Mycoplasma genitalium promotes epithelial crossing and peripheral blood mononuclear cell infection by HIV-1

Background Mycoplasma genitalium co-infection in HIV-infected individuals has been reported to increase the shedding of HIV in the urogenital region of females. To better understand this relationship, we investigated the influence of M. genitalium on the transmission and replication of HIV using an in vitro model. Methods The Transwell co-culture system was employed to assess the crossing of an endocervical cell barrier by HIV-1. Immunocytochemistry and confocal microscopy were used to assess the distribution of the nectin-1 molecule on M. genitalium -infected epithelial cells of the End1/E6E7 endocervical cell line, grown as monolayers in the insert wells. Peripheral blood mononuclear cells (PBMC) were cultured in the bottom wells to assess the effects of M. genitalium , passing through the semipermeable culturing membrane, on subsequent HIV infection of susceptible target cells. Results Infection of the endocervical cells with the adhesion-positive M. genitalium G37 strain (wild-type) significantly elevated the passage of HIV across the epithelial cell barrier relative to HIV transfer across endocervical cells infected with the adhesion-negative M. genitalium JB1 strain. Immunostaining of the M. genitalium -G37-infected epithelial cells disclosed capping and internalization of the junctional regulatory protein nectin-1, in association with reduced transepithelial resistance (TER) in the cell monolayer. When PBMC were cultured beneath insert wells containing M. genitalium -G37-infected epithelial cell monolayers, we observed significantly enhanced infectivity and replication of HIV added afterward to the cultures. Conclusions M. genitalium influences events on both sides of a cultured mucosal epithelial monolayer: (1) by infecting the epithelial cells and reducing the integrity of the barrier itself, and (2) by activating HIV target cells below it, thereby promoting HIV infection and progeny virus production

Detection, isolation, and characterization of human rotavirus (HRV) isolated from patients at two hospitals on Oahu

Typescript.Thesis (Ph. D.)--University of Hawaii at Manoa, 1990.Includes bibliographical references (leaves 189-208)Microfiche.xv, 208 leaves, bound ill. (some col.) 29 c

WRRCTR No.161 Replication of Human Rotavirus in Tissue Culture: Recovery and Detection in Fecal, Sewage, and Natural Water Samples

Human rotavirus is the major cause of gastroenteritis among young children. To replicate this virus, sensitive methods using standard tissue culture systems are required. The project goal was to develop laboratory capability to recover and detect this infectious rotavirus in fecal, sewage, and natural water samples. Using simian rotavirus (SA-II) as a model system and an enzyme-linked immunosorption (ELISA) test capable of detecting high concentrations of rotavirus, the protamine sulfate method was determined as superior to the aluminum chloride precipitation and polymer two-phase methods for recovering rotavirus from sewage. The ELISA method was very effective in detecting rotavirus in stool samples of children. Stools from children not displaying clinical symptoms of rotavirus infection were negative for rotavirus, whereas 43 to 58% of stools from children displaying clinical symptoms of rotavirus infection was positive for rotavirus. The results suggested an association of increased rotavirus infections during the winter months in the state of Hawaii. Stools positive for rotavirus by the ELISA test were used as inoculum to develop methods to replicate human rotavirus in the cell culture system. After many unsuccessful attempts, human rotavirus was cultured after two passages in primary cynomolgus monkey kidney cells. Human rotavirus which replicated in the primary monkey kidney cells was shown to be capable of replicating in continuous monkey kidney cell lines such as the MA-104.U.S. Department of the Interior Grant/Contract No. CT371300; 37130

Regulation of cyclin T1 during HIV replication and latency establishment in human memory CD4 T cells

Abstract Background The regulatory cyclin, Cyclin T1 (CycT1), is a host factor essential for HIV-1 replication in CD4 T cells and macrophages. The importance of CycT1 and the Positive Transcription Elongation Factor b (P-TEFb) complex for HIV replication is well-established, but regulation of CycT1 expression and protein levels during HIV replication and latency establishment in CD4 T cells is less characterized. Methods To better define the regulation of CycT1 levels during HIV replication in CD4 T cells, multiparameter flow cytometry was utilized to study the interaction between HIV replication (intracellular p24) and CycT1 of human peripheral blood memory CD4 T cells infected with HIV in vitro. CycT1 was further examined in CD4 T cells of human lymph nodes. Results In activated (CD3+CD28 costimulation) uninfected blood memory CD4 T cells, CycT1 was most significantly upregulated in maximally activated (CD69+CD25+ and HLA.DR+CD38+) cells. In memory CD4 T cells infected with HIV in vitro, two distinct infected populations of p24+CycT1+ and p24+CycT1- cells were observed during 7 days infection, suggestive of different phases of productive HIV replication and subsequent latency establishment. Intriguingly, p24+CycT1- cells were the predominant infected population in activated CD4 T cells, raising the possibility that productively infected cells may transition into latency subsequent to CycT1 downregulation. Additionally, when comparing infected p24+ cells to bystander uninfected p24- cells (after bulk HIV infections), HIV replication significantly increased T cell activation (CD69, CD25, HLA.DR, CD38, and Ki67) without concomitantly increasing CycT1 protein levels, possibly due to hijacking of P-TEFb by the viral Tat protein. Lastly, CycT1 was constitutively expressed at higher levels in lymph node CD4 T cells compared to blood T cells, potentially enhancing latency generation in lymphoid tissues. Conclusions CycT1 is most highly upregulated in maximally activated memory CD4 T cells as expected, but may become less associated with T cell activation during HIV replication. The progression into latency may further be predicated by substantial generation of p24+CycT1- cells during HIV replication

The pulsations of PG 1351+489

PG 1351+489 is one of the 20DBVs – pulsating helium-atmospherewhite dwarf stars – known and has the simplest power spectrum for this class of star, making it a good candidate to study cooling rates. We report accurate period determinations for the main peak at 489.334 48 s and two other normal modes using data from the Whole Earth Telescope (WET) observations of 1995 and 2009. In 2009, we detected a new pulsation mode and the main pulsation mode exhibited substantial change in its amplitude compared to all previous observations. We were able to estimate the star’s rotation period, of 8.9 h, and discuss a possible determination of the rate of period change of (2.0 ± 0.9) × 10−13 s s-ˡ, the first such estimate for a DBV