Natural SIV infection of sooty mangabeys (SMs) is nonprogressive despite chronic virus replication. Strikingly, it is characterized by low levels of immune activation, while pathogenic SIV infection of rhesus macaques (RMs) is associated with chronic immune activation. To elucidate the mechanisms underlying this intriguing phenotype, we used high-density oligonucleotide microarrays to longitudinally assess host gene expression in SIV-infected SMs and RMs. We found that acute SIV infection of SMs was consistently associated with a robust innate immune response, including widespread upregulation of IFN-stimulated genes (ISGs) in blood and lymph nodes. While SMs exhibited a rapid resolution of ISG expression and immune activation, both responses were observed chronically in RMs. Systems biology analysis indicated that expression of the lymphocyte inhibitory receptor LAG3, a marker of T cell exhaustion, correlated with immune activation in SIV-infected RMs but not SMs. Our findings suggest that active immune regulatory mechanisms, rather than intrinsically attenuated innate immune responses, underlie the low levels of immune activation characteristic of SMs chronically infected with SIV.
Naturally SIV-infected sooty mangabeys (SMs) do not progress to AIDS despite high-level virus replication. We previously showed that the fraction of CD4+CCR5+ T-cells is lower in SMs compared to humans and macaques. Here we found that, after in vitro stimulation, SM CD4+ T-cells fail to up-regulate CCR5, and that this phenomenon is more pronounced in CD4+ central-memory T-cells (TCM). CD4+ T-cell activation was similarly uncoupled from CCR5 expression in SMs in vivo during (i) acute SIV infection and (ii) following antibody-mediated CD4+ T-cell depletion. Remarkably, CD4+ TCM of SMs that express low levels of CCR5 demonstrated reduced susceptibility to SIV infection both in vivo and in vitro when compared to CD4+ TCM of RMs. These data suggest that low CCR5 expression on SM CD4+ T-cells favors the preservation of CD4+ T-cell homeostasis and promotes an AIDS-free status by protecting CD4+ TCM from direct virus infection.
SUMMARY Streptococcus pneumoniae and Haemophilus influenzae are members of the normal human nasal microbiota with the ability to cause invasive infections. Bacterial invasion requires translocation across the epithelium; however, mechanistic understanding of this process is limited. Examining the epithelial response to murine colonization by S. pneumoniae and H. influenzae, we observed the TLR-dependent downregulation of claudins 7 and 10, tight junction components key to the maintenance of epithelial barrier integrity. When modeled in vitro, claudin downregulation was preceded by upregulation of SNAIL1, a transcriptional repressor of tight junction components, and these phenomena required p38 MAPK and TGF-β signaling. Consequently, downregulation of SNAIL1 expression inhibited bacterial translocation across the epithelium. Furthermore, disruption of epithelial barrier integrity by claudin 7 inhibition in vitro or TLR stimulation in vivo promoted bacterial translocation. These data support a general mechanism for epithelial opening exploited by invasive pathogens to facilitate movement across the epithelium to initiate disease.
Natural host sooty mangabeys (SM) infected with simian immunodeficiency virus SIVsmm do not develop AIDS despite high viremia. SM and other natural hosts express very low levels of CCR5 on CD4 ؉ T cells, and we recently showed that SIVsmm infection and robust replication occur in vivo in SM genetically lacking CCR5, indicating the use of additional entry pathways. SIVsmm uses several alternative coreceptors of human origin in vitro, but which molecules of SM origin support entry is unknown. We cloned a panel of putative coreceptors from SM and tested their ability to mediate infection, in conjunction with smCD4, by pseudotypes carrying Envs from multiple SIVsmm subtypes. smCXCR6 supported efficient infection by all SIVsmm isolates with entry levels comparable to those for smCCR5, and smGPR15 enabled entry by all isolates at modest levels. smGPR1 and smAPJ supported low and variable entry, whereas smCCR2b, smCCR3, smCCR4, smCCR8, and smCXCR4 were not used by most isolates. In contrast, SIVsmm from rare infected SM with profound CD4 ؉ T cell loss, previously reported to have expanded use of human coreceptors, including CXCR4, used smCXCR4, smCXCR6, and smCCR5 efficiently and also exhibited robust entry through smCCR3, smCCR8, smGPR1, smGPR15, and smAPJ. Entry was similar with both known alleles of smCD4. These alternative coreceptors, particularly smCXCR6 and smGPR15, may support virus replication in SM that have restricted CCR5 expression as well as SM genetically lacking CCR5. Defining expression of these molecules on SM CD4 ؉ subsets may delineate distinct natural host target cell populations capable of supporting SIVsmm replication without CD4 ؉ T cell loss. HIV-1 infection of humans and simian immunodeficiency virus SIVmac infection of non-natural host rhesus macaques (RM) result in high viral loads, peripheral CD4 ϩ T cell loss, and progression to AIDS. In contrast, SIVsmm infection of natural host sooty mangabeys (SM) rarely leads to peripheral CD4 ϩ T cell loss or disease despite viral loads comparable to those measured in non-natural hosts. Identifying the mechanisms responsible for the different outcomes of infection in natural hosts compared with non-natural simian or recent human hosts has become a central focus of efforts to understand AIDS pathogenesis. Although many models of natural host infection exist, SIVsmm is unique because cross-species transmission of SIVsmm from SM to humans and RM gave rise to pathogenic HIV-2 and SIVmac, respectively (2, 28). Similarly, experimental transmission of SIVsmm from infected SM hosts results in AIDS in non-natural host RM (40). While the factors regulating pathogenic versus nonpathogenic outcomes of infection are complex and not fully understood (9), comparison of infected SM with RM recently revealed differential targeting of CD4 ϩ T cell subsets (45). Thus, an understanding of SIVsmm cellular tropism may identify cells in natural host SM that maintain viral replication without leading to CD4 ϩ T cell depletion or AIDS.Classically, SIVsmm was thought to exclus...
Expression of the human epidermal growth factor receptor 2 (HER2) is amplified in 25 – 30% of breast cancers and has been associated with an unfavorable prognosis. Here we report the construction, purification, and characterization of Affitoxin – a novel class of HER2-specific cytotoxic molecules combining HER2-specific Affibody molecule as a targeting moiety and PE38KDEL, which is a truncated version of Pseudomonas exotoxin A (PE), as a cell killing agent. It is highly soluble and does not require additional refolding, oxidation, or reduction steps during its purification. Using Surface Plasmon Resonance (SPR) technology and competitive binding assays, we have shown that Affitoxin binds specifically to HER2 with nanomolar affinity. We have also observed a high correlation between HER2 receptor expression and retention of Affitoxin bound to the cell surface. Affitoxin binding and internalization is followed by PE activity domain-mediated ADP-ribosylation of translation elongation factor 2 (eEF2) and, consequently, inhibition of protein synthesis as shown by protein expression analysis of HER2-positive cells treated with Affitoxin. Measured IC50 value for HER2-negative cells MDA-MB468 (65±2.63 pM) was more than 20 times higher than the value for low HER2 level-expressing MCF7 cells (2.56±0.1 pM), and almost three orders of magnitude higher for its HER2-overexpressing derivative MCF7/HER2 (62.7±5.9 fM). These studies suggest that Affitoxin is an attractive PE38-based candidate for treatment of HER2-positive tumors.
Natural-host sooty mangabeys (SM) infected with simian immunodeficiency virus (SIV) exhibit high viral loads but do not develop disease, whereas infection of rhesus macaques (RM) causes CD4؉ T cell loss and AIDS. Several mechanisms have been proposed to explain these divergent outcomes, including differences in cell targeting, which have been linked to low expression of the canonical SIV entry receptor CCR5 on CD4 ؉ T cells of SM and other natural hosts. We previously showed that infection and high-level viremia occur even in a subset of SM that genetically lack functional CCR5, which indicates that alternative entry coreceptors are used by SIV in vivo in these animals. We also showed that SM CXCR6 is a robust coreceptor for SIV smm in vitro. Here we identify CXCR6 as a principal entry pathway for SIV in SM primary lymphocytes. We show that ex vivo SIV infection of lymphocytes from CCR5 wild-type SM is mediated by both CXCR6 and CCR5. In contrast, infection of RM lymphocytes is fully dependent on CCR5. These data raise the possibility that CXCR6-directed tropism in CCR5-low natural hosts may alter CD4 ؉ T cell subset targeting compared with that in nonnatural hosts, enabling SIV to maintain high-level replication without leading to widespread CD4 ؉ T cell loss.
The interaction between HIV gp120 and galactose-containing cell surface glycolipids such as GalCer or Gb3 is known to facilitate HIV binding to both CD4+ as well as CD4- cells. In an effort to develop small molecule HIV-1 entry inhibitors with improved solubility and efficacy, we have synthesized a series of C-glycoside analogs of GalCer and tested their anti HIV-1 activity. The analogs were tested for gp120 binding using a HIV-1 (IIIB) V3-loop specific peptide. Two of the six analogs that interfered with gp120 binding also inhibited HIV Env-mediated cell-to-cell fusion and viral entry in the absence of any significant cytotoxicity. Analogs with two side chains did not show inhibition of fusion and/or infection under identical conditions. The inhibition of virus infection seen by these compounds was not coreceptor dependent, as they inhibited CXCR4, CCR5 as well as dual tropic viruses. These compounds showed inhibition of HIV entry at early steps in viral infection since the compounds were inactive if added post viral entry. Temperature-arrested state experiments showed that the compounds act at the level of virus attachment to the cells likely at a pre-CD4 engagement step. These compounds also showed inhibition of VSV glycoprotein-pseudotyped virus. The results presented here show that the glycoside derivatives of GalCer with simple side chains may serve as a novel class of small molecule HIV-1 entry inhibitors that would be active against a number of HIV isolates as well as other enveloped viruses.
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