The effects of proton pump inhibition on patient-reported severity of dyspepsia when receiving dual anti-platelet therapy with clopidogrel and low-dose aspirin: analysis from the Clopidogrel and the Optimization of Gastrointestinal Events Trial.

BACKGROUND: Dual anti-platelet therapy with clopidogrel and low-dose aspirin increases the risk for gastrointestinal clinical events. Omeprazole has been shown to significantly reduce these events without compromising cardiovascular safety in patients treated with dual anti-platelet therapy. Whether or not omeprazole improves patient-reported outcomes is undetermined. AIM: To assess the impact of prophylactic omeprazole with background dual anti-platelet therapy on patient-reported symptoms of dyspepsia compared to placebo. METHODS: We analysed results of the Severity of Dyspepsia Assessment questionnaires collected in the Clopidogrel and the Optimization of Gastrointestinal Events Trial. RESULTS: Patient-reported outcome data from 3759 subjects were available for analysis. At 4 weeks, the mean scores of pain intensity and nonpain symptoms were lower in the omeprazole group (5.61 ± 0.17 vs. 6.40 ± 0.17, P = 0.001, and 10.61 ± 0.07 vs. 11.00 ± 0.07, P < 0.001 respectively). These differences were maintained at 24 weeks (5.91 ± 0.35 vs. 7.10 ± 0.37, P = 0.020 for pain intensity; 10.36 ± 0.12 vs. 10.93 ± 0.13, P = 0.001 for nonpain symptoms). After adjusting for covariates there were no statistically significant differences between the groups in the percent of patients with dyspepsia during follow-up. CONCLUSIONS: In addition to reducing the risk of gastrointestinal bleeding, statistically significant benefits with prophylactic omeprazole use on both pain and nonpain symptoms were evident at 4 weeks and sustained through 24 weeks. The clinical significance of these overall results is unclear, but greater in patients with pain at baseline.

HIV-1 Vpr enhances viral burden by facilitating infection of tissue macrophages but not nondividing CD4+ T cells.

Prior experiments in explants of human lymphoid tissue have demonstrated that human immunodeficiency virus type 1 (HIV-1) productively infects diverse cellular targets including T cells and tissue macrophages. We sought to determine the specific contribution of macrophages and T cells to the overall viral burden within lymphoid tissue. To block infection of macrophages selectively while preserving infection of T cells, we used viruses deficient for viral protein R (Vpr) that exhibit profound replication defects in nondividing cells in vitro. We inoculated tonsil histocultures with matched pairs of congenic viruses that differed only by the presence of a wild-type or truncated vpr gene. Although these viruses exhibited no reduction in the infection or depletion of T cells, the ability of the Vpr-deficient R5 virus to infect tissue macrophages was severely impaired compared with matched wild-type R5 virus. Interestingly, the Vpr-deficient R5 virus also exhibited a 50% reduction in overall virus replication compared with its wild-type counterpart despite the fact that macrophages represent a small fraction of the potential targets of HIV-1 infection in these tissues. Collectively, these data highlight the importance of tissue macrophages in local viral burden and further implicate roles for CC chemokine receptor 5, macrophages, and Vpr in the life cycle and pathogenesis of HIV-1.

HIV-1 actively replicates in naive CD4(+) T cells residing within human lymphoid tissues.

Although HIV-1 gene expression is detected in naive, resting T cells in vivo, such cells are resistant to productive infection in vitro. However, we found that the endogenous microenvironment of human lymphoid tissues supports de novo infection and depletion of this population. Cell cycle analysis and DNA labeling experiments established that these cells were definitively quiescent and thus infected de novo. Quantitation of the "burst size" within naive cells further demonstrated that these cells were productively infected and contributed to the local viral burden. These findings demonstrate that lymphoid tissues support active HIV-1 replication in resting, naive T cells. Moreover, these cells are not solely reservoirs of latent virus but are permissive hosts for viral replication that likely targets them for elimination.

Soluble type II transforming growth factor-beta (TGF-beta) receptor inhibits TGF-beta signaling in COLO-357 pancreatic cancer cells in vitro and attenuates tumor formation.

UNLABELLED: Human pancreatic ductal adenocarcinomas overexpress transforming growth factor-betas (TGF-betas). This overexpression has been correlated with decreased patient survival. TGF-betas bind to a type II TGF-beta receptor (TbetaRII) dimer, which heterotetramerizes with a type I TGF-beta receptor (TbetaRI) dimer, thereby activating downstream signaling. PURPOSE AND EXPERIMENTAL DESIGN: To determine whether blocking TGF-beta actions would suppress pancreatic cancer cell growth in vivo, we expressed a soluble TbetaRII, encoding amino acids 1-159 of the extracellular domain in COLO-357 human pancreatic cancer cells. This cell line expresses all of the three mammalian TGF-beta isoforms and is growth inhibited by TGF-beta in vitro. RESULTS: COLO-357 clones expressing soluble TbetaRII were no longer growth inhibited by exogenous TGF-beta1 and exhibited a marked decrease in their invasive capacity in vitro. When injected s.c. into athymic mice, these clones exhibited attenuated growth rates and angiogenesis and decreased levels of plasminogen activator inhibitor-1 mRNA as compared with tumors formed by sham-transfected cells. CONCLUSIONS: These results indicate that endogenous TGF-betas can confer a growth advantage in vivo to a pancreatic cancer cell line that is growth inhibited in vitro and suggest that a soluble receptor approach can be used to block these tumorigenic effects of TGF-betas.

Cytopathicity of human immunodeficiency virus type 1 primary isolates depends on coreceptor usage and not patient disease status.

It has been hypothesized that human immunodeficiency virus type 1 (HIV-1) evolves toward increased cytopathicity in conjunction with disease progression in infected patients. A viral property known to evolve in some but not all patients is coreceptor utilization, and it has been shown that a switch in coreceptor utilization is sufficient for the development of increased cytopathicity. To test the hypothesis that the evolution of other viral properties also contributes to accelerating cytopathicity in vivo, we used human lymphoid tissue explants to assay the cytopathicity of a panel of primary HIV-1 isolates derived from various stages of disease characterized by the presence or absence of changes in coreceptor preference. We found no evidence of coreceptor-independent increases in cytopathicity in isolates obtained either before coreceptor preference changes or from patients who progressed to AIDS despite an absence of coreceptor evolution. Instead, the cytopathicity of all HIV-1 isolates was determined solely by their coreceptor utilization. These results argue that HIV-1 does not evolve toward increased cytopathicity independently of changes in coreceptor utilization.

Susceptibility of rat-derived cells to replication by human immunodeficiency virus type 1.

Progress in developing a small animal model of human immunodeficiency virus type 1 (HIV-1) disease would greatly facilitate studies of transmission, pathogenesis, host immune responses, and antiviral strategies. In this study, we have explored the potential of rats as a susceptible host. In a single replication cycle, rat cell lines Rat2 and Nb2 produced infectious virus at levels 10- to 60-fold lower than those produced by human cells. Rat-derived cells supported substantial levels of early HIV-1 gene expression, which was further enhanced by overexpression of human cyclin T1. Rat cells displayed quantitative, qualitative, and cell-type-specific limitations in the late phase of the HIV-1 replication cycle including relative expression levels of HIV-1 Gag proteins, intracellular Gag processing, and viral egress. Nb2 cells were rendered permissive to HIV-1 R5 viruses by coexpression of human CD4 and CCR5, indicating that the major restriction on HIV-1 replication was at the level of cellular entry. We also found that primary rat lymphocytes, macrophages, and microglia expressed considerable levels of early HIV-1 gene products following infection with pseudotyped HIV-1. Importantly, primary rat macrophages and microglia, but not lymphocytes, also expressed substantial levels of HIV-1 p24 CA and produced infectious virions. Collectively, these results identify the rat as a promising candidate for a transgenic small animal model of HIV-1 infection and highlight pertinent cell-type-specific restrictions that are features of this species.

Folate receptor-alpha is a cofactor for cellular entry by Marburg and Ebola viruses.

Human infections by Marburg (MBG) and Ebola (EBO) viruses result in lethal hemorrhagic fever. To identify cellular entry factors employed by MBG virus, noninfectible cells transduced with an expression library were challenged with a selectable pseudotype virus packaged by MBG glycoproteins (GP). A cDNA encoding the folate receptor-alpha (FR-alpha) was recovered from cells exhibiting reconstitution of viral entry. A FR-alpha cDNA was recovered in a similar strategy employing EBO pseudotypes. FR-alpha expression in Jurkat cells facilitated MBG or EBO entry, and FR-blocking reagents inhibited infection by MBG or EBO. Finally, FR-alpha bound cells expressing MBG or EBO GP and mediated syncytia formation triggered by MBG GP. Thus, FR-alpha is a significant cofactor for cellular entry for MBG and EBO viruses.

Primary and recombinant HIV type 1 strains resistant to protease inhibitors are pathogenic in mature human lymphoid tissues.

Preserved peripheral CD4+ T cell counts despite virologic failure in patients undergoing protease inhibitor (PI)-containing antiviral regimens are a frequent occurrence in human immunodeficiency virus (HIV) disease. One hypothesis to explain the relative sparing of CD4+ T cells is that HIV strains exhibiting PI resistance concomitantly are attenuated in terms of cytopathicity for mature T cells. To test this hypothesis, we used a three-dimensional human tonsil histoculture microenvironment to assess the pathogenic potential of a panel of primary and recombinant HIV-1 strains derived from patients experiencing PI failure. All the viruses tested replicated efficiently in these cultures and, in some cases, better than comparable wild-type viral isolates. Furthermore, the PI-resistant strains depleted CD4+ T cells potently and comparably with wild-type isolates in these ex vivo lymphoid tissues. These results demonstrate that PI-resistant viruses are not inherently less pathogenic for mature T cells. Therefore, the sustained peripheral lymphocyte counts in patients with selective virologic failure may be due to specific defects in viral replication in other cell compartments or to an undefined host adaptation to viral infection during PI therapy.

Human immunodeficiency virus type 1 clade A and D neurotropism: molecular evolution, recombination, and coreceptor use.

Human immunodeficiency virus type 1 (HIV-1) non-B clade viral infections of the brain have not been studied to date. Among nine AIDS patients from Nairobi, Kenya, infected with HIV-1 A (N = 5) or D (N = 4) clade strains, brain-derived HIV-1 env sequences displayed greater evolutionary distance than B clade brain-derived viruses (P < 0.001). Similarly, molecular diversity between matched brain and spleen env clones was clade-dependent and concentrated in the hypervariable V4 region (P < 0.001), with phylogenetic clustering of sequences derived from the same organ. Brain-derived A and D clade sequences displayed significantly lower ratios of nonsynonymous/synonymous substitution rates (d(N)/d(S)) compared to matched spleen-derived clones and brain-derived B clade viruses. Interclade recombination events were infrequently observed among the present env sequences. A chimeric virus containing the C2V3 region from an A clade brain-derived sequence preferentially used CD4 and CCR5 for infection. These findings demonstrate that differences in molecular diversity in brain-derived sequences were dependent on the individual clade and domain within the env gene, but both B and non-B clade brain-derived viruses exhibit a preference for CCR5 as a coreceptor.

Molecular function of the CD4 D1 domain in coreceptor-mediated entry by HIV type 1.

The surface molecule CD4 plays a key role in initiating cellular entry by the human immunodeficiency virus type 1 (HIV-1), and it is now recognized as acting synergistically with select chemokine receptors (coreceptors) in the infection process. The present study was undertaken to determine whether the extracellular region of CD4 is sufficient to induce fusion of HIV-1 virions with target cells in the absence of its anchoring function. Using pseudotype reporter viruses to quantitate infection, soluble CD4 (sCD4) was tested for its ability to induce fusion by viruses utilizing CCR5 as their coreceptor. We found that sCD4 was competent to replace membrane-bound CD4 to trigger infection mediated by several HIV-1 envelopes. Furthermore, in a comparison of the envelopes of HIV-1 NL4-3 and a chimera containing the gp120 V3 loop of Ba-L, the V3 region was found to be one factor affecting susceptibility to induction by sCD4. In addition, using truncated and mutant derivatives of sCD4, the amino-terminal D1 domain of CD4 was found to be necessary and sufficient for induction of fusion and to require an intact gp120-binding site for this activity. These results delineate determinants on CD4 and gp120 required for fusion induction in collaboration with a coreceptor, and suggest a mechanism whereby CD4 may contribute to viral infection in trans.

Cytopathicity of human immunodeficiency virus type 2 (HIV-2) in human lymphoid tissue is coreceptor dependent and comparable to that of HIV-1.

Epidemiological studies have shown that human immunodeficiency virus type 2 (HIV-2) is markedly less pathogenic than HIV-1 in vivo. Individuals infected with HIV-2 exhibit a remarkably slow rate of disease development, and these clinical properties have been attributed presumptively to an "attenuated" phenotype of HIV-2 itself. Here, we investigated the impact of coreceptor usage on the cytopathicity of HIV-2 and compared its pathogenic potential with that of HIV-1 in a unique human lymphoid histoculture model. We found that HIV-2 strains, as well as closely related simian immunodeficiency viruses (SIV), displayed mildly or highly aggressive cytopathic phenotypes depending on their abilities to use the coreceptor CCR5 or CXCR4, respectively. A side-by-side comparison of primary X4 HIV-1 and HIV-2 strains revealed similar, high degrees of cytopathicity induced by both HIV types. Furthermore, we found that HIV-2 coreceptor specificity for CCR5 and CXCR4 determined the target cell population for T-cell depletion in lymphoid tissue. Finally, utilization of the alternate coreceptors BOB and Bonzo did not significantly increase the cytopathic properties of HIV-2. These findings demonstrate that coreceptor preference is a key regulator of target cell specificity and the cytopathic potential of HIV-2, with indistinguishable rules compared with HIV-1. Moreover, HIV-2 strains are not characterized by an intrinsically lower cytopathicity than HIV-1 strains. Therefore, direct cytopathic potential per se does not explain the unique behavior of HIV-2 in people, highlighting that other unknown factors need to be elucidated as the basis for their lesser virulence in vivo.

Human immunodeficiency virus type 1 coreceptor preferences determine target T-cell depletion and cellular tropism in human lymphoid tissue.

The present study sought to determine how usage of coreceptors by human immunodeficiency virus type 1 dictates cell tropism and depletion of CD4(+) T cells in human lymphoid tissues cultured ex vivo. We found that coreceptor preferences control the marked, preferential depletion of coreceptor-expressing CD4(+) lymphocytes. In addition, there was a strong, but not absolute, preference shown by CXCR4-using strains for lymphocytes and by CCR5-using strains for macrophages.

Distinct mechanisms of entry by envelope glycoproteins of Marburg and Ebola (Zaire) viruses.

Since the Marburg (MBG) and Ebola (EBO) viruses have sequence homology and cause similar diseases, we hypothesized that they associate with target cells by similar mechanisms. Pseudotype viruses prepared with a luciferase-containing human immunodeficiency virus type 1 backbone and packaged by the MBG virus or the Zaire subtype EBO virus glycoproteins (GP) mediated infection of a comparable wide range of mammalian cell types, and both were inhibited by ammonium chloride. In contrast, they exhibited differential sensitivities to treatment of target cells with tunicamycin, endoglycosidase H, or protease (pronase). Therefore, while they exhibit certain functional similarities, the MBG and EBO virus GP interact with target cells by distinct processes.

Viral entry through CXCR4 is a pathogenic factor and therapeutic target in human immunodeficiency virus type 1 disease.

The chemokine receptors CCR5 and CXCR4 function as the principal coreceptors for human immunodeficiency virus type 1 (HIV-1). Coreceptor function has also been demonstrated for a variety of related receptors in vitro. The relative contributions of CCR5, CXCR4, and other putative coreceptors to HIV-1 disease in vivo have yet to be defined. In this study, we used sequential primary isolates and recombinant strains of HIV-1 to demonstrate that CXCR4-using (X4) viruses emerging in association with disease progression are highly pathogenic in ex vivo lymphoid tissues compared to CXCR4-independent viruses. Furthermore, synthetic receptor antagonists that specifically block CXCR4-mediated entry dramatically suppressed the depletion of CD4(+) T cells by recombinant and clinically derived X4 HIV-1 isolates. Moreover, in vitro specificity for the additional coreceptors CCR3, CCR8, BOB, and Bonzo did not augment cytopathicity or diminish sensitivity toward CXCR4 antagonists in lymphoid tissues. These data provide strong evidence to support the concept that adaptation to CXCR4 specificity in vivo accelerates HIV-1 disease progression. Thus, therapeutic intervention targeting the interaction of HIV-1 gp120 with CXCR4 may be highly valuable for suppressing the pathogenic effects of late-stage viruses.

Genomic cloning, structure, and regulatory elements of the 1 alpha, 25(OH)2D3 down-regulated gene for cyclic AMP-dependent protein kinase inhibitor.

The cyclic AMP-dependent protein kinase inhibitor (PKI) mRNA and protein are negatively and tissue-specifically regulated in the kidney by 1 alpha, 25(OH)2D3. A 17-kb PKI clone, isolated from a chick genomic library, revealed that the PKI gene consists of two exons separated by a 4.5-kb intron. A 411-bp upstream region (constituting 93 bp upstream and 318 bp downstream from the transcriptional start site) containing a putative negative VDRE (nVDRE) fused to the luciferase gene was used for transient transfections of primary cultures of chick kidney cells. Luciferase activity was significantly down-regulated in response to 1 alpha, 25(OH)2D3. This result suggests that the promoter region containing the putative nVDRE plays a pivotal role in the negative regulation of PKI gene transcription.

Genetic evidence for an additional factor required for erythropoietin-induced signal transduction.

Erythropoietin (EPO) and its receptor (EPOR) are required for the development of mature erythrocytes. After binding of ligand, the EPOR activates a variety of signaling pathways that ultimately control cellular proliferation, survival, and specific gene expression. Although erythroid progenitors appear to be the principal EPO-responsive cell type in vivo due to the restricted expression of the EPOR, many growth factor-dependent cell lines expressing the EPOR can respond to EPO by activating many or all of these pathways. In the present study, we have identified a cellular context (the interleukin-2 [IL-2]-dependent HT-2 line) in which the EPO stimulation of the EPOR fails to support cellular proliferation, STAT-5 induction, or MAPK activation, despite efficient phosphorylation of the EPOR and JAK2 and inhibition of apoptosis after withdrawal of IL-2. Interestingly, when we fused HT-2 cells expressing the EPOR with Ba/F3 cells in a complementation assay, the resulting hybridomas proliferated and potently activated STAT-5 and MAPK in response to EPO. These data indicate that an unidentified cellular factor is needed to mediate signaling by the EPOR. Moreover, Ba/F3 cells apparently express this factor(s) and somatic fusions can, therefore, confer EPO-responsiveness to HT-2 cells that lack this factor.

A trans-receptor mechanism for infection of CD4-negative cells by human immunodeficiency virus type 1.

Chemokine receptors, particularly CCR5 and CXCR4, act as essential coreceptors in concert with CD4 for cellular entry by human immunodeficiency virus type 1 (HIV-1; reviewed in [1]). But infection of CD4(-) cells has also been encountered in various tissues in vivo, including astrocytes, neurons and microvascular endothelial cells of the brain [2] [3] [4] [5] [6], epithelial cells [5] [7], CD4(-) lymphocytes and thymocytes [8] [9], and cardiomyocytes [10]. Here, we present evidence for the infection of CD4(-) cell lines bearing coreceptors by well-known HIV-1 strains when co-cultured with CD4(+) cells. This process requires contact between the coreceptor-bearing and CD4(+) cells and supports the full viral replication cycle within the coreceptor-bearing target cell. Furthermore, CD4 provided in trans facilitates infection of primary human cells, such as brain-derived astrocytes. Although the pathobiological significance of infection of CD4(-) cells in vivo remains to be elucidated, this trans-receptor mechanism may facilitate generation of hidden reservoirs of latent virus that confound antiviral therapies and that contribute to specific AIDS-associated clinical syndromes.

Shared and unique determinants of the erythropoietin (EPO) receptor are important for binding EPO and EPO mimetic peptide.

We have shown previously that Phe93 in the extracellular domain of the erythropoietin (EPO) receptor (EPOR) is crucial for binding EPO. Substitution of Phe93 with alanine resulted in a dramatic decrease in EPO binding to the Escherichia coli-expressed extracellular domain of the EPOR (EPO-binding protein or EBP) and no detectable binding to full-length mutant receptor expressed in COS cells. Remarkably, Phe93 forms extensive contacts with a peptide ligand in the crystal structure of the EBP bound to an EPO-mimetic peptide (EMP1), suggesting that Phe93 is also important for EMP1 binding. We used alanine substitution of EBP residues that contact EMP1 in the crystal structure to investigate the function of these residues in both EMP1 and EPO binding. The three largest hydrophobic contacts at Phe93, Met150, and Phe205 and a hydrogen bonding interaction at Thr151 were examined. Our results indicate that Phe93 and Phe205 are important for both EPO and EMP1 binding, Met150 is not important for EPO binding but is critical for EMP1 binding, and Thr151 is not important for binding either ligand. Thus, Phe93 and Phe205 are important binding determinants for both EPO and EMP1, even though these ligands share no sequence or structural homology, suggesting that these residues may represent a minimum epitope on the EPOR for productive ligand binding.

V3 recombinants indicate a central role for CCR5 as a coreceptor in tissue infection by human immunodeficiency virus type 1.

Binding of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 to both CD4 and one of several chemokine receptors (coreceptors) permits entry of virus into target cells. Infection of tissues may establish latent viral reservoirs as well as cause direct pathologic effects that manifest as clinical disease such as HIV-associated dementia. We sought to identify the critical coreceptors recognized by HIV-1 tissue-derived strains as well as to correlate these coreceptor preferences with site of infection and dementia diagnosis. To reconstitute coreceptor use, we cloned HIV-1 envelope V3 sequences encoding the primary determinants of coreceptor specificity from 13 brain-derived and 6 colon-derived viruses into an isogenic (NL4-3) viral background. All V3 recombinants utilized the chemokine receptor CCR5 uniformly and efficiently as a coreceptor but not CXCR4, BOB/GPR15, or Bonzo/STRL33. Other receptors such as CCR3, CCR8, and US28 were inefficiently and variably used as coreceptors by various envelopes. CCR5 without CD4 present did not allow for detectable infection by any of the tested recombinants. In contrast to the pathogenic switch in coreceptor specificity frequently observed in comparisons of blood-derived viruses early after HIV-1 seroconversion and after onset of AIDS, the characteristics of these V3 recombinants suggest that CCR5 is a primary coreceptor for brain- and colon-derived viruses regardless of tissue source or diagnosis of dementia. Therefore, tissue infection may not depend significantly on viral envelope quasispeciation to broaden coreceptor range but rather selects for CCR5 use throughout disease progression.