Effects of hepatitis B virus infection on human sperm chromosomes

AIM: To evaluate the level of sperm chromosome aberrations in male patients with hepatitis B, and to directly detect whether there are HBV DNA integrations in sperm chromosomes of hepatitis B patients.METHODS: Sperm chromosomes of 14 tested subjects (5healthy controls, 9 patients with HBV infection, including 1with acute hepatitis B, 2 with chronic active hepatitis B, 4with chronic persistent hepatitis B, 2 chronic HBsAg carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free golden hamster ova and human spermatozoa, and the frequencies of aberration spermatozoa were compared between subjects of HBV infection and controls. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes.RESULTS: The total frequency of sperm chromosome aberrations in HBV infection group (14.8%, 33/223) was significantly higher than that in the control group (4.3%,5/116). Moreover, the sperm chromosomes in HBV infection patients commonly presented stickiness, clumping, failure to staining, etc, which would affect the analysis of sperm chromosomes. Specific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis. In 9 (9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots, others presented 2 to 4signals. There was significant difference of fluorescence intensity among the signal spots. The distribution of signal sites among chromosomes was random.CONCLUSION: HBV infection can bring about mutagenic effects on sperm chromosomes. Integrations of viral DNA into sperm chromosomes which are multisites and nonspecific, can further increase the instability of sperm chromosomes. This study suggested that HBV infection can create extensively hereditary effects by alteration genetic constituent and/or induction chromosome aberrations, as well as the possibility of vertical transmission of HBV via the germ line to the next generation.

Construction and analysis of SSH cDNA library of human vascular endothelial cells related to gastrocarcinoma

AIM: To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database.METHODS: Human VECs related to gastric adenocarcinoma and corresponding normal tissue were separated by magnetic beads coupled with antibody CD31 (Dynabeads CD31). A few amount of total RNA were synthesized and amplified by SMARTTM PCR cDNA Synthesis Kit. Then, using SSH and T/A cloning techniques, cDNA fragments of differentially expressed genes in human VECs of gastric adenocarcinoma were inserted into JM109 bacteria. One hundred positive bacteria clones were randomly picked and identified by colony PCR method. To analyze cDNA libraries of gastrocarcinoma and VECs in CGAP database, the tools of Library Finder,cDNA xProfiler, Digital GENE Expression Displayer (DGED),and Digital Differential Display (DDD) were used.RESULTS: Forward and reverse subtraction cDNA libraries of human VECs related to gastrocarcinoma were constructed successfully with SSH and T/A cloning techniques. Analysis of CGAP database indicated that no appropriate library of VECs related to carcinoma was constructed.CONCLUSION: Construction of subtraction cDNA libraries of human VECs related to gastrocarcinoma was successful and necessary, which laid a foundation for screening and cloning new and specific genes of VECs related to gastrocardnoma.

Inhibition of the activating signals in NK92 cells by recombinant GST-sHLAG1α chain

The soluble HLA-G1 (sHLA-G1) isoform was found to be secreted by trophoblast cells at the matemo-fetal interface, which suggests that it may act as an immunomodulator during pregnancy. In this paper, we reported that GST-sHLA-G1α chain could bind to its receptor ILT-2 on NK92 cells and then the latter recruited Src homology 2 domaincontaining tyrosine phosphatase-1 (SHP-1), which consequently dephosphorylated some important protein tyrosine kinases and blocked the activation of downstream molecules such as MEK and ERK so that the cytotoxicity of natural killer (NK) cells was inhibited. These results indicated that GST-sHLA-G1α chain might be exploited in new immunotherapy strategies aiming at inducing immunotolerance during allograft, xenograft and autoimmune situations. In addition, we found that modification of O-linked β-N-acetylglucosamine (O-GlcNAc) was involved in NK cells' activating and inhibitory signals. This may provide a novel molecular target for inducing immunotolerance but needs further study.