Variable, only the medians were used for statistical analysis.ResultsIn an ex vivo cervical tissue system we analyzed biological properties of eight HIV-1 constructs that contained env sequences derived from mucosally transmitted T/F HIV-1 and three constructs that contained envelopes derived from control reference HIV-1 variant (C/R) viruses: NL-SF162.ecto, NL-YU-2.ecto, and NL-BaL.ecto. All env sequences were expressed in otherwise isogenic NL4-3-based backbones [4]. Also, in several experiments we used two full-length T/F viruses, CH077.t and RHPA.c [6]and the laboratory-adapted HIV-1BaL isolate, which we used as the reference. Earlier, we had shown that the HIV-1BaL isolate and the Env-IMC cognate NL-BaL.ecto were similar in cellular tropism and virus replication in various primary target cells ([6] and unpublished]). Cervical tissue blocks were inoculated with virus as described earlier [5] and infection was evaluated by determining the fraction of infected T cells as well as the amount of p24 released into the culture medium. Overall we performed experiments with cervical tissues from 37 donors. Each donor tissue was infected with at least one C/R virus and at least one T/F virus. According to our optimized protocol for cervical tissue infection, for any given virus stock, 16 tissue blocks per donor per condition have to be inoculated. The amount of cervical tissue obtained from individual donor did not allow for the infection of tissue from each donor with all the used viruses while keeping the number of replicates dictated by the protocol. Therefore, to increase the statistical power we pooled data from 1480666 58 infections with T/F HIV-1 variants and compared them with pooled data from 39 infections with C/R HIV-1 variants. In some experiments, we also compared the data for one T/F HIV-1 variant, NL-1051.TD12.ecto with the data for the control HIV-1 variant NL-SF162.ecto, but replicating in donor-matched cervical tissues. In order to distinguish 1676428 de novo HIV-1 production from the release of virus or free p24 merely adsorbed at inoculation, we treated infected tissues with the RT inhibitor 3TC. For reliably determining that the infection was productive, based on our prior experience, we defined infection to be SPDB biological activity productive if the difference between the total amount of p24 released into the medium by inoculated tissue exceeds that of the matched 3TC- treated one by at least 100 pg. Using this criterion, there were no significant differences between the fractions of tissues that supported productive infection with C/R Env-IMC, T/F Env-IMC, and T/F full length viruses (66.67 , 41.18 , and 45.45 , respectively, likelihood ratio p = 0.39).Cervical TissuesTissues obtained from routine hysterectomy through the National Disease Research Interchange (Philadelphia, PA) were cultured and infected as previously described with slight modifications [5,9]. Briefly, mucosal epithelium and JWH 133 site underlying stroma of both ecto- and endo-cervix were separated from muscular tissue, dissected into approximately 2-mm3 blocks, and infected in 1.5mL conical tubes containing 16 tissue blocks per experimental condition and 0.5 mL of viral stock. After 2 hours of incubation at 37uC, tissue blocks were gently washed three times with phosphate-buffered saline (PBS), placed on top of a collagen sponge gel into a 12 well-plate (8 blocks/well) and cultured for 12 days, with a change of medium every third day. Thus in our cultures endo- and exo-cervical tissue blocks were mixe.Variable, only the medians were used for statistical analysis.ResultsIn an ex vivo cervical tissue system we analyzed biological properties of eight HIV-1 constructs that contained env sequences derived from mucosally transmitted T/F HIV-1 and three constructs that contained envelopes derived from control reference HIV-1 variant (C/R) viruses: NL-SF162.ecto, NL-YU-2.ecto, and NL-BaL.ecto. All env sequences were expressed in otherwise isogenic NL4-3-based backbones [4]. Also, in several experiments we used two full-length T/F viruses, CH077.t and RHPA.c [6]and the laboratory-adapted HIV-1BaL isolate, which we used as the reference. Earlier, we had shown that the HIV-1BaL isolate and the Env-IMC cognate NL-BaL.ecto were similar in cellular tropism and virus replication in various primary target cells ([6] and unpublished]). Cervical tissue blocks were inoculated with virus as described earlier [5] and infection was evaluated by determining the fraction of infected T cells as well as the amount of p24 released into the culture medium. Overall we performed experiments with cervical tissues from 37 donors. Each donor tissue was infected with at least one C/R virus and at least one T/F virus. According to our optimized protocol for cervical tissue infection, for any given virus stock, 16 tissue blocks per donor per condition have to be inoculated. The amount of cervical tissue obtained from individual donor did not allow for the infection of tissue from each donor with all the used viruses while keeping the number of replicates dictated by the protocol. Therefore, to increase the statistical power we pooled data from 1480666 58 infections with T/F HIV-1 variants and compared them with pooled data from 39 infections with C/R HIV-1 variants. In some experiments, we also compared the data for one T/F HIV-1 variant, NL-1051.TD12.ecto with the data for the control HIV-1 variant NL-SF162.ecto, but replicating in donor-matched cervical tissues. In order to distinguish 1676428 de novo HIV-1 production from the release of virus or free p24 merely adsorbed at inoculation, we treated infected tissues with the RT inhibitor 3TC. For reliably determining that the infection was productive, based on our prior experience, we defined infection to be productive if the difference between the total amount of p24 released into the medium by inoculated tissue exceeds that of the matched 3TC- treated one by at least 100 pg. Using this criterion, there were no significant differences between the fractions of tissues that supported productive infection with C/R Env-IMC, T/F Env-IMC, and T/F full length viruses (66.67 , 41.18 , and 45.45 , respectively, likelihood ratio p = 0.39).Cervical TissuesTissues obtained from routine hysterectomy through the National Disease Research Interchange (Philadelphia, PA) were cultured and infected as previously described with slight modifications [5,9]. Briefly, mucosal epithelium and underlying stroma of both ecto- and endo-cervix were separated from muscular tissue, dissected into approximately 2-mm3 blocks, and infected in 1.5mL conical tubes containing 16 tissue blocks per experimental condition and 0.5 mL of viral stock. After 2 hours of incubation at 37uC, tissue blocks were gently washed three times with phosphate-buffered saline (PBS), placed on top of a collagen sponge gel into a 12 well-plate (8 blocks/well) and cultured for 12 days, with a change of medium every third day. Thus in our cultures endo- and exo-cervical tissue blocks were mixe.
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