Detection of candidate nectin gene mutations in infertile men with severe teratospermia.

PURPOSE: Approximately 40% of infertile men have an abnormal semen analysis, resulting from either abnormalities of sperm production (defective spermatogenesis) or sperm shape (defective spermiogenesis). This latter process is dependent upon the function of Sertoli cells, which maintain specialized junctional complexes with germ cells. Nectins, members of the immunoglobulin superfamily, participate in formation of these dynamic complexes. Male mice in which the nectin-2 or nectin-3 gene is knocked out are sterile. Their spermatozoa exhibit severe teratospermia, altered motility, and an impaired ability to fertilize eggs. We asked whether mutations in the protein coding regions of the nectin-2 (aka PVRL2) and nectin-3 (aka PVRL3) genes could be detected in men from infertile couples whose semen analysis revealed unimpaired sperm production, judged by normal sperm concentration, but severe abnormalities in sperm shape. METHODS: Ejaculates were snap frozen in liquid nitrogen and later submitted for Sanger analysis of these two genes, to detect mutations in their protein coding regions. RESULTS: Eighty-nine of 455 ejaculates (19.5%) met the inclusion criteria for study. Two of the 56 samples that were successfully analyzed for nectin-2 (3.6%) and one of 73 (1.3%) analyzed for nectin-3 possessed possibly damaging mutations. CONCLUSIONS: Despite the small-scale nature of the study, at least two low-frequency deleterious variants were identified. These results suggest the need for a larger-scale study of sequence variants in the nectins in severe teratospermia.

Transcript profiling of human platelets using microarray and serial analysis of gene expression (SAGE).

Platelets are anucleated cells that are generated from megakaryocytes via thrombopoiesis. They lack genomic DNA but have a pool of individual mRNA transcripts. Taken together, these mRNAs constitute a platelet transcriptome. Platelets have a unique and reproducible transcript profile, which includes approximately 1,600-3,000 individual transcripts. In this chapter, we will focus on platelet purification and on transcript profiling using an Affymetrix microarray platform and serial analysis of gene expression (SAGE). Platelet purification is described in detail. Large-scale platelet purification schema is designed to purify platelets from apheresis platelet bags (approximately 3-5 x 10(11) platelets/bag). Modification of this schema --small-scale platelet purification--is designed to isolate platelets from 20 ml of peripheral blood. This chapter provides detailed protocols for microarray and SAGE transcript profiling. We also discuss peculiarities of platelet purification, RNA isolation, and transcript profiling.

Zika Virus Persistently Infects and Is Basolaterally Released from Primary Human Brain Microvascular Endothelial Cells.

Zika virus (ZIKV) is a mosquito-borne Flavivirus that has emerged as the cause of encephalitis and fetal microencephaly in the Americas. ZIKV uniquely persists in human bodily fluids for up to 6 months, is sexually transmitted, and traverses the placenta and the blood-brain barrier (BBB) to damage neurons. Cells that support persistent ZIKV replication and mechanisms by which ZIKV establishes persistence remain enigmatic but central to ZIKV entry into protected neuronal compartments. The endothelial cell (EC) lining of capillaries normally constrains transplacental transmission and forms the BBB, which selectively restricts access of blood constituents to neurons. We found that ZIKV (strain PRVABC59) persistently infects and continuously replicates in primary human brain microvascular ECs (hBMECs), without cytopathology, for >9 days and following hBMEC passage. ZIKV did not permeabilize hBMECs but was released basolaterally from polarized hBMECs, suggesting a direct mechanism for ZIKV to cross the BBB. ZIKV-infected hBMECs were rapidly resistant to alpha interferon (IFN-α) and transiently induced, but failed to secrete, IFN-ß and IFN-λ. Global transcriptome analysis determined that ZIKV constitutively induced IFN regulatory factor 7 (IRF7), IRF9, and IFN-stimulated genes (ISGs) 1 to 9 days postinfection, despite persistently replicating in hBMECs. ZIKV constitutively induced ISG15, HERC5, and USP18, which are linked to hepatitis C virus (HCV) persistence and IFN regulation, chemokine CCL5, which is associated with immunopathogenesis, as well as cell survival factors. Our results reveal that hBMECs act as a reservoir of persistent ZIKV replication, suggest routes for ZIKV to cross hBMECs into neuronal compartments, and define novel mechanisms of ZIKV persistence that can be targeted to restrict ZIKV spread.IMPORTANCE ZIKV persists in patients, crossing placental and neuronal barriers, damaging neurons, and causing fetal microencephaly. We found that ZIKV persistently infects brain endothelial cells that normally protect neurons from viral exposure. hBMECs are not damaged by ZIKV infection and, analogous to persistent HCV infection, ZIKV constitutively induces and evades antiviral ISG and IFN responses to continuously replicate in hBMECs. As a result, hBMECs provide a protective niche for systemic ZIKV spread and a viral reservoir localized in the normally protective blood-brain barrier. Consistent with the spread of ZIKV into neuronal compartments, ZIKV was released basolaterally from hBMECs. Our findings define hBMEC responses that contribute to persistent ZIKV infection and potential targets for clearing ZIKV infections from hBMECs. These results further suggest roles for additional ZIKV-infected ECs to facilitate viral spread and persistence in the protected placental, retinal, and testicular compartments.