CXCR4-dependent HIV-1 infection of differentiated epithelial cells

Epithelial cells constitute a physical barrier to sexual transmission of HIV, but are also a source of cytokines that could alter infection efficiency. We studied HIV infection of the human colonic epithelial cell line HCT116, which is a model for differentiation of intestinal mucosal epithelium. Differentiated HCT116 cells had increased expression of cell surface C-X-C chemokine receptor type-4 (CXCR4) that mediated HIV entry, despite the apparent absence of cell surface CD4. HIV infection in differentiated HCT116 cells increased the levels of IL-1, and IFN- mRNA even though only 1% of cells had integrated provirus. The inefficient, CXCR4-mediated infection of differentiated HCT116 cells supports the view that epithelial cells are a barrier and not a portal for HIV transmission. However, low level infection of epithelial cells could trigger the release of cytokines that indirectly increase the transmission rate.     

Molecular subtyping of the HIV-1 V3 loop sequences detected in HIV-1-positive patients in southern Taiwan

Polymerase chain reaction and nucleotide sequence analysis were performed to amplify and determine the V3 loop sequences of human immunodeficiency virus type 1 (HIV-1) from ten seropositive patients at National Cheng Kung University Hospital, Tainan. The nucleotide sequences and the deduced amino acid (a. a.) sequences of these V3 regions were compared with those of known HIV-1 prototypes. The V3 loop a. a. sequences detected in eight individuals belong to subtype B which predominates in North America and Europe, whereas two individuals were infected with HIV-1 subtype E which is mainly found in the heterosexual populations of Thailand. Sequence analysis of these variant HIV-1 strains revealed a number of interesting features and a phylogenetic tree was also constructed according to the V3 loop nucleotide sequences of these variant strains and HIV-1 isolates from other parts of the world. Furthermore, our results suggest that the north vs south geographical separation in terms of HIV-1 epidemiology in Taiwan is insignificant.     

Glucosamine suppresses interleukin-8 production and ICAM-1 expression by TNF-alpha-stimulated human colonic epithelial HT-29 cells

Intestinal epithelial cells play an important role in the mucosal immune reaction in inflammatory bowel diseases via the production and expression of chemokines and adhesion molecules, such as interleukin-8 (IL-8) and intercellular adhesion molecule-1 (ICAM-1), which are involved in the neutrophil infiltration and tissue damage in the inflamed colon. Notably, glucosamine, a naturally-occurring amino monosaccharide, has been shown to exhibit an anti-inflammatory action by inhibiting neutrophil functions. In the present study, to evaluate the anti-inflammatory action of glucosamine on intestinal epithelial cells, we examined the effects of glucosamine on the activation of a human colonic epithelial cell line HT-29. The results revealed that glucosamine suppressed the IL-8 production and ICAM-1 expression by TNF-alpha-activated HT-29 cells. Furthermore, glucosamine inhibited the TNF-alpha-induced phosphorylation of p38MAPK and NF-kappaB p65, and the nuclear translocation of NF-kappaB in the cells. Thus, glucosamine demonstrates inhibitory actions on the inflammatory and signaling molecules (IL-8, ICAM-1, p38MAPK and NF-kappaB) in intestinal epithelial cells. However, glucosamine did not essentially affect the binding of TNF-alpha to its receptor on HT-29 cells. Together, these observations suggest that glucosamine may have the potential to exhibit an anti-inflammatory action on intestinal epithelial cells, by possibly interfering with the activation signaling downstream of the ligand/receptor binding.     

Sequence variation and consensus sequence of V3 loop on HIV-1 gp120

Mutation in the V3 loop of HIV-1 gp120 could affect syncytium formation, virus infectivity and neutralization. To acquire more information of the V3 loop mutation, we analyzed amino acid sequences of the V3 loop of 24504 isolates from most HIV-1 clades (including A, B, C, D, E, F, G and H clades). The consensus sequence of the V3 loop of each subtype with the highest frequency emerging on each position is constituted and the conservation of each amino acid in this region is also calculated. Exploring the restricted mutation of the V3 region could help to understand mechanism of HIV entry and to develop new strategy against HIV-1.     

Ultrastructural study of Mycobacterium avium infection of HT-29 human intestinal epithelial cells

Mycobacterium avium is a common pathogen in AIDS patients and, in a large percentage of those patients, M. avium infection appears to be acquired via the gastrointestinal tract. M. avium is able to bind to and enter human and murine intestinal epithelial cells in vitro and in vivo. The invasion by and intracellular fate of M. avium in the HT-29 intestinal epithelial cell line was examined in an ultrastructural study. Bacterial contact with polarised cells was observed 10-15 min after monolayer infection and in polarised monolayers this always occurred in areas lacking microvilli. Contact with HT-29 cells did not appear to take place in a preferential area on the bacterial cell. Following invasion, M. avium was encountered within vacuoles containing either single or multiple bacteria; the latter evolved to contain only an individual bacterium. Vacuoles containing more than one bacterium were seen early in the infection and eventually underwent segmentation, with each bacterium occupying a vacuole. No bacteria were observed outside vacuoles up to 5 days after infection.     

Stromal cell-derived factor-1 from biliary epithelial cells recruits CXCR4-positive cells: implications for inflammatory liver diseases

Although stromal cell-derived factor-1 (SDF-1) plays an important role in hematopoiesis in the fetal liver, the role after birth remains to be clarified. We investigated the role of SDF-1 and its receptor, CXCR4, in 75 patients; this included controls and patients with viral hepatitis, liver cirrhosis, primary biliary cirrhosis, primary sclerosing cholangitis, and autoimmune hepatitis. Interestingly, SDF-1 appeared up-regulated in biliary epithelial cells (BEC) of inflammatory liver disease. Furthermore, in inflammatory liver diseases, SDF-1 was expressed by BEC of interlobular and septal bile ducts and by proliferated bile ductules. The message expression of SDF-1 in BEC was confirmed at a single-cell level by RT-PCR and laser capture microdissection. The plasma levels of SDF-1 were significantly higher in patients with liver diseases than in normal controls. Flow cytometric analysis of the surface expression of CXCR4 showed that most liver-infiltrating lymphocytes express CXCR4 and the intensity was up-regulated more significantly in liver-infiltrating lymphocytes than in peripheral blood lymphocytes. These results suggest that increased SDF-1 production by BEC may play an important role in the recruitment of CXCR4-positive inflammatory cells into the diseased livers. These data are significant because modulation of the SDF-1/CXCR4 interaction has therapeutic implications for inflammatory liver diseases.     

Accessory cell function of a human colonic epithelial cell line HT-29 for bacterial superantigens

The expression and up-regulation of cell adhesion molecules on a human colonic epithelial cell line HT-29, and the peripheral blood T lymphocyte proliferation responses to bacterial superantigens presented by this cell line were investigated, compared with peripheral blood monocytes. In HT-29 cells, there was constitutive expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-3 (LFA-3) at a low level, but no constitutive expression of HLA-DR, LFA-1, B7-1 and B7-2 molecules. After stimulation with the supernatants of staphylococcal enterotoxin B (SEB)-stimulated peripheral blood mononuclear cells for 48 h, there was significant up-regulation of HLA-DR and ICAM-1 molecules (both >90% positive). However, this stimulation had no effect on the expression of LFA-1, B7-1, B7-2 and LFA-3 molecules. In the presence of all tested superantigens SEB, toxic shock syndrome toxin-1, and streptococcal pyogenic exotoxin A, stimulated HT-29 cells caused significant T cell proliferation. When monocytes were used as antigen-presenting cells (APC), the MoAbs against HLA-DR, B7-2 and LFA-3 showed a significant inhibition of SEB-induced T cell proliferation. Anti-ICAM-1 MoAb had no effect on this response. On the other hand, when stimulated HT-29 cells were used as APC, the MoAbs against HLA-DR and ICAM-1 significantly inhibited SEB-induced T cell proliferation. In contrast to monocytes, anti-B7-2 and anti-LFA-3 had no effect on this response. SEB could not induce HT-29 cells to produce IL-8 directly; however, SEB significantly induced the stimulated HT-29 cells to produce IL-8 in the presence of T cells. Thus these data demonstrate that the products of superantigen-stimulated T cell activation can increase the expression of HLA-DR and ICAM-1 molecules on HT-29 cells significantly. Stimulated HT-29 cells can serve as APC to bacterial superantigens. This response is an HLA-DR- and ICAM-1-dependent, but B7-2- and LFA-3-independent process, which was different from professional APC monocytes.     

CD4/CXCR4 co-expression allows productive HIV-1 infection in canine kidney MDCK cells

The Madin-Darby canine kidney (MDCK) cell line has become the prototypic cell type for studying the mechanisms involved in viral glycoproteins transport and viral assembly in polarized cells. This cell line has been used in our laboratories for studying human immunodeficiency virus (HIV-1), despite the fact that MDCK cells cannot be infected by HIV. In transfected MDCK cells, HIV-1 glycoproteins are specifically transported to the basolateral cell surface where viral budding also mostly occurs. However, this model is of limited use when viral propagation, infection of most cells, or larger production of virions, is needed. The initial objective of this work was thus to establish an MDCK-derived cell line that could be productively infected by HIV-1, in order to pursue our studies on the polarization of viral budding. Expression of both receptor and co-receptor for T-tropic strains of the virus showed that canine cells are rendered permissive once virus binding and entry is allowed. In addition, a reduced infectivity of the viral particles released from the basolateral surface was observed. This observation most likely reflects the interference mediated by CD4 molecules that accumulate at the basolateral domain. Accordingly, this effect was largely prevented when using viruses that down-regulate cell surface CD4 by expression of both viral accessory proteins Vpu and Nef. This is a further evidence that the function of different viral proteins depends of the site of viral budding, which is itself determined by the presence of targeting signal(s) harbored by viral envelope glycoproteins.     

V3 loop region of the HIV-1 gp120 envelope protein is essential for virus infectivity.

The mechanism by which HIV-1 mediates cell fusion and penetrates target cells, subsequent to receptor (CD4) binding, is not well understood. However, neutralizing antibodies, which recognize the principal neutralizing determinants of the gp120 envelope protein (the V3 loop region, residues 296 to 331), have been shown to effectively block cell fusion and virus infectivity independent of the initial gp120-CD4 binding. To investigate the role of the V3 loop in an HIV infection, a series of site-specific mutations were introduced into the HIV-1 envelope gene. Specifically, each residue (312 to 315) in the strongly conserved tetrapeptide sequence, GPGR, which is positioned in the center of the V3 loop domain was individually altered. The processing, transport, and CD4 binding properties of the mutant envelope proteins were comparable to those of the wild-type protein, however, none of the mutants were able to form syncytia in the HeLa-T4 assay. Molecular HIV-1 clones containing mutations altering the G312, G314, or R315 residues produced noninfectious virions, whereas a clone with a P313A mutation was found to be infectious. These results demonstrate that certain V3 loop mutations can be lethal and clearly indicate that this region of the HIV-1 gp120 protein is essential for virus infectivity.     

Genetic characteristics of the V3 region associated with CXCR4 usage in HIV-1 subtype C isolates

CXCR4 coreceptor usage appears to occur less frequently among HIV-1 subtype C viruses. The aim of this study was to investigate the genetic determinants within the V3 region of subtype C isolates able to use CXCR4. Thirty-two subtype C isolates with known phenotypes (16 R5, 8 R5X4 and 8 X4 isolates) were assessed. A subtype C-specific V3 heteroduplex tracking assay (HTA) was used to determine sample complexity, and nucleotide sequencing analysis was used to compare characteristics associated with CCR5 and CXCR4-using isolates. There were sufficient genetic differences to discriminate between R5 viruses and those able to use CXCR4. In general, R5 isolates had an HTA mobility ratio >0.9 whereas CXCR4-using isolates were usually <0.9. Multiple bands were more frequently seen among the dualtropic isolates. Sequence analysis of the V3 region showed that CXCR4-using viruses were often associated with an increased positive amino acid charge, insertions and loss of a glycosylation site, similar to HIV-1 subtype B. In contrast, where subtype B consensus V3 has a GPGR crown motif irrespective of coreceptor usage, all 16 subtype C R5 viruses had a conserved GPGQ sequence at the tip of the loop, while 12 of the 16 (75%) CXCR4-using viruses had substitutions in this motif, most commonly arginine (R). These findings were confirmed using a larger published data set. We therefore suggest that changes within the crown motif of subtype C viruses might be an additional pathway to utilise CXCR4 and thus GPGQ may limit the potential for the development of X4 viruses.     

趋化因子RANTES和SDF-1细胞内共表达抑制HIV-1感染的研究

目的　观察HIV 1辅受体CCR5和CXCR4的配体在细胞内共表达抑制HIV 1感染的作用。方法　应用磷酸钙沉淀法共转染HIV 1辅受体及其配体的质粒 ,制成辅受体表型剔除的靶细胞 ,与转染HIV 1膜蛋白质粒的细胞混合 ,观察合胞体形成并记数 ;脂质体介导法将含有报告基因CAT而缺失HIV包膜蛋白的质粒与HIV包膜蛋白质粒共转染 2 93细胞 ,包装成具有一次感染活性的假病毒 ,感染转化pCMV R K S K、pCMV R K、pCMV S K或pCMV的PM 1细胞 ,采用同位素薄层层析分析法检测CAT活性。结果　pCMV R K S K转染可以显著抑制M及T嗜性HIV膜蛋白诱导的合胞体形成 ;CAT检测发现与pCMV转染组相比 ,当两种嗜性重组病毒感染pCMV R K S K转染组PM 1细胞时 ,仅检测到背景水平的CAT活性。结论　HIV 1辅受体CCR5 CXCR4表型剔除可以明显抑制M和T嗜性HIV 1病毒进入靶细胞     

Increased CXCR4-dependent HIV-1 fusion in activated T cells: role of CD4/CXCR4 association

Activation of peripheral CD4+ T cells resulted in augmented fusion with X4 human immunodeficiency virus type 1 (HIV-1) envelope-expressing cells without parallel increases in the surface expression of CD4 or CXC chemokine receptor 4 (CXCR4). Our study used biochemical methods and biological assays to correlate the increased fusion potential of activated T cells with changes in CXCR4 isoforms and CD4-CXCR4 association. Western blot analyses of CXCR4, precipitated from resting T cells, identified several CXCR4 species with molecular weights of 47, 50, 62, and 98 kDa. After 24 h stimulation with phytohemagglutinin/interleukin-2, a marked reduction was seen in the 47-kDa, with a concomitant increase in the amounts of 50 and 62-64 kDa CXCR4. T cell activation also induced an increase in the coprecipitation of CXCR4 with CD4. The 62-kDa CXCR4 predominantly coprecipitated with CD4 and was shown to be ubiquitinated. Stripping of CD4 from the cell surface with pronase treatment prior to cell lysis only partially reduced coprecipitation of CD4 with the 62-kDa CXCR4, revealing a pool of intracellular CD4-CXCR4 complexes. Coprecipitation of CXCR4 with CD4 was reduced in activated cells treated with Brefeldin A and Monensin, suggesting that late endosomes play a role in intracellular association of CXCR4 with CD4. Confocal microscopy confirmed the colocalization of CD4 and CXCR4 within CD63+ endocytic compartments. These findings demonstrated a correlation between the enhanced susceptibility of activated T cells to HIV-1 fusion and accumulation of ubiquitinated 62-64 kDa CXCR4 species, which preferentially associated with CD4. The CD4-CXCR4 complexes may shuttle between late endosomes and the cell surface.     

淋巴细胞HIV-1辅受体表型剔除阻断病毒感染的研究

目的　观察HIV 1辅受体CCR5和CXCR4表型剔除对HIV 1DP1株感染的阻断作用。方法　用含有pLNCX R K S K的重组逆转录病毒液感染原代人PBLs(外周血淋巴细胞 ) ,抗体 免疫磁珠法分离筛选转化成功PBLs,流式细胞仪检测筛选效率 ;HIV 1DP1株攻击转化PBLs ,进行合胞体形成和p2 4抗原分泌检测。结果　抗 NGFR 免疫磁珠法获得了转化成功的PBLs ,流式细胞仪检测发现pLNCX R K S K转染组 77.4%的PBLsNGFR(神经生长因子受体 )标记物为阳性 ;HIV 1DP1株攻击后 ,未转染组和pLNCX转染组可以见到明显的合胞体形成 ,而在pLNCX R K S K转化PBLs没有见到合胞体形成 ;pLNCX R K S K转染组在感染后第 4、7和 10天皆可发现显著的p2 4抗原分泌抑制 ,抑制率分别为 15%、43 %、19%。结论　细胞内趋化因子通过与CCR5和CXCR4细胞内结合 ,使HIV 1两类主要辅受体难以在PBLs表面表达 ,从而可以达到阻断HIV 1病毒感染的目的     

CXCR4 overexpression during the course of HIV-1 infection correlates with the emergence of X4 strains

The factors that determine the emergence of X4 isolates in some HIV-1-infected subjects are unknown. As the level of expression of CXCR4 could favor an R5 to X4 switch, quantitative flow cytometry was used to measure CXCR4 density on CD4 T cells in 200 HIV-1-positive adults, and this was compared with CD4 counts, interleukin-7 (IL-7), and RANTES (regulated on activation, normal T expressed and secreted) plasma levels and the R5/X4 virus phenotype. CD4 T-cell surface CXCR4 densities were increased in infected subjects and inversely correlated with CD4 T-cell count (r=-0.548, P<0.001). Yet, in vitro infection with either R5 or X4 strains and in vivo increases in viral load following interruption of antiretroviral treatment did not induce CXCR4 overexpression. The plasma levels of IL-7 and RANTES, 2 cytokines able to induce CXCR4 expression, did not correlate with CXCR4 density. Finally, higher CXCR4 densities were observed in patients harboring X4 strains (3300, 95% CI 2431-4169 CXCR4 molecules per cell) than in patients harboring only R5 strains (2406, 95% CI 2135-2677, P=0.027). These data suggest that CXCR4 overexpression during the course of the disease in some patients could favor the emergence of X4 strains.     

The gastrointestinal tract is critical to the pathogenesis of acute HIV-1 infection

It has become evident that the gastrointestinal tract is preferentially and profoundly depleted of CD4+ T cells during acute HIV-1 infection. The enhanced susceptibility of gastrointestinal lymphoid tissue to HIV-1 is in part due to the large complement of CCR5+ memory CD4+ T cells resident at this site. Here we summarize the recent findings demonstrating that the gastrointestinal tract plays a critical role in the pathogenesis of acute HIV-1 and simian immunodeficiency virus infections. Ongoing work in this field is likely to have a significant effect on HIV research in the near future.     

人源性抗HIV—1 V3环噬菌体抗体的基因克隆与序列分析

以ＨＩＶ－１ ｇｐ１２０Ｖ３环的合成环肽为抗原,从人的噬菌体抗体组合文库中,筛选出与ＨＩＶ－１ Ｖ３肽具有结合活性的人源性噬菌体抗体,用ＥＬＩＳＡ测定其活性,竞争抑制实验证实了该噬菌体抗体的特异性。序列分析表明,重链基因的ＩｇＧ１亚类,可变区属ＶＨ Ｉ亚组,与胚系基因ＤＰ－８８的同源性最高。Ｄ区为Ｄ３－３,Ｊ区为ＪＨ５,轻链为ｋ亚型,可变区属ＶＬ ＩＶ亚群,Ｄ区为ＤＰＫ２２,Ｊ链为ＪＫ４胚系。     

HIV-1 infection in the female reproductive tract: role of interactions between HIV-1 and genital epithelial cells

Despite recent progress in understanding the mucosal transmission of human immunodeficiency virus (HIV)-1, the immediate events following transmission in the female genital tract are incompletely understood. Recent in vivo studies in primate models indicate that HIV-1 transmission may occur in the upper or lower genital tract and the initial HIV-1 replication occurs primarily in the target T cells and in some subsets of DCs localized in the genital tract. However, the principal mechanism(s) that allow the virus to cross the primary barrier of genital epithelial cells (GECs) are still unclear. A number of pathways have been proposed as possible ways that HIV-1 could use to cross the epithelium. However, little attention has been paid to the response of GECs to HIV-1. We recently demonstrated that exposure to HIV-1 rapidly upregulates a wide array of pro-inflammatory cytokine production by GECs. Among these cytokines, tumour necrosis factor (TNF)-α impaired the tight junction barrier allowing HIV-1 and luminal bacteria to translocate across the epithelium. This study illustrated that GECs are dynamically active cells that mount rapid host responses to HIV-1, independent of viral replication. Cytokine responses of GECs could play a critical role in HIV transmission and replication. Further understanding of GEC responses to HIV-1 and their regulation could be critical to understanding HIV-1 transmission dynamics during heterosexual transmission.