Most simian–human immunodeficiency viruses (SHIVs) bearing envelope (Env) glycoproteins from primary HIV-1 strains fail to infect rhesus macaques (RMs). We hypothesized that inefficient Env binding to rhesus CD4 (rhCD4) limits virus entry and replication and could be enhanced by substituting naturally occurring simian immunodeficiency virus Env residues at position 375, which resides at a critical location in the CD4-binding pocket and is under strong positive evolutionary pressure across the broad spectrum of primate lentiviruses. SHIVs containing primary or transmitted/founder HIV-1 subtype A, B, C, or D Envs with genotypic variants at residue 375 were constructed and analyzed in vitro and in vivo. Bulky hydrophobic or basic amino acids substituted for serine-375 enhanced Env affinity for rhCD4, virus entry into cells bearing rhCD4, and virus replication in primary rhCD4 T cells without appreciably affecting antigenicity or antibody-mediated neutralization sensitivity. Twenty-four RMs inoculated with subtype A, B, C, or D SHIVs all became productively infected with different Env375 variants—S, M, Y, H, W, or F—that were differentially selected in different Env backbones. Notably, SHIVs replicated persistently at titers comparable to HIV-1 in humans and elicited autologous neutralizing antibody responses typical of HIV-1. Seven animals succumbed to AIDS. These findings identify Env–rhCD4 binding as a critical determinant for productive SHIV infection in RMs and validate a novel and generalizable strategy for constructing SHIVs with Env glycoproteins of interest, including those that in humans elicit broadly neutralizing antibodies or bind particular Ig germ-line B-cell receptors.

A primary obstacle to an HIV-1 cure is long-lived viral reservoirs, which must be eliminated or greatly reduced. Cure strategies have largely focused on monitoring changes in T cell reservoirs in peripheral blood (PB), even though the lymphoid tissues (LT) are primary sites for viral persistence. To track and discriminate viral reservoirs within tissue compartments we developed a specific and sensitive next-generation in situ hybridization approach to detect vRNA, including vRNA+ cells and viral particles (“RNAscope”), vDNA+ cells (“DNAscope”) and combined vRNA and vDNA with immunohistochemistry to detect and phenotype active and latently infected cells in the same tissue section. RNAscope is highly sensitive with greater speed of analysis compared to traditional in situ hybridization. The highly sensitive and specific DNAscope detected SIV/HIV vDNA+ cells, including duplexed detection of vDNA and vRNA or immunophenotypic markers in the same section. Analysis of LT samples from macaques prior to and during combination antiretroviral therapy demonstrated that B cell follicles are an important anatomical compartment for both latent and active viral persistence during treatment. These new tools should allow new insights into viral reservoir biology and evaluation of cure strategies.

The nuclear factor-erythroid 2-related factor 2 (Nrf2) plays a critical role in protecting various tissues against inflammation, which is a potential risk factor for colorectal and other cancers. Our previously published mouse model work showed that Nrf2 helps protect against dextran sulfate sodium (DSS)-induced colitis/inflammation, and others have shown that Nrf2 helps protect against inflammation-associated colorectal carcinogenesis (aberrant crypt foci). The present study extended these important earlier findings by exploring the role of Nrf2 in colitis-associated colorectal cancer in a mouse model involving azoxymethane/ DSS-induced colorectal carcinogenesis in Nrf2 knockout mice. Azoxymethane/DSS-treated Nrf2 knockout mice had increased incidence, multiplicity, and size of all colorectal tumors, including adenomas, versus treated wild-type (WT) mice, and the proportion of tumors that were adenocarcinoma was much higher in knockout (80%) versus WT (29%) mice. Compared with WT mice, knockout mice also had increased markers of inflammation in tumor tissue (cyclooxygenase-2 and 5-lipoxygenase expressions and prostaglandin E 2 and leukotriene B 4 levels) and in inflamed colonic mucosa (nitrotyrosine expression), supporting the association of knockout mouse tumor formation with inflammation. The phase II detoxifying/ antioxidant enzymes NAD(P)H-quinone reductase 1 and UDP-glucurosyltransferase 1A1 were elevated in the normal mucosa of WT, but not Nrf 2 knockout, mice treated with azoxymethane/DSS. Our findings show that Nrf2 plays a critical role in protecting against inflammation-associated colorectal cancer.

Nonhuman primate natural hosts for simian immunodeficiency viruses (SIV) develop a nonresolving chronic infection IntroductionSimian immunodeficiency viruses (SIVs) belong to the group of lentiviruses that in the wild infect nonhuman primates (NHPs). The lentiviruses that cause immunodeficiencies in humans and Asian macaques originated from cross-species transmission of viruses that naturally infect NHPs in Africa. 1 Despite persistent infection, SIV-infected natural hosts generally do not progress to AIDS but live healthy life spans despite continual viral replication and high levels of plasma SIV viremia. In contrast, SIV infection of Asian macaques and HIV-1 infection of humans result in chronic infection, and the majority of infected persons progress to AIDS. Dissecting the mechanisms underlying the nonprogressive nature of natural SIV infection will lead to a better understanding of the aspects of HIV infection responsible for the progressive nature of the disease in humans. 2,3 Previous studies have demonstrated that natural hosts do not avoid disease progression by immunologic control of the virus because SIV-infected natural hosts can maintain high levels of viremia. [4][5][6] Moreover, experimental depletion of CD8 ϩ T cells does not affect plasma viremia, 7,8 and natural hosts do not exhibit more stringent cellular 8 or humoral 9 control of viremia compared with HIV-infected humans or SIV-infected rhesus macaques (RMs). The lentiviruses that infect African green monkeys (AGMs) and sooty mangabeys (SMs), 2 prototypic natural SIV host species studied, have been shown to be cytopathic to CD4 ϩ T cells from these species with short life spans of infected cells in vivo. [10][11][12] Finally, these viruses can be pathogenic when used to infect other NHPs. Specifically, SIVagm isolates, which naturally infect AGMs, can be used to infect pigtail macaques that subsequently manifest simian AIDS, 13 and isolates of SIVsmm can also cause progressive infection in RMs. [14][15][16] The mechanism underlying the natural hosts' maintenance of high levels of plasma viremia without progression to AIDS are unclear, but modulation of viral receptors to protect the critical central memory CD4 ϩ T cell population appears to be part of the answer. 17,18 Recent studies have demonstrated that CD4 ϩ T cells from AGMs down-regulate the CD4 receptor 19 as CD4 ϩ T cells enter the memory pool and that CCR5 is differentially expressed by SM CD4 ϩ T cells compared with CD4 ϩ T cells from Asian macaques. 20,21 Modulation of receptors for SIV by CD4 ϩ T cells in natural hosts suggests that cellular targets for viral replication may differ between natural hosts and non-natural hosts of primate immunodeficiency lentiviruses and that natural hosts have adapted to resist disease in part by shifting the infection to cell populations that may be less essential to the survival of the host. 21 Here we have used in situ hybridization with immunohistochemistry, flow cytometric sorting, and quantitative, real-time PCR to study the infection freque...

Pak4 is a member of the B group of p21-activated (Pak) kinases, originally identified as an effector protein for Cdc42. Although Pak4 is expressed at low levels in most adult tissues, it is highly overexpressed in tumor cell lines. Here, we show that Pak4 is also overexpressed in primary tumors, including colon, esophageal, and mammary tumors. Overexpression of Pak4 also leads to tumor formation in athymic mice, whereas deletion of Pak4 inhibits tumorigenesis. Although a constitutively active Pak4 mutant was previously shown to promote oncogenic transformation in cultured cells, our results are the first to show that Pak4 also promotes tumorigenesis in experimental animals. Furthermore, these results show for the first time that not only constitutively active Pak4, but also wild-type Pak4, is transforming, when experimental animals are used. These results are highly significant because wild-type Pak4, rather than activated Pak4, is overexpressed in tumor cells. Our results suggest that overexpression or activation of Pak4 is a key step in oncogenic transformation, due to its ability to promote cell survival and subsequent uncontrolled proliferation. The finding that Pak4 is up-regulated in so many types of cancers indicates that Pak4 may play a vital role in a wide range of different types of cancer. This makes it an attractive candidate for drug therapy for different types of cancer.

The present study investigated the inhibitory effects of Polyphenon E [a standardized green tea polyphenol preparation containing 65% (À)-epigallocatechin-3-gallate] and caffeine on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumor progression from adenoma to adenocarcinoma. Female A/J mice were treated with a single dose of NNK (103 mg/kg body weight, i.p.) and kept for 20 weeks for the mice to develop lung adenomas. The mice were then given a solution of 0.5% Polyphenon E or 0.044% caffeine as the sole source of drinking fluid until week 52. Both treatments significantly decreased the number of visible lung tumors. Histopathologic analysis indicated that Polyphenon E administration significantly reduced the incidence (by 52%) and multiplicity (by 63%) of lung adenocarcinoma. Caffeine also showed marginal inhibitory effects in incidence and multiplicity of adenocarcinoma (by 48% and 49%, respectively). Markers of cell proliferation, apoptosis, and related cell signaling were studied by immunohistochemistry, and the labeling index and staining intensity were quantified by the Image-Pro system. Polyphenon E and caffeine treatment inhibited cell proliferation (by 57% and 50%, respectively) in adenocarcinomas, enhanced apoptosis in adenocarcinomas (by 2.6-and 4-fold, respectively) and adenomas (both by 2.5-fold), and lowered levels of c-Jun and extracellular signal-regulated kinase (Erk) 1/2 phosphorylation. In the normal lung tissues, neither agent had a significant effect on cell proliferation or apoptosis. The results show that tea polyphenols (and perhaps caffeine) inhibit the progression of NNK-induced lung adenoma to adenocarcinoma. This effect is closely associated with decreased cell proliferation, enhanced apoptosis, and lowered levels of c-Jun and Erk1/2 phosphorylation. (Cancer Res 2006; 66(23): 11494-501)

CD4 + T cells play a central role in the immunopathogenesis of HIV/AIDS, and their depletion during chronicHIV infection is a hallmark of disease progression. However, the relative contribution of CD4 + T cells as mediators of antiviral immune responses and targets for virus replication is still unclear. Here, we have generated data in SIV-infected rhesus macaques (RMs) that suggest that CD4 + T cells are essential in establishing control of virus replication during acute infection. To directly assess the role of CD4 + T cells during primary SIV infection, we in vivo depleted these cells from RMs prior to infecting the primates with a pathogenic strain of SIV. Compared with undepleted animals, CD4 + lymphocyte-depleted RMs showed a similar peak of viremia, but did not manifest any post-peak decline of virus replication despite CD8 + T cell-and B cell-mediated SIV-specific immune responses comparable to those observed in control animals. Interestingly, depleted animals displayed rapid disease progression, which was associated with increased virus replication in non-T cells as well as the emergence of CD4-independent SIV-envelopes. Our results suggest that the antiviral CD4 + T cell response may play an important role in limiting SIV replication, which has implications for the design of HIV vaccines.

Mucosal damage to the gastrointestinal (GI) tract with resulting microbial translocation is hypothesized to significantly contribute to the heightened and persistent chronic inflammation and immune activation characteristic to HIV infection. Here we employ a non-human primate model of chemically induced colitis in SIV-uninfected rhesus macaques that we developed using dextran sulfate sodium (DSS), to directly test this hypothesis. DSS treatment results in GI barrier damage with associated microbial translocation, inflammation and immune activation. The progression and severity of colitis are longitudinally monitored by a magnetic resonance imaging approach. DSS treatment of SIV-infected African green monkeys, a natural host species for SIV that does not manifest GI tract damage or chronic immune activation during infection, results in colitis with elevated levels of plasma SIV RNA, sCD14, LPS, CRP and mucosal CD4+ T-cell loss. Together these results support the hypothesis that GI tract damage leading to local and systemic microbial translocation, and associated immune activation, are important determinants of AIDS pathogenesis.