Tsun-Mei Lin

Failure to Detect Human Papillomavirus DNA in Malignant Epithelial Neoplasms of Conjunctiva by Polymerase Chain Reaction

To elucidate the putative role of human papillomavirus (HPV) infection in the etiology of conjunctival tumors, 44 formalin-fixed, paraffin-embedded specimens of conjunctival tumors (24 patients with papillomas and 20 patients with dysplastic and/or malignant tumors) were screened for HPV infection using 4 different polymerase chain reactions (PCRs). Of the 24 samples of papilloma, 14 (58%) displayed positive results by applying nested PCR using primer sets of HPV consensus L1 region. HPV type 6 or 11 was detected in 9 cases of papilloma by type-specific primer sets, but none of them were positive for HPV type 16 or 18. However, by using the highly sensitive PCR technique, we failed to demonstrate the HPV DNA of HPV types 6, 11, 16, and 18 in any of the 20 malignant epithelial tumors of conjunctiva. We conclude that HPV-6 or HPV-11 is present in a substantial percentage of conjunctival papillomas, which is in accordance with findings of previously reported studies. In contrast, malignant conjunctival carcinomas are not associated with HPV infection; other pathogenic mechanisms, such as UV light, probably are more important in the cause of these malignant lesions.

RHD 1227A Is an Important Genetic Marker for RhDel Individuals

Approximately 30% of apparently Rh- Taiwanese people actually were RhD(el), a rare variant of the Rh system that might carry a grossly intact RHD gene. Several studies have indicated that the RhD(el) trait might be generated by multiple molecular mechanisms. In this study, a total of 294 Taiwanese serologically RhD- blood donors were tested for Rh phenotypes and RHD genotypes. Among them, total RHD deletion, partial RHD gene, and RhD(el) were found in 185 (62.9%), 15 (5.1%), and 94 (32.0%), respectively. The 1227A allele and exon 9 of the RHD gene were found in all 94 RhD(el) donors. The Ccee was the most prevalent phenotype in the RhD(el) group (78/94 [83%]), and the ccee phenotype was highly prevalent in the true D- group (87.6%). RHD 1227A can be used as an important and useful genetic marker for RhD(el). It can be detected easily by a simple, rapid, specific sequence primer-polymerase chain reaction method.

TLR7 and TLR8 Gene Variations and Susceptibility to Hepatitis C Virus Infection

Toll-like receptors (TLRs) play pivotal roles in the innate immune system and control inflammatory responses and adaptive immunity. We previously evaluated associations between TLR7 and TLR8 gene SNPs and susceptibility to hepatitis C virus (HCV) infection. Our results suggested that TLR7IVS2-151G and TLR8-129G alleles were present at higher frequency in males of an HCV-infected group as compared to a control group (24.1% vs. 14.4%, p = 0.028; 17.6% vs. 6.8%, p = 0.004, respectively). Based upon their recognition of single stranded viral RNA, this suggested that TLR7 and TLR8 played a significant role in anti-HCV immune responses. Here, we studied the functional effects of these polymorphisms by analyzing the mRNA expressions of TLR7 and TLR8 and cytokine production induced ex vivo by TLR7- and TLR8-specific agonists using whole blood of subjects with different genotypes. The percentage of CD14+ cells from those with an AG haplotype that expressed TLR7 and TLR8 was significantly lower, but higher in intensity compared to cells from those with GG and AC haplotypes. Cells from those with an AG haplotype produced more IFN-α and less amounts of pro-inflammatory cytokines upon stimulation. This suggests that variations in TLR7 and TLR8 genes might impair immune responses during HCV infection.

4G/5G promoter polymorphism of plasminogen activator inhibitor-1, lipid profiles, and ischemic stroke.

The 4G allele of common 4G/5G polymorphism in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene is associated with increased PAI-1 transcription and has been proposed as a candidate genetic risk factor for thrombotic diseases. We investigated the relationship between this polymorphism and lipid profiles and stroke risk. One hundred patients with ischemic stroke and 150 age- and sex-matched control subjects were enrolled. PAI-1 genotype was determined with the use of polymerase chain reaction and restriction-length analysis. Genotype distribution in the stroke group was 40% 4G/4G, 46% 4G/5G, and 14% 5G/5G; in the control group it was 38.7% 4G/4G, 45.3% 4G/5G, and 16% 5G/5G. The allele and genotype frequencies of 4G/5G polymorphism were not different between the stroke and control groups. Control subjects who were homozygous for the 4G allele had significantly lower high-density lipoprotein (HDL) cholesterol levels than did those carrying the 5G allele (51.2 +/- 11.8 vs 58.4 +/- 15.8 mg/dL; P =.002). In the control group, regression analysis revealed a significant contribution of 4G/4G genotype to increased triglyceride (P =.042) and to decreased HDL cholesterol (P <.001) levels. Our findings suggest that PAI-1 4G/5G promoter polymorphism alone is not associated with ischemic stroke. However, this polymorphism influences lipid levels, and the underlying mechanism must be determined.

Promoter polymorphism of the CD14 endotoxin receptor gene is associated with biliary atresia and idiopathic neonatal cholestasis.

Objective.To investigate whether single-nucleotide polymorphisms in the promoter regions of endotoxin-responsive genes CD14 and tumor necrosis factor-α (TNF-α) are associated with biliary atresia (BA) and idiopathic neonatal cholestasis (INC). Methods.We obtained genomic DNA from 90 patients with established diagnosis of BA and 28 patients with INC. Forty-two adult patients with hepatitis B–related cirrhosis and 143 healthy children served as control populations. The genotypes of CD14/C(−159)T and TNF-α/G(−308)A (G allele, TNF*1; A allele, TNF*2) were determined by using a restriction enzyme–based assay. Plasma soluble CD14 levels were determined in different disease stages and genotypes of BA. Results.The frequencies of T allele and T/T homozygosity of the CD14/−159 promoter polymorphism were significantly higher in patients with BA (T allele: 61.7%; T/T genotype: 42.2%) and in patients with INC (T allele: 67.9%; T/T genotype: 53.6%) but not in control populations. Decrease of plasma soluble CD14 from the early stage of BA when the patients received a Kasai operation to the late stage of liver cirrhosis was observed in carriers of the T/T and T/C genotypes but not in carriers of the C/C genotype. The TNF-α/−308 promoter polymorphisms (TNF*1 and TNF*2) were not associated with BA. Conclusion.These findings show that the single-nucleotide polymorphism at CD14/−159 is associated with the development of BA and INC. Endotoxin susceptibility may play a role in the pathogenesis of infantile cholestasis.

Decrease of fibrinolytic activity in human endothelial cells by arsenite

Blackfoot disease (BFD) is an endemic peripheral vascular occlusive disease that occurred in the southwest coast of Taiwan. It is believed that arsenic in the drinking water from artesian wells plays an important role in the development of the disease. We have previously shown that BFD patients had significant lower tissue-type plasminogen activator (t-PA) antigen level and higher plasminogen activator inhibitor, Type 1 (PAI-1) antigen level than normal controls. The purpose of this study was to investigate the effects of arsenite on the fibrinolytic and anticoagulant activities of cultured macrovascular and microvascular endothelial cells. Incubation of human microvascular endothelial cells (HMEC-1), but not human umbilical vein endothelial cells (HUVECs), with arsenite caused a decrease of t-PA mRNA level, a rise of both PAI-1 mRNA level and PAI activity. Arsenite could also inhibit the thrombomodulin (TM) mRNA expression and reduce the TM antigen level in HMEC-1. In conclusion, arsenite had a greater effect on HMEC-1 as compared to HUVECs in lowering the fibrinolytic activity and may be responsible for the reduced capacity of fibrinolysis associated with BFD.

Susceptibility of endothelial cells to bovine herpesvirus type 4 (BHV-4)

The sensitivity of two different types of cells to bovine herpesvirus type 4 (BHV-4) was compared by median tissue culture infectious dose (TCID50) assays. The bovine arterial endothelial (BAE) cell culture derived from bovine carotid arteries was 100-1000 times more sensitive to two strains of BHV-4, Movar 33/63 and DN 599, than Madin Darby bovine kidney (MDBK) cell line commonly used for the propagation of these viruses. BAE cell cultures infected with BHV-4 displayed cytopathic effects (CPE) earlier and more prominently than the MDBK cells infected with the same viruses. BAE cells were also more sensitive than MDBK cells in conventional plaque assays in that the former developed well characterized and easily recognized plaques after infection with the viruses. BAE cells, which are proved to be exceptionally susceptible to BHV-4, can be used in the detection and quantitation of BHV-4.

The binding of plasminogen fragments to cultured human umbilical vein endothelial cells

Glu-plasminogen, kringle 1-5, kringle 1-3, and miniplasminogen exhibited strong binding to human umbilical vein endothelial cells (HUVEC). On the other hand, no significant binding was obtained with microplasminogen and kringle 4. Kringle 1-5 and miniplasminogen, which both contained kringle 5, specifically inhibited the binding of plasminogen to HUVEC while kringle 1-3 did not. The results implied plasminogen molecule contained at least two binding sites, with which it interacted HUVEC. The stronger binding site was located in kringle 5 and the weaker one was in kringle 1-3. Kringle 4 and the active site domain exhibited no significant binding to HUVEC. The interaction of plasminogen with HUVEC is mainly through binding site on kringle 5.

Aberrant RNA splicing in RHD 7-9 exons of DEL individuals in Taiwan: a mechanism study.

BACKGROUND The Rh blood D group provides a clinically important model of aberrant splicing with skipped exons. Approximately 30% of serologically D-negative Chinese individuals have an intact RHD gene (DEL phenotype) and induce allo-immunization in transfusions. The RHD1227GNA polymorphism occurs in >95% DEL phenotype of Asian descent. The effects of RHD 1227A and a novel allele on exon 9 splicing were examined. RESULTS Amplified DEL RNA products revealed that 3 transcripts involved skipping of exons 8-9, exon 9, or exon 9 with an inserted 170-bp cryptic exon located between exons 7 and 8. A novel, single nucleotide polymorphism was identified in the 7th intron, (IVS7) 923C>T, and present in all DEL patients. The odds ratio of RHD1227G>A allele with DEL phenotype was 2711. Splicing analysis of transcripts from minigenes containing the 1227GNA allele, but not the (IVS7) 923C>T allele, demonstrated aberrant exon 9 skipping. CONCLUSIONS A combined haplotype of 1227G>A and IVS7 923C>T alleles was apparent in >95% DEL Chinese individuals. RHD1227A mutation significantly increased aberrant mRNA splicing, producing a hybrid RHD mRNA lacking exon 9. These results provide a molecular basis of the DEL phenotype in the Chinese population.

Persistent infection of bovine herpesvirus type 4 in bovine endothelial cell cultures

Herpesviruses can establish a persistent infection in the cells and tissues of their natural hosts and thus may produce diseases due to cytolytic infections. We have isolated a herpesvirus from a bovine vascular endothelial cell culture after continuous subculturing. Typical cytopathic changes were observed in bovine endothelial cell cultures 2 days after inoculation of the virus. The virus had an icosahedral nucleocapsid of 100-150 nm in diameter and an envelope. The sequences of some DNA fragments of the virus were highly homologous to those of the bovine herpesvirus type 4 (BHV-4) strains. The DNA restriction maps of the virus and the reference strains of BHV-4, DN 599 and Movar 33/63 were very similar but not identical. Therefore, the newly isolated virus has been designated Taiwan strain. The presence of BHV-4 DNA in apparently normal bovine endothelial cell cultures was shown by Southern blot hybridization with the BamHI fragment of the newly isolated BHV-4 and was further confirmed by digestion of the DNA with BamHI plus AccI. In conclusion, we have demonstrated that BHV-4 persisted in the bovine endothelial cell cultures and continuous subcultures could lead to the production of infectious viral particles.