Molecular Epidemiological Investigation of Thalassemia in the Chengdu Region, Sichuan Province, Southwest China.

Thalassemia is the most common inherited disease in southern China. However, this disorder is usually ignored by the health system in the Sichuan Province due to the lack of epidemiological data. To provide basic epidemiological data for thalassemia screening, genetic counseling, and prenatal diagnosis (PND) in the Chengdu region, a total of 3262 healthy subjects were assessed by complete blood count (CBC), reverse dot-blot gene chip, gap-polymerase chain reaction (gap-PCR), and PCR-DNA sequencing. A frequency of heterozygous thalassemia of 3.43% (112/3262) was found, of which 2.21% (72/3262) patients carried α-thalassemia (α-thal), 1.19% (39/3262) ß-thalassemia (ß-thal) and 0.3% (1/3262) hereditary persistence of fetal hemoglobin (Hb) (HPFH)/δß-thalassemia (δß-thal). Four types of α-thal mutations were found, the most prevalent being - -(SEA) (68.06%), followed by -α(3.7) (rightward deletion, 25.0%), Hb Quong Sze (Hb QS; HBA2: c.377 T > C) (4.17%), and -α(4.2) (leftward deletion, 2.78%). The seven ß-thal mutations included: codons 41/42 (-TTCT), HBB: c.126_129delCTTT (13/39, 33.33%); codon 17 (A > T), HBB: c.52 A > T (11/39, 28.95%); IVS-II-654 (C > T), HBB: c.316-197 C > T (9/39, 23.68%); -28 (A > G), HBB: c.-78 A > G (3/39, 7.69%); -29 (A > G), HBB: c.-79 A > G (1/39, 2.56%); codons 27/28 (+C), HBB: c.84_85insC (1/39, 2.56%), and the rare IVS-II-850 (G > T), HBB: c.316-1 G > T (1/39, 2.56%). Only one case of the Southeast Asian HPFH deletion was found. This is the first detailed molecular epidemiological survey of thalassemia in the Chengdu region, Sichuan Province, People's Republic of China (PRC).

[The mechanism of accumulative oxidative injury by hydrogen peroxide on human retinal pigment epithelial cells].

OBJECTIVE: To explore the effects of oxidative injury induced by hydrogen peroxide on human retinal pigment epithelial (RPE) cells. METHODS: Cultured human RPE cells were treated by 600 micromol/L hydrogen peroxide (H2O2) for 1, 6, 12, 24 and 72 hours. Cell viability was assessed by the MTT assay. Apoptosis was assessed by Annexin V-fluorescein isothiocyanate/Propidium iodium (Annexin V-FITC/PI) staining. The expression of clusterin was assessed by Western blot. RESULTS: (1) The treatment of RPE cells with 600 micromol/L H2O2 caused a time-dependent decrease of cellular viability. (2) Apoptosis was detected in cultured human RPE cells treated with 600 micromol/L H2O2 for 6 hours. The number of apoptotic cells reached a maximum at 24th hour after being exposed to 600 micromol/L H2O2 (P < 0.05). (3) Western blot showed the expression of clusterin protein was demonstrated in 6 hours exposure to 600 micromol/L H2O2, a significantly increasing of clusterin expression was observed overtime (P < 0.01). Thereafter the expression of clusterin protein decreased at 24th hour after being exposed to 600 micromol/L H2O2. At 72nd hour, the expression of clusterin protein was quite weak. CONCLUSION: Hydrogen peroxide can inhibits RPE proliferation and induces apoptosis and aging gene expression;the result suggest that accumulative oxidative injury induced by hydrogen peroxide in RPE in vitro may be similar to the aging changes in vivo.

[The protective effect of hypoxic preconditioning against light-induced photoreceptor degeneration in mice].

OBJECTIVE: To study the protective roles of hypoxic preconditioning in light induced retinal injury in a mice model and investigate the possible mechanism of related gene regulation. METHODS: 54 BALB/c mice were randomly divided into simple light exposure group (SL), hypoxic pretreatment group (HP) and control group (CON). The mice of SL and HP were continually exposed to light for 3 h, which built model of light-induced damage. Morphologic changes of photoreceptor cells in different group were examined by light microscope and the apoptosis was detected by TdT-mediated dUTP nick end labeling. Different expressions of c-fos and caspase-1 gene were examined by immunohistochemical staining. RESULTS: In group SL, the photic injury appeared very obviously and early. Different changes appeared in the outer nuclear layer after light exposure. Photic injury was aggravated following the increased light exposure. Positive staining of c-fos and caspase-1 could be seen in the outer nuclear layer. In group HP, the changes of retinal morphology appeared slightly and lately. Compared with group SL, caspsae-1 expression was decreased obviously, while no difference was seen in the expression of c-fos. The retinal structures was normal in the mice of control group and c-fos and caspase-1 were stained negative. CONCLUSION: Hypoxic preconditioning has neuroprotective effect on photoreceptor cells by inhibiting the expression of apoptosis-related genes in photic injured mice model. caspase-1 may be involved in the protective mechanism.

Gene therapies for pulmonary hypertension-from experimental trials to bedside aspects.

Accompanying the continuously deepening understanding of the mechanism of pulmonary hypertension that many genes are found to be aetiologically involved in its development, burgeoning literature manifest that gene therapies aimed at correcting these genetic defects have the ability to restore deficient pulmonary gene expression, over-express biologically active gene products, reverse established disease and regenerate pulmonary vasculature, and may constitute a promising therapeutic strategy for pulmonary hypertension. Therefore, to provide new information to basic scientists and clinical investigators, we present a review that attempts a clear description of the therapeutic potential of gene therapy in the treatment of pulmonary hypertension.

[Sequence analysis of the HA1 regions of hemagglutinin genes of influenza viruses (H3N2) isolated from children in Beijing from 1998 - 2004].

OBJECTIVE: To characterize the HA1 regions of hemagglutinin gene of influenza viruses (H3N2) isolated from children in Beijing from 1998 - 2004. METHODS: The HA1 regions of hemagglutinin gene were amplified by RT-PCR from the viruses isolated and identified as A3 (H3N2) from clinical samples collected from infants and children during the peak seasons of influenza between 1998 and 2004. PCR products were sequenced or cloned into T-A vector and were analyzed after being sequenced. RESULTS: The HA1 regions of hemagglutinin genes amplified from those isolates were 987 bp in length, encoding a protein of 329 amino acids in length. The identities of nucleotides and amino acids among these H3N2 isolates in Beijing and vaccines strains from 1998 - 2004 were 95.5% - 100.0% and 93.0% - 100.0%, respectively. The homology of the HA1 regions were related to the date of virus isolation, meaning the homology was higher among those strains isolated in nearer dates than others. Seven potential N-linked glycosylation sites in the HA1 regions located at amino acid positions 8, 22, 38, 63, 126, 165 and 285 were conserved in all the viruses analyzed. Two sites at 122 and 133 were inserted in those virus isolated after 1997, and another site at 144 appeared in those isolated after 1999. More amino acid substitutions located in the five putative antigenic sites or receptor binding sites were found more in the isolates than the isolates from previous year. Phylogenetic analysis showed new branches appeared continuously during 1998 - 2004. The strains isolated during winter in 2004 belonged to different branches, suggesting the appearance of new variants. CONCLUSION: Amino acid substitutions continuously occurred in the HA1 regions of hemagglutinin genes in influenza virus (H3N2) isolated from children in Beijing from 1998 - 2004, which might have resulted in antigenic drift and led to the appearance of new variants.

[Application of molecular biological techniques in the surveillance of influenza viruses in infants and young children].

OBJECTIVE: To establish a rapid, specific and effective technique for identifying subtyping A(1), A(3) and B of influenza virus isolates and clinical specimens as well as to analyze the sequences of nucleotides and deduced amino acids of HA1 regions from isolates of influenza virus A(3) isolated from 1996 to 2002. METHODS: Six inner and outer sets of oligonucleotide primers were designed to detect, type and subtype human influenza A and B. The first two corresponding sets differentiate type A and B of matrix (M) gene while, the second two corresponding sets identify the H(1) and H(3) subtypes of type A virus HA gene. To type and subtype influenza viruses in clinical isolates, a mixture of inner primer sets specific for H(1), H(3) and B were used in a single PCR reaction tube. To detect influenza viruses in clinical specimens, a mixture of the outer primer sets were used in a single primary PCR tube, and the inner ones in a single second PCR reaction tube. Amplified products were visualized in 1.2% agrose gel containing ethidium bromide. HA1 regions of hemagglutinin of 5 field strains (H3N2) isolated from 1996 to 2002 in Beijing were amplified by RT-PCR and sequenced directly. RESULTS: There was 100% correlation between multiplex RT-PCR and culture to type and subtype influenza viruses from clinical isolates. For typing and subtyping, 76.9%, 57.1% and 86.5% were positive for A(1), A(3) and B by multiplex nested-PCR compared within virus isolation on culture, respectively. The sequence data of HA1 of A(3) strains showed that there was a high homology of nucleotide and amino acid, and the closer the date of isolating was, the higher homology showed. CONCLUSIONS: Multiplex RT-PCR and nested-PCR for influenza viruses could provide a useful alternative to existing methods of influenza detected and identified from clinical isolate and specimens. There were certain, continuous mutations and increasing glycosylated sites which might cause the antigen drift in the A(3) strains during 1996-2002 in Beijing area.

Effects of a bioartificial liver support system on acetaminophen induced acute liver failure canines.

AIM:To evaluate the safety and efficacy of the bioartificial liver support system in canines with acute liver failure (ALF).METHODS:Nine canines with acute liver failure by acetaminophen-induced received TECA-I bioartificial liver support system (BALSS) from Hong Kong TECA LTD Co. Blood was perfused through a hollow fiber tube containing (1-2)X10(10) the porcine hepatocytes.In contrast, another 10 canines with acute liver failure by Acetaminophen received drugs. Each treatment lasted 6 hours.RESULTS:BALSS treatment resulted in beneficial effects for acetaminophen-induced ALF canines with survival and with the recovery of the liver functions and tissues, and plasma ammonia decreased from 135.9&mgr;mol/L plus minus 17.5&mgr;mol/L to 65.7&mgr;mol/L plus minus 22.0&mgr;mol/L, 32.5&mgr;mol/L plus minus 8.8&mgr;mol/L, GPT from 97.8U/L plus minus 8.7U/L to 64.8U/L plus minus 11.9U/L, 19.0U/L plus minus 6.3U/L, GOT from 103.0U/L plus minus 16.7U/L to 75.7U/L plus minus 19.6U/L, 26.5U/L plus minus 5.0U/L, and AKP from 158.3U/L plus minus 12.1U/L to 114.5U/L plus minus 19.8U/L, 43.8U/L plus minus 5.6U/L during and after the treatment. In contrast, 10 ALF canines in both the drug and control groups died 1 or 2 days after treatment.CONCLUSION: TECA-1 artificial liver support system is safe and efficacious for canines with acute liver failure.