Volume 111  ·   Issue 06  ·  1998

Chronic hepatitis B (HBV) infection is a serious global health problem affecting around 300 million individuals. Of these, many will die of cirrhosis or hepatocellular carcinoma. To date, interferon has been widely used to treat chronic hepatitis B infection. Unfortunately, the use of interferon is not satisfactory in Chinese, because most infections are vertical, leading to long-lasting immune tolerant state.

OBJECTIVE To dissect the molecular mechanism of toxic neuropathy induced by allyl chloride (AC). METHODS Fluorescence molecular probe (Fura-2/AM), electron probe X-ray microprobe analysis (EPMA) and biochemical methods were used to determine the concentrations of cytosolic free Ca2+, the contents of intracellular Ca2+ percentage, Ca2+-free calmodulin (CaM), the activity of Ca2+/CaM-dependent protein kinase Ⅱ (Ca2+/CaM-PK Ⅱ), and cytoskeletal protein synthesis in chicken embryo brain cells induced by AC. RESULTS The contents of Ca2+ percentage, the concentrations of cytosolic free Ca2+, and the activities of Ca2+/CaM-PK Ⅱ in the cells were increased significantly as AC was added (P<0.01). However, the content of Ca2+-free CaM and the synthesis of cytoskeletal proteins were markedly decreased (P<0.01). CONCLUSIONS The results suggest that one of the mechanism of AC-induced cytoskeletal injury in vitro might be related to the elevation of intracellular Ca 2+, activated CaM and Ca2+/CaM-PK Ⅱ.

OBJECTIVE To elucidate the molecular changes of bone collagen during the development of postmenopausal osteoporosis and to investigate the molecular effects of estrogen replacement. METHODS An adult ovariotomy rat model was used. Type-I collagen and matrix metalloproteinase-9 (MMP-9) expressions in bone tissues of rats treated by sham surgery (SH), bilateral ovariotomy (OVX) and OVX with estradiol (OVX-E2) were analysed at mRNA level by using dot blot technique. The distribution of mRNA of these two genes in bone tissues was studied by in situ hybridization. RESULTS The expression levels of both type-I collagen and MMP-9 in bone tissues of OVX rats were higher than those of SH group, while treated with estradiol, the expression of both genes declined to some degree. In situ hybridization showed that type-I collagen mRNA located in osteoblasts, whereas MMP-9 was mainly expressed in osteoclasts, some lining cells on bone surface, and some mononuclear cells in bone marrow. CONCLUSIONS The reduction of high bone turnover in osteoporotic bone tissues induced by estrogen replacement may result from alterations in gene expression related to bone formation and bone resorption. These alterations are consistent with the changes observed previously by histomorphometry and biochemical markers of bone metabolism on OVX animals and postmenopausal osteoporosis.

OBJECTIVE To further understand the role of growth regulation of human ovarian cancer cells by transforming growth factor (TGF) β1. METHODS The cell proliferation, cAMP synthesis, gene expression, and induction of programmed cell death (PCD) in human epithelial ovarian cancer cell line HO-8910 cells exposed to TGF β1 in vitro were studied. RESULTS TGF β1 inhibited cell growth and DNA synthesis, and induced G0/G1 arrest in cell cycle. It could also trigger PCD in cells. This induction of PCD may occur within G0/G1 phase. Meanwhile, the assay also showed that TGF β1 could inhibit the mRNA expression of c-myc, EGFR and TGF β1 genes in cells. CONCLUSIONS TGF β1 can not only act as an autocrine to inhibit cell proliferation, but also trigger PCD in HO-8910 cells. These functions may be fulfilled through some specific signal transduction pathways.

OBJECTIVE To investigate the value of cine magnetic resonance(MR) in determination of cardiac function by using four chamber/multiple slice multiple phase (4CH/MSMP) sequence. METHODS In 18 healthy subjects, several indices of left ventricular function were measured by using cine MR 4CH/MSMP sequence, and the correlation of the results was compared with ultrasound cardiography by t test. RESULTS In the measurement of left ventricular short diameter, left ventricular posterior wall thickness, internal ventricular septum thickness, left ventricular end diastolic and end systolic volume, the values of cine MR 4CH/MSMP sequence correlated well with those of echocardiography. The gamma value was between 0.88 and 0.99. CONCLUSIONS The cine MR 4CH/MSMP sequence may be considered an important noninvasive modality for accurate assessment of cardiac function.

OBJECTIVE To evaluate modified laparoscopic retroperitoneal lymph node dissection (LRPLND) performed for stage I nonseminomatous germ cell tumor. METHODS Between December 1993 and January 1996, modified unilateral laparoscopic retroperitoneal lymph node dissection was performed on 13 patients with nonseminomatous germ cell tumor of the testis. In 8 patients, the tumor was on the right side, and in 5, on the left. RESULTS The procedure was completed in 12 patients at a mean operating time of 292 min (range: 210-400 min). The mean estimated blood loss was 250 ml. The mean hospital stay was 6.4 days. Conversion to open surgery was required in one patient because of an uncontrollable venous bleeding. No other major complications occurred. Antegrade ejaculation was preserved in 10 patients, and the last 3 patients have not yet been addressed now. CONCLUSIONS Our results demonstrate that LRPLND is an accurate and reliable staging procedure with low morbidity, which allows quick patient discharge and rapid return to normal activity. For further evaluation, long follow-up in larger groups of patients is required.

OBJECTIVE To produce an enzyme linked immunosorbent assay (ELISA) to detect antibodies against Epstein Barr virus (EBV) specified nuclear antigen 1 (EBNA 1) and the 18 kD EBV matrix protein, and to determine and optimize its sensitivity and specificity for the diagnosis of nasopharyngeal carcinoma (NPC). METHODS We used a combination of highly purified glutathione transferase fusion proteins of the 40 kD carboxy domain of EBNA1 and the 18 kD EBV matrix protein for coating ELISA plates. In three separate studies, we tested for IgA antibodies in serum specimens from 28 EBV seronegative donors, 284 EBV seropositive donors and 160 newly diagnosed NPC patients. By comparing the sensitivity and specificity of diagnosis obtained for different cutoff values, we derived several quantitative parameters to evaluate assay performance, establish objective diagnostic criteria which optimize the intrinsic diagnostic capability of the assay and assess the significance of individual test results, respectively. Optimum cutoff optical density (OD) is defined as the cutoff OD where sensitivity of the assay equals its specificity, and resolution of the assay is indicated by the value of sensitivity (or specificity) determined at the optimum cutoff OD. Diagnosis of NPC was achieved by setting a cutoff zone at±20% of this value. RESULTS All the EBV seronegative donors tested were not reactive, and most of the EBV seropositive donors were weakly reactive, while the majority of NPC patients were moderately or strongly reactive. While the assay was thus shown to be specific for EBV, there was an overlap in the level of these serum antibodies between few individuals of the two latter groups. It was shown that the assay performed equally well in two separate studies conducted under different testing conditions and using different collections of sera in that assay resolution determined on these occasions were 86% and 87% respectively. Diagnosis of NPC can be achieved at the same expected sensitivity of 89% and 83% determined at the lower and upper limits of the cutoff zones, with the corresponding values of specificity being 78% and 91%. It was further shown in the third study that resolution of the assay can be increased to 90% using an assay produced with a higher concentration of the same antigens, and that diagnosis of NPC can be achieved at a higher sensitivity ranging between 86% and 95% at a corresponding specificity of 93% and 86%. CONCLUSIONS After optimization and standardization, the ELISA can achieve a sensitivity ranging from 86% to 95%, with corresponding specificities of 93% and 86% respectively for the diagnosis of NPC.

OBJECTIVE To establish the polymorphism of DXS102 locus from Xq26.3-27.1 in Chinese population for the gene diagnosis in Hemophilia B family. METHODS DNA was extracted from blood samples obtained from Shanghai unrelated volunteer donors with phenol-chloroform method. A total of 23x chromosomes (154 from females, 80 from males) were studied. A hemophilia B family in which a hemophilia B patient has received gene therapy was analyzed. The polymorphism of DXS102 locus in Chinese population was determined with amplified fragment length polymorphisms assay (Amp-FLP), denaturing polyacrylamide gel electrophoresis, silver stain detection. Short tandem repeats (STRs) linkage analysis was used to conduct gene diagnosis in hemophilia B family. RESULTS Eight alleles were found at DXS102 locus, of which two alleles were first reported. The repeated number of AC dinucleotide ranges from 13 to 21. And the values of the observed heterozygosity, calculated heterozygosity and polymorphism information content(PIC) were 0.87, 0.80, 0.80 respectively. It was also found that the difference of the allele frequencies of DXS102 in Chinese and European populations was significant. By using the linkage analysis of the DXS102 locus, a family with a hemophilia B patient receiving gene therapy in 1994 was analyzed and meanwhile a carrier in that family was then detected. CONCLUSIONS The polymorphism of DXS102 locus reveals significant difference between Chinese and European populations. DXS102 locus can be used as a promising marker for gene diagnosis in hemophilia B family.

OBJECTIVE To investigate the primary autoantigens which contribute to the production of anti-DNA antibodies. These antibodies are serological hallmark and pathogenic factor of systemic lupus erythematosus (SLE). METHODS Nonautoimmune predisposed BALB/c mice were immunized with concanavalin A (Con A) activated, lipopolysaccharide (LPS) activated and nonactivated syngeneic spleen cells. Nuclei and chromatin from activated/nonactivated lymphocytes were isolated and syngeneic mice were immunized. Sera were taken after the third immunization. IgG anti-dsDNA antibody was determined by ELISA (calf thymus DNA treated with S1 nuclease was used as the coated antigen). The glomerular IgG deposition was observed by immunofluorescence one month after the third immunization. RESULTS Con A activated T cells and LPS activated B cells induced anti-double stranded (ds) DNA antibody in syngeneic nonautoimmune BALB/c mice and formed the glomerular IgG deposition. Further studies showed that active chromatin isolated from activated lymphocytes induced anti-ds DNA antibody, but not resting chromatin isolated from nonactivated lymphocytes. CONCLUSIONS Activated lymphocytes and their active chromatin could be the autoimmunogen(s) driving the anti-dsDNA antibodies. The change of chromatin's antigenicity by environmental factors and genetic background may be the common pathway to SLE pathogenesis.

OBJECTIVE To study the replicative competency and pathogenicity of precore gene mutants of duck hepatitis B virus (DHBV) in the duck model. METHODS Three site-directed point mutations in the precore region of cloned DHBV were constructed. Head-to-tail dimers were formed. The three plasmids were named: pEDM1-2 (initiation codon ATG mutated to TTG), pEDM2-2 (an "A" was inserted down stream of codon 12, leading to frame shift in the distal end of precore region), pEDM3-2 (codon 38 was changed from TAT to TAA, leading to a stop codon at the 3'-end). Mutants and wild-type cloned DNA dimers were first separately used to transfect LMH cells (a chicken hepatoma cell line) and viruses were collected from supernatant and used to infect 6 one-day-old ducklings per group. Serum duck hepatitis B surface antigen (DHBsAg) and DHBV DNA were assayed. Six weeks after infection, ducks were killed and liver tissues were studied for histopathological changes. RESULTS After transfection, pEDM1-2, pEDM2-2 and pEDM3-2 expressed similar level of DHBsAg. Replication of pEDM1-2 and pEDM3-2 was similar to that of the wild type clone, while pEDM2-2 replicated at a significantly decreased level. Infection study employing the supernatant of transfected cells was as follows: pEDM1-2 infected 5/6 ducklings, pEDM2-2 non infected, pEDM3-2 infected 2/6 ducklings, wild type virus infected 6/6 ducklings. Positive serum samples from both pEDM1-2 and pEDM3-2 were at a lower serum DHBV level compared to that of the wild type virus. Pathological changes were more significant in pEDM3-2 infected duck livers, with numerous inflammatory cells in portal tract and infiltration into parenchyma. CONCLUSIONS Mutations in the initiating codon or generation of a stop codon at the 3'-end of the precore region resulted in decreased replication competency of DHBV, while frame-shift mutation of the precore region, covering the epsilon encapsidation signal abolished the replication of DHBV. When the mutants replicated in hosts, more severe pathological changes were observed in ducks infected with mutant harboring a stop codon at the 3'-end. Data suggest that replicative-competent DHBV precore mutant can be more pathogenic than wild-type DHBV.

OBJECTIVE To test the hypothesis that in a rat model of acute intestinal ischemia/reperfusion injury, endotoxemia from the gastrointestinal tract would play a role in mediating tumor necrosis factor (TNF) response and remote organ dysfunction. METHODS Wistar rats underwent 45 minutes of superior mesenteric artery occlusion followed by 6 hours of reperfusion. The rats were treated intravenously with either TNF monoclonal antibody (MoAb) or saline solution 90 minutes prior to the onset of ischemia. RESULTS Significant elevation of plasma TNF level in both portal and systemic circulation was detected immediately after the onset of reperfusion. The level peaked at two hours after reperfusion (P<0.01). Similarly, a remarkable TNF mRNA expression in the intestine in controls was detected at 0.5 hour post-reperfusion, and sustained a marked elevation throughout the observation period (P<0.05-0.01). TNF elevation was found associated with gut-origin endotoxemia, and the maximal TNF response occurred approximately 0.5-2 hours after the initial appearance of endotoxin in the portal vein. Concomitantly, multiple organ dysfunction in response to local ischemic insult was also observed in untreated controls upon reperfusion, but it was significantly attenuated by pretreatment with MoAb against TNF. CONCLUSIONS Intestinal injury can result in the gut becoming a cytokine-liberating organ. The escape of endotoxin and bacteria from the gut may be responsible for the TNF expression and release, which would be an important mechanism underlying pathophysiological alterations associated with intestinal ischemia/reperfusion injury.

Escherichia coli has seldom been reported to cause pseudobacteremia. The investigation of an outbreak of amoxycillin-clavulanic acid-resistant E. coli pseudobacteremia is described. Seventeen cases occurred over a five-day period. The source of the E. coli was traced to the blood culture specimen of a patient (index patient) with genuine bacteremia as a result of urinary tract infection. The other 16 case-patients had pseudobacteremia which was found to be the result of cross-contamination during subculture of blood specimens. The E. coli strain was carried over from the culture bottle of the index patient, through the contaminated gloved hands of a technician to the culture bottles of the other 16 cases. Although the pseudobacteremia occurred over a five-day period, they all resulted from cross-contamination during blood culture processing within one day. An early outbreak investigation was prompted by the unusual finding of amoxycillin-clavulanic acid resistance in the case E. coli isolates in a short period. The relatedness of the E. coli strains from the 17 cases was confirmed by arbitrary-primed polymerase chain reaction. Clinicians should be alerted to the possibility of a blood E. coli isolate being a contaminant despite its predominant role as a true pathogen.

In this paper it is intended to unify the standard lead and the augmented unipolar limb lead (aV lead) system into a single lead system. Viewing from the angle of lead axes, the standard leads and the aV leads are not only reflecting the frontal plane vector, but are also actually complementary to each other. By now it has become quite obvious that they not only can, but also should be unified into a single system. Viewing these six leads (from standard lead and aV lead systems), starting from aVL, lead Ⅰ, reversed aVR, lead Ⅱ, aVF to lead Ⅲ, an arc including approximately 150 degrees is formed. It is suggested that this unified system may be called "F" system with each of the above leads renamed as F1-F6 leads, respectively, since they are all reflecting the vectors of the frontal plane. In this F-lead system each lead is approximately 30 degrees apart. If such a system would be adopted it is only necessary to time the amplitude of the P, QRS and T of aV lead by 1.15 (a figure calculated by vector analysis). If this suggestion is widely adopted there will be only a F lead system for the frontal vectors and the present V system for the "horizontal" vectors in the electrocardiogram.