratins in the liver is useful in establishing the cellular origin of neoplasms and understanding the pathogenesis of liver diseases.     

Generation of alphabeta T-cell receptor+ CD4- CD8+ cells in major histocompatibility complex class I-deficient mice upon activation of the aryl hydrocarbon receptor by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Gene-targeted mice lacking the beta2 microglobulin gene (beta2m-/- mice), and hence functional major histocompatibility complex (MHC) class I molecules, do not develop CD4- CD8+ cells. We show here that both in vitro and in vivo treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a trans-activating ligand of the endogenous aryl hydrocarbon receptor (Ah-R), bypasses the need for MHC class I molecules for selection into the CD4- CD8+ cell pool. When beta2m-/- dams were given a single dose of 50 microg of TCDD, approximately 13% of CD4- CD8+ thymocytes could be detected in their newborn pups. In TCDD-exposed fetal thymus organ cultures of beta2m-/- mice, approximately 35% CD4- CD8+ thymocytes were detectable. About 16% of these CD4- CD8+ cells bore the alpha beta T-cell receptor (TCR) and approximately 33% bore CD3. Only a minority of the CD8+ cells were heat-shock antigen positive. The cells possessed killing activity as shown using the 51Cr-release assay comprising gamma delta TCR- CD4- CD8+ thymocytes from 3 to 4-day-old b2m-/- mice. Thus, TCDD leads to a significant increase of mature CD4- CD8+ thymocytes in relative and absolute numbers. High numbers of CD4- CD8+ thymocytes developed also in organ cultures from thymi, lacking both MHC class I and class II molecules, exposed to TCDD. A 10-fold transient increase of Notch1 mRNA in thymocytes from fetal thymus organ culture, exposed for 4 days to TCDD, was detected in CD4+ CD8+ cells compared with controls. We suggest that TCDD affects thymic selection and directs the lineage commitment of CD4+ CD8+ thymocytes towards CD4- CD8+ cells, possibly via up-regulation of the Notch1 gene.     

T-lymphocyte activation in IgA nephropathy: Serum-soluble interleukin 2 receptor level,interleukin 2 production,and interleukin 2 receptor expression by cultured lymphocytes

The present study was undertaken to examine the T-lymphocyte activation in IgA nephropathy. Serum-soluble interleukin 2 receptor (sIL2R) levels were studied in 29 IgA nephritic patients, 17 patients with chronic glomerulonephritis (non-IgA nephropathy), and 30 healthy controls during an infection-free period. No difference in serum sIL2R level was demonstrated among these three groups of subjects. However, the serum sIL2R levels of IgA nephritic patient rose significantly during clinical exacerbation with synpharyngitic macroscopic hematuria and the serum sIL2R levels fell when hematuria subsided. Mitogen-stimulated cellular interleukin 2 receptor (IL2R) expression, sIL2R release, and interleukin 2 (IL2) production were also examined in peripheral blood mononuclear cells (PBMC) cultured for 24–48 hr in 21 patients with IgA nephropathy, 17 patients with chronic glomerulonephritides, and 17 healthy controls. The total cellular IL2R expression and sIL2R release did not differ among these three groups of subjects. However, the individual T-cell subsets bearing IL2R were distinctly different between IgA nephritic patients and the other two groups of controls. IgA nephritic patients had increased activated CD4+ lymphocytes and reduced activated CD8+ lymphocytes. Furthermore, IL2 production in response to phytohemagglutinin and pokeweed mitogen stimulation was increased in lymphocytes from patients with IgA nephropathy. The IL2 production did not correlate with the quantities of cellular and sIL2R yet the cellular IL2R expression paralleled the sIL2R released by cultured lymphocytes. Our present study suggests that the T lymphocytes from patients with IgA nephropathy have a defect in overproduction of IL2 and increased activated T helper-cell subset upon mitogenic stimulation. Serum measurement of sIL2R could potentially be useful in monitoring the disease activity.     

Class II defective avian sarcoma viruses: Comparative analysis of genome structure

The genomes of class II avian sarcoma viruses PRCII, PRCII-p, PRCIV, and Fujinami sarcoma virus (FSV), were studied by oligonucleotide fingerprinting, heteroduplex mapping, and nucleic acid hybridization. All of these viruses are genetically defective and have a small RNA genome between 4.5 and 6.1 kilobases (kb) in length. They contain helper-related sequences at both the 5′- and 3′-ends, but most of the retroviral sequences in the middle of the genome are deleted. In place of this deleted information, a contiguous stretch of transformation-specific sequences, termed fps, is found. These putative oncogenic sequences are about 1.2 kb in PRCII, and those in PRCII-p and PRCIV are roughly 2.9 kb. From the analysis of oligonucleotides, it appears that the fps sequences of PRCII represent a subset of those of PRCII-p. Most of the additional sequences present in PRCII-p but absent from PRCII are at the 5′-half of fps. The helper-related sequences in PRCII and PRCII-p are almost indistinguishable, except that PRCII-p contains slightly more retroviral information at the 3′-end of the genome. Therefore, it is possible that PRCII has been derived by deletion from PRCII-p. By contrast, PRCII-p and PRCIV were found to contain identical fps sequences, but their helper-related sequences have diverged substantially. These two sarcoma viruses either represent two independent isolates or, if derived from a single isolate, they have undergone extensive mutation and recombination with diverse avian retroviruses. FSV was found to differ to a greater extent from other class II sarcoma viruses in both helper-related and fps sequences. The difference in fps sequences is localized in the 5′-half of that region. Considering the variation in fps among all members of class II avian sarcoma viruses, it appears that the 3′-half of that genetic region is more conserved than the 5′-half.     

Impact of cadaveric organ donation on Taiwanese donor families during the first 6 months after donation

OBJECTIVE: Organ donation is a complex decision for family members of Asian donors. The impact of cadaveric organ donation on both Chinese and Western donor families has not been well investigated within a cultural framework. The purposes of this study were to follow Chinese family members' appraisal of their decision to donate organs, to explore the possible negative and positive impacts of organ donation on their family life, and to determine what help they expected from healthcare providers during the first 6 months after donation. METHODS: Twenty-two family members (10 men and 12 women) of cadaveric organ donors who signed consent forms at an organ transplant medical center in Taiwan participated in this project and completed in-depth interviews during the sixth month after donation. RESULTS: Participants were 25 to 56 years old (mean = 48.15 +/- 8.31 years). The type of kinship of the participants included the donor's parents, older sister, and spouse. Subjects reported several negative impacts: worry about the donor's afterlife (86%), stress due to controversy among family members over the decision to donate (77%), and stress due to others' devaluation of the donation (45%). Positive impacts reported by the subjects included having a sense of reward for helping others (36%), having an increased appreciation of life (32%), having closer family relationships (23%), and planning to shift life goals to the study of medicine (9%). Subjects expected the transplant team to provide information about organ recipients (73%), to submit the necessary documents so that family members could receive healthcare payments from the insurance company (68%), to help resolve legal proceedings and settlements associated with accidents (64%), and to not overly publicize their decision to donate (64%). CONCLUSIONS: Although all of the subjects reported that organ donation was the right decision, the decision to donate did not protect Taiwanese donor families from negative psychocognitive bereavement. The impacts of organ donation were affected by the subject's social cultural, spiritual, and legal context and the nature of their bereavement.     

Adeno-associated virus-mediated vascular endothelial growth factor gene therapy in skeletal muscle before transplantation promotes revascularization of regenerating muscle

Successful clinical transplantation of whole skeletal muscles can be limited by impaired muscle revascularization and regeneration. The aim of this study was to enhance the revascularization (and hence speed of regeneration) of transplanted whole muscles by transducing muscles with the vascular endothelial growth factor (VEGF) gene before transplantation, using a recombinant adeno-associated virus (rAAV). The rAAV encoding VEGF and green fluorescent protein (GFP) (rAAV.VEGF.GFP) was injected into the tibialis anterior muscles of adult BALB/c mice. One month after injection whole muscle autotransplantation was performed. Muscles were sampled 7 days after autografting. GFP expression was examined as an indicator of persistent transgene expression after grafting, and immunohistochemistry was used to identify VEGF, blood vessels, and newly formed myotubes. After grafting, GFP expression persisted only in a few surviving myofibers in the periphery of rAAV.VEGF.GFP-pretreated muscles, although abundant VEGF expression was seen in myogenic cells in all grafted muscles. Quantitative analysis demonstrated that, although only small numbers of rAAV.VEGF.GFP-transduced myofibers were present, whole muscle grafts preinjected with rAAV.VEGF.GFP were significantly more vascular than saline-injected and uninjected control muscle grafts. Furthermore, rAAV.VEGF.GFP-injected whole muscle transplants were further advanced in terms of regeneration (myotube formation) compared with the uninjected control muscle transplants. This study clearly shows that rAAV-mediated VEGF expression persists only in myofibers that survive the necrosis induced by muscle transplantation; however, this amount of VEGF results in significantly increased revascularization and regeneration of whole muscle transplants.