Dely utilized control reference viruses, as well as the cognate isolates themselves, taken from different biological compartments: HIV-1BaL (isolated from lung), and HIV-1SF162 (isolated from cerebrospinal fluid). We argued that the use of these C/R laboratory-adapted HIV-1 variants as controls should maximize the chance of detecting unique phenotypic characteristics of T/F viruses. Nevertheless, our study did not reveal striking differencesbetween C/R and T/F HIV-1 envelopes in infection of cervical tissue that pointed towards a T/F phenotype related gatekeeping mechanism(s). Although the rate of HIV-1 transmission ex vivo is much higher than that in vivo, not every cervical tissue inoculated with HIV-1 supported productive infection. Why some cervical tissues were not infected by HIV-1 remains to be studied and may be related to the stage of menstrual cycle at which they were isolated [G. Poli, personal communication] and/or the expression of innate restriction factors. Whichever the gatekeeping mechanisms that protect the tissues from infection are, the rates of transmission of C/R and T/F HIV-1 variants were not different in our model system. Using p24 release into the culture medium as a read-out, some tissues may support replication of HIV-1 at a level that we did not consider reliably indicative of de novo virus production since the p24 amount measured may merely represent a slow release of virions adsorbed during inoculation. To exclude these tissues from further analysis, we established a formal criterion: tissue was considered to support productive HIV-1 infection if the amount of the released virus 1480666 exceeded the amount of the released adsorbed virus by 100 pg. Although this criterion is somewhat arbitrary, in our experience the total amount of cumulative production of virus over 12 days of culture should be not lower than this amount. To determine the cumulative de novo production, we blocked HIV-1 infection with the NRTI 3TC and measured the amount of virusTransmission of Founder HIV-1 to Cervical Explantsreleased. The amount of 3TC we applied seems to block HIV-1 infection, as neither CD4 T cell depletion nor CD4 T cell activation were observed in these tissues. In tissues that were productively infected we evaluated the efficiency of this infection by measuring the release of p24 in the culture medium and by enumerating p24+ CD4 T cells with flow cytometry. By both these criteria there were no statistically significant differences between tissues inoculated with C/R and T/F HIV-1 variants. T cell depletion is a hallmark of HIV-1 infection. All HIV-1 variants employed here significantly deplete cervical tissue of CD4 T cells, and with similar efficiency. As expected the magnitude of T cell depletion is proportional to the efficiency of infection, in our case to the number of infected cells in the tissue. Neither when we compared CD4 T cell depletion in NL-SF162.ecto?and NL1051.TD12.ecto nfected donor matched tissues, nor when we compared all T/F and C/R HIV-1 variants as groups, were there statistically significant differences. It is known that Lixisenatide activated CD4 T cells 56-59-7 web preferentially support productive HIV-1 infection and that HIV-1 infection may activate bystander cells [15]. This was confirmed in this study: there were more activated cells (as evaluated by the expression of various activation markers) among HIV-1 infected T cells than in controls. Both T/F and C/R HIV-1 variants replicated predominantly in these activated cel.Dely utilized control reference viruses, as well as the cognate isolates themselves, taken from different biological compartments: HIV-1BaL (isolated from lung), and HIV-1SF162 (isolated from cerebrospinal fluid). We argued that the use of these C/R laboratory-adapted HIV-1 variants as controls should maximize the chance of detecting unique phenotypic characteristics of T/F viruses. Nevertheless, our study did not reveal striking differencesbetween C/R and T/F HIV-1 envelopes in infection of cervical tissue that pointed towards a T/F phenotype related gatekeeping mechanism(s). Although the rate of HIV-1 transmission ex vivo is much higher than that in vivo, not every cervical tissue inoculated with HIV-1 supported productive infection. Why some cervical tissues were not infected by HIV-1 remains to be studied and may be related to the stage of menstrual cycle at which they were isolated [G. Poli, personal communication] and/or the expression of innate restriction factors. Whichever the gatekeeping mechanisms that protect the tissues from infection are, the rates of transmission of C/R and T/F HIV-1 variants were not different in our model system. Using p24 release into the culture medium as a read-out, some tissues may support replication of HIV-1 at a level that we did not consider reliably indicative of de novo virus production since the p24 amount measured may merely represent a slow release of virions adsorbed during inoculation. To exclude these tissues from further analysis, we established a formal criterion: tissue was considered to support productive HIV-1 infection if the amount of the released virus 1480666 exceeded the amount of the released adsorbed virus by 100 pg. Although this criterion is somewhat arbitrary, in our experience the total amount of cumulative production of virus over 12 days of culture should be not lower than this amount. To determine the cumulative de novo production, we blocked HIV-1 infection with the NRTI 3TC and measured the amount of virusTransmission of Founder HIV-1 to Cervical Explantsreleased. The amount of 3TC we applied seems to block HIV-1 infection, as neither CD4 T cell depletion nor CD4 T cell activation were observed in these tissues. In tissues that were productively infected we evaluated the efficiency of this infection by measuring the release of p24 in the culture medium and by enumerating p24+ CD4 T cells with flow cytometry. By both these criteria there were no statistically significant differences between tissues inoculated with C/R and T/F HIV-1 variants. T cell depletion is a hallmark of HIV-1 infection. All HIV-1 variants employed here significantly deplete cervical tissue of CD4 T cells, and with similar efficiency. As expected the magnitude of T cell depletion is proportional to the efficiency of infection, in our case to the number of infected cells in the tissue. Neither when we compared CD4 T cell depletion in NL-SF162.ecto?and NL1051.TD12.ecto nfected donor matched tissues, nor when we compared all T/F and C/R HIV-1 variants as groups, were there statistically significant differences. It is known that activated CD4 T cells preferentially support productive HIV-1 infection and that HIV-1 infection may activate bystander cells [15]. This was confirmed in this study: there were more activated cells (as evaluated by the expression of various activation markers) among HIV-1 infected T cells than in controls. Both T/F and C/R HIV-1 variants replicated predominantly in these activated cel.