Cell Surface Expression Of CD4 Research Articles

Differential HIV-1 replication in neonatal and adult blood mononuclear cells is influenced at the level of HIV-1 gene expression

The majority of HIV-1-infected neonates and infants have a higher level of viremia and develop AIDS more rapidly than infected adults, including differences seen in clinical manifestations. To determine the mechanisms of HIV-1 infection in neonates vs. adults, we compared the replication kinetics of HIV-1 in neonatal (cord) and adult blood T lymphocytes and monocyte-derived macrophages (MDM) from seven different donors. We found that HIV-1 replicated 3-fold better in cord blood T lymphocytes compared with adult blood T lymphocytes and 9-fold better in cord MDM than adult MDM. We also show that this differential HIV-1 replication did not depend on differences in cell proliferative capabilities, cell surface expression of CD4, CXCR4, and CCR5, or in the amount of PCR products of reverse transcription, DNA synthesis, and translocation of preintegration complex into the nucleus in cord and adult T lymphocytes and MDM. Furthermore, using a single-cycle replication competent HIV-1-NL4-3-Env(-) luciferase amphotropic virus, which measures HIV-1 transcriptional activity independent of receptor and coreceptor expression, we found there was a 3-fold increase of HIV-1 LTR-driven luciferase expression in cord T lymphocytes compared with adult T lymphocytes and 10-fold in cord MDM than in adult MDM. The HIV-1 LTR-driven luciferase expression correlated with HIV-1 LTR transcription, as measured by ribonuclease protection assay. These data suggest that the increased replication of HIV-1 in cord blood compared with adult blood mononuclear cells is regulated at the level of HIV-1 gene expression, resulting in a higher level of viremia and faster disease progression in neonates than adults.

Preclinical pharmacokinetics, pharmacodynamics, and activity of a humanized anti-CD40 antibody (SGN-40) in rodents and non-human primates.

1. Cell-surface expression of CD40 in B-cell malignancies and multiple solid tumors has raised interest in its potential use as a target for antibody-based cancer therapy. SGN-40, a humanized monoclonal anti-CD40 antibody, mediates antibody-dependent cytotoxicity and inhibits B-cell tumor growth in vitro, properties of interest for the treatment of cancers, and is currently in Phase I clinical trials for B-cell malignancies. In this study, we determined in vivo activity and pharmacokinetics properties of SGN-40. 2. Effect of SGN-40 in xenograft model of CD40-expressing B-cell lymphoma in severe-combined immune deficiency mice and its in vivo pharmacokinetics properties in normal mice, rats and cynomolgus monkeys were studied. 3. Treatment with SGN-40 significantly increased the survival of mice xenografted with human B-cell lymphoma cell line. SGN-40 exhibited nearly 100% bioavailability in mice and it cleared faster when given at a low dose. In monkeys, clearance of SGN-40 was also much faster at low dose, suggesting nonlinear pharmacokinetics in these species. In rats, however, SGN-40 clearance at all tested doses was similar, suggesting that pharmacokinetics were linear in this dose range in rats. Administration of SGN-40 to monkeys also produced marked, dose-dependent, and persistent depletion of peripheral CD20(+) B lymphocytes. 4. Data presented in this report suggest that SGN-40 is active in in vivo, and based upon interspecies scaling, SGN-40 clearance in humans is predicted to be similar to observed SGN-40 clearance in monkeys. These data suggest that SGN-40 has appropriate pharmacokinetic properties that support its clinical use.

Optimal combination chemotherapy and chemoradiotherapy with etoposide for advanced cervical squamous cancer cells in vitro

Patients with unresectable advanced carcinoma of the uterine cervix are usually treated with chemotherapy or chemoradiotherapy. In the present study, the optimal administration protocol for etoposide in chemotherapy and chemoradiotherapy for advanced cervical cancer patients was investigated in vitro using the radio-sensitive and anticancer drug-sensitive human cervical squamous cell carcinoma cell line ME180. Therapeutic doses of concurrent irradiation reduced the cellular etoposide sensitivity in a dose-dependent manner, while postirradiation-surviving subclones established from repeatedly irradiated ME180 cells showed significantly higher etoposide sensitivities than the non-irradiated parent cells. Of the 6 monoclonal etoposide-resistant subclones established from ME180 cells, 5 were significantly radioresistant. Although the etoposide-resistant subclones were also significantly resistant to other anticancer drugs, such as cisplatin, carboplatin, nedaplatin, pirarubicin, paclitaxel and docetaxel, they were more sensitive to 5-fluorouracil, mitomycin C and SN38 than the parent cells. Flow cytometric analyses revealed that the etoposide-resistant subclones showed significantly increased cell surface expression of CD40 compared to the parent cells, which expressed undetectable levels of CD40. However, the expression of some integrin receptor subunits, such as CD29, CD49a and CD49f, was apparently reduced in the etoposide-resistant subclones. These results indicate that etoposide should be administered to advanced cervical squamous cancer patients after the completion of radiotherapy, rather than as a concurrent chemoradiotherapy. In order to kill surviving etoposide-resistant cancer cells more effectively, 5-fluorouracil, mitomycin C and irinotecan may be candidate combination drugs for use with etoposide. Differential expression of integrin receptors and CD40 may be involved in the acquisition of etoposide resistance by cervical squamous cancer cells.

CD4+CD25+Regulatory T Cells and Their Therapeutic Potential

The human immune system mounts specific responses to a vast array of antigens. Although this is clearly beneficial in fighting off harmful infections and cancerous cells, the system must be carefully controlled to ensure that normal self-antigens are not targeted. A recently characterized subset of T cells, identified by their cell surface expression of CD4 and CD25, is critical in regulating the function of other immune cells and preventing potentially harmful autoimmune responses. This article reviews what is currently known about these so-called regulatory T cells and discusses the therapeutic potential of these cells to modulate human immune-based diseases.

3. Cytokines and chemokines

Cytokines and chemokines are secreted proteins with growth, differentiation, and activation functions that regulate the nature of immune responses. Cytokines are involved in nearly every facet of immunity and inflammation, from induction of the innate immune response to the generation of cytotoxic T cells and the development of antibodies by the humoral immune system. The combination of cytokines that are produced in response to an immune insult determines which arm of the immune system will be activated. For this update, recent advances in our understanding of cytokines will be discussed, which includes the IL-10, IL-17, and IL-27 families.

Targeted infection of HIV-1 Env expressing cells by HIV(CD4/CXCR4) vectors reveals a potential new rationale for HIV-1 mediated down-modulation of CD4

BackgroundEfficient targeted gene transfer and cell type specific transgene expression are important for the safe and effective expression of transgenes in vivo. Enveloped viral vectors allow insertion of exogenous membrane proteins into their envelopes, which could potentially aid in the targeted transduction of specific cell types. Our goal was to specifically target cells that express the T cell tropic HIV-1 envelope protein (Env) using the highly specific interaction of Env with its cellular receptor (CD4) inserted into the envelope of an HIV-1-based viral vector.ResultsTo generate HIV-1-based vectors carrying the CD4 molecule in their envelope, the CD4 ectodomain was fused to diverse membrane anchors and inserted together with the HIV-1 coreceptor CXCR4 into the envelopes of HIV-1 vector particles. Independent of the type of CD4 anchor, all chimeric CD4 proteins inserted into HIV-1 vector envelopes and the resultant HIV(CD4/CXCR4) particles were able to selectively confer neomycin resistance to cells expressing the fusogenic T cell tropic HIV-1 Env protein. Unexpectedly, in the absence of Env on the target cells, all vector particles carrying the CD4 ectodomain anchored in their envelope adhered to various cell types without infecting these cells. This cell adhesion was very avid. It was independent of the presence of Env on the target cell, the type of CD4 anchor or the presence of CXCR4 on the particle. In mixed cell populations with defined ratios of Env+/Env- cells, the targeted transduction of Env+ cells by HIV(CD4/CXCR4) particles was diminished in proportion to the number of Env- cells.ConclusionVector diversion caused by a strong, non-selective cell binding of CD4+-vector particles effectively prevents the targeted transduction of HIV-1 Env expressing cells in mixed cell populations. This Env-independent cell adhesion severely limits the effective use of targeted HIV(CD4/CXCR4) vectors designed to interfere with HIV-1 replication in vivo. Importantly, the existence of this newly described and remarkably strong CD4-dependent cell adhesion suggests that the multiple viral efforts to reduce CD4 cell surface expression may, in part, be to prevent cell adhesion to non-target cells and thereby to increase the infectivity of viral progeny. Preventing CD4 down-modulation by HIV-1 might be an effective component of a multi-faceted antiviral strategy.

HIV-1 Nef mutations abrogating downregulation of CD4 affect other Nef functions and show reduced pathogenicity in transgenic mice

HIV-1 Nef has the ability to downmodulate CD4 cell surface expression. Several studies have shown that CD4 downregulation is required for efficient virus replication and high infectivity. However, the pathophysiological relevance of this phenomenon in vivo, independently of its role in sustaining high virus loads, remains unclear. We studied the impact of the CD4 downregulation function of Nef on its pathogenesis in vivo, in the absence of viral replication, in the CD4C/HIV transgenic (Tg) mouse model. Two independent Nef mutants (RD35/36AA and D174K), known to abrogate CD4 downregulation, were tested in Tg mice. Flow cytometry analysis showed that downregulation of murine CD4 was severely decreased or abrogated on Tg T cells expressing respectively Nef RD35/36AA and Nef D174K. Similarly, the severe depletion of double-positive CD4 +CD8 + and of single-positive CD4 +CD8 − thymocytes, usually observed with Nef Wt, was not detected in Nef RD35/36AA and Nef D174K Tg mice. However, both mutant Tg mice showed a partial depletion of peripheral CD4 + T cells. This was accompanied, as previously reported for Net Wt Tg mice, by the presence of an activated/memory-like phenotype (CD69 +, CD25 +, CD44 +, CD45RB Low, CD62 Low) of CD4 + T cells expressing Nef RD35/36AA and to a lesser extent Nef D174K. In addition, both mutants retained the ability to block CD4 + T cell proliferation in vitro after anti-CD3 stimulation, but not to enhance apoptosis/death of CD4 + T cells. Therefore, it appears that Nef-mediated CD4 downregulation is associated with thymic defects, but segregates independently of the activated/memory-like phenotype, of the partial depletion and of the impaired in vitro proliferation of peripheral CD4 + T cells. Histopathological assessment revealed the total absence of or decrease severity and frequency of organ AIDS-like diseases (lung, heart and kidney pathologies) in respectively Nef RD35/36AA and Nef D174K Tg mice, relative to those developing in Nef Wt Tg mice. Our data suggest that the RD35/36AA and D174K mutations affect other Nef functions, namely those involved in the development of lung and kidney diseases, in addition to their known role in CD4 downregulation. Similarly, in HIV-1-infected individuals, loss of CD4 downregulation by Nef alleles may reflect their lower intrinsic pathogenicity, independently of their effects on virus replication.

A sea urchin lectin, SUL-1, from the Toxopneustid sea urchin induces DC maturation from human monocyte and drives Th1 polarization in vitro

The sea urchin Toxopneustes pileolus belonging to the family Toxopneustidae, they have well-developed globiferous pedicellariae with pharmacologically active substances. We have purified a novel sea urchin lectin-1 (SUL-1) from the large globiferous pedicellariae of T. pileolus. Dendritic cells (DC) are professional APC and play a pivotal role in controlling immune responses. This study investigated whether SUL-1 can drive DC maturation from human immature monocyte-derived DC in vitro. Human monocytes were cultured with GM-CSF and IL-4 for 6 days followed by another 1 day in the presence of SUL-1 or LPS. DC harvested on day 7 were examined using functional assays. The expression levels of CD1a, CD80, CD83, CD86 and HLA-DR as expressed by mean fluorescence intensity (MFI) on DC differentiated from immature DC after culture with 1.0 microg/ml of SUL-1 for 1 day were enhanced and decreased endocytic activity. SUL-1-treated DC also displayed enhanced T cell stimulatory capacity in an MLR, as measured by T cell proliferation. Cell surface expression of CD80, CD83 and CD86 on SUL-1-treated DC was inhibited by anti-DC-SIGN mAb, while anti-DC-SIGN mAb had no influence on allogeneic T cell proliferation by SUL-1-treated DC. DC differentiated with SUL-1 induced the differentiation of naïve T cell towards a helper T cell type 1 (Th1) response at DC/T (1:5) cells ratio depending on IL-12 secretion. In CTL assay, the production of IFN-gamma and 51Cr release on SUL-1-treated DC were more augmented than of immature DC or LPS-treated DC. SUL-1-treated DC expressed CCR7 and had a high migration to MIP-3beta. Intracellular Ca2+ mobilization in SUL-1-treated DC was also induced by MIP-3beta. These results suggest that SUL-1 bindings to DC-SIGN on surface of immature DC may lead to differentiate DC from immature DC. Moreover, it suggests that SUL-1 may be used on DC-based vaccines for cancer immunotherapy.

A PSP94-derived Peptide PCK3145 inhibits MMP-9 Secretion and Triggers CD44 Cell Surface Shedding: Implication in Tumor Metastasis

PCK3145 is a synthetic peptide corresponding to amino acids 31-45 of prostate secretory protein 94, which can reduce experimental skeletal metastases and prostate tumor growth in vivo. Part of its biological action involves the reduction of circulating plasma matrix metalloproteinase (MMP)-9, a crucial mediator in extracellular matrix (ECM) degradation during tumor metastasis and cancer cell invasion. The antimetastatic mechanism of action of PCK3145 is however, not understood. HT-1080 fibrosarcoma cells were treated with PCK3145, and cell lysates used for immunoblot analysis of small GTPase RhoA and membrane type (MT)1-MMP protein expression. Conditioned media was used to monitor soluble MMP-9 gelatinolytic activity by zymography and protein expression by immunoblotting. RT-PCR was used to assess RhoA, MT1-MMP, MMP-9, RECK, and CD44 gene expression. Flow cytometry was used to monitor cell surface expression of CD44 and of membrane-bound MMP-9. Cell adhesion was performed on different purified ECM proteins, while cell migration was specifically performed on hyaluronic acid (HA). We found that PCK3145 inhibited HT-1080 cell adhesion onto HA, laminin-1, and type-I collagen suggesting the common implication of the cell surface receptor CD44. In fact, PCK3145 triggered the shedding of CD44 from the cell surface into the conditioned media. PCK3145 also inhibited MMP-9 secretion and binding to the cell surface. This effect was correlated to increased RhoA and MT1-MMP gene and protein expression. Our data suggest that PCK3145 may antagonize tumor cell metastatic processes by inhibiting both MMP-9 secretion and its potential binding to its cell surface docking receptor CD44. Such mechanism may involve RhoA signaling and increase in MT1-MMP-mediated CD44 shedding. Together with its beneficial effects in clinical trials, this is the first demonstration of PCK3145 acting as a MMP secretion inhibitor.

Human Tumor Antigen MUC1 Is Chemotactic for Immature Dendritic Cells and Elicits Maturation but Does Not Promote Th1 Type Immunity

The immunostimulatory outcome of the interactions of many pathogens with dendritic cells (DCs) has been well characterized. There are many fewer examples of similar interactions between DCs and self-molecules, especially the abnormal self-proteins such as many tumor Ags, and their effects on DC function and the immune response. We show that human epithelial cell Ag MUC1 mucin is recognized in its aberrantly glycosylated form on tumor cells by immature human myeloid DCs as both a chemoattractant (through its polypeptide core) and a maturation and activation signal (through its carbohydrate moieties). On encounter with MUC1, similar to the encounter with LPS, immature DCs increase cell surface expression of CD80, CD86, CD40, and CD83 molecules and the production of IL-6 and TNF-alpha cytokines but fail to make IL-12. When these DCs are cocultured with allogeneic CD4+ T cells, they induce production of IL-13 and IL-5 and lower levels of IL-2, thus failing to induce a type 1 response. Our data suggest that, in vivo in cancer patients, MUC1 attracts immature DCs to the tumor through chemotaxis and subverts their function by negatively affecting their ability to stimulate type 1 helper T cell responses important for tumor rejection.

Scrambling of the amino acids within the transmembrane domain of Vpu results in a simian-human immunodeficiency virus (SHIV TM) that is less pathogenic for pig-tailed macaques

Previous studies have shown that the transmembrane (TM) domain of the subtype B Vpu enhances virion release from cells and some studies have shown that this domain may form an oligomeric structure with properties of an ion channel. To date, no studies have been performed to assess the role of this domain in virus pathogenesis in a macaque model of disease. Using a pathogenic molecular clone of simian human immunodeficiency virus (SHIV KU-1bMC33), we have generated a novel virus in which the transmembrane domain of the Vpu protein was scrambled but maintained hydrophobic in nature (SHIV TM), which presumably would disrupt any ion channel TM properties of this protein. Vectors expressing the Vpu as a fusion protein with the enhanced green fluorescent protein (Vpu TMEGFP) indicate that it was transported to the same intracellular compartment as the unmodified Vpu protein but did not down-regulate cell surface expression of CD4. To assess the pathogenicity of SHIV TM, three pig-tailed macaques were inoculated with the SHIV TM and monitored for 6–8 months for CD4 + T cell levels, viral loads and the stability of the sequence of the vpu gene. Our results indicated that unlike the parental SHIV KU-1bMC33, inoculation of macaques with SHIV TM did not cause a severe CD4 + T cell loss over the course of their infections. Sequence analysis of the vpu gene analyzed from sequential PBMC samples derived from macaques revealed that the scrambled TM was stable during the course of infection. At necropsy, examination of tissues revealed low viral loads and none of the pathology commonly observed in lymphoid and non-lymphoid tissues following inoculation with the pathogenic parental SHIV KU-1bMC33 virus. Thus, these results show for the first time that the TM domain of Vpu contributes to the pathogenicity of SHIV KU-1bMC33 in pig-tailed macaques.

Interleukin-15 increases neutrophil adhesion onto human respiratory epithelial A549 cells and attracts neutrophilsin vivo

Interleukin-15 (IL-15) is a neutrophil agonist that plays a role in inflammatory disorders, including a variety of pulmonary diseases. Adhesion of neutrophils onto pulmonary cells is a major event leading to development of inflammation. Recently, elevated levels of IL-15 have been associated with different pulmonary diseases. There is no clear evidence that IL-15 modulates cell surface expression of adhesion molecules in neutrophils, or that IL-15 is involved in neutrophil adhesion onto pulmonary cells. Also, it is not clear if IL-15 induces a neutrophilic inflammation in vivo. This study was aimed at elucidation of these issues. Neutrophils were treated with IL-15 and cell surface expression of CD11a, CD11b, CD11c and CD18 was monitored by flow cytometry. The human respiratory epithelial A549 cell line was used as a substrate for the neutrophil adhesion assay and cell surface expression of CD50, CD54 and CD106 was monitored in IL-15-induced A549 cells. The murine air pouch model was used for investigating potential neutrophilic inflammation induced by IL-15 in vivo. IL-15 significantly increased neutrophil cell surface expression of CD11b and CD18 and up-regulated A549 cell surface expression of CD54. Moreover, A549 cells were found to express IL-15R components and adhesion of neutrophils onto A549 cells was increased when neutrophils or A549 cells were treated with IL-15. Finally, IL-15 induced neutrophilic inflammation in vivo and concentrations of IL-6 and CXCL2/MIP-2 were increased in IL-15-induced pouches. IL-15 might participate in inflammatory pulmonary diseases by attracting neutrophils, modulating cell surface expression molecules and increasing neutrophil adhesion onto pulmonary cells.

Increased expression of plasma and cell surface co-stimulatory molecules CTLA-4, CD28 and CD86 in adult patients with allergic asthma

The co-stimulatory interactions of the B7 family molecules CD80 and CD86 on antigen-presenting cells, together with their T cell counter receptors CD28 and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), modulate T lymphocyte-mediated immune responses in a reciprocal manner. To investigate whether there is altered expression and the clinical significance of soluble co-stimulatory molecules in asthmatic patients, plasma concentrations of sCTLA-4, sCD28, sCD80 and sCD86 in 51 adult allergic asthmatic adults with or without steroid treatment, and 35 sex- and age-matched control subjects were measured by enzyme-linked immunosorbent assay (ELISA). Cell surface expression of CTLA-4 and CD28 on peripheral blood mononuclear cells (PBMC) were analysed by flow cytometry. Results showed that the plasma sCTLA-4 concentration was significantly higher in all asthmatic patients while sCD28 and sCD86 concentrations were significantly higher in steroid and non-steroid treated asthmatic patients, respectively, compared with control subjects (all P < 0.01). Significantly increased cell surface expression of CD28 but not CTLA-4 on PBMC was found in asthmatic patients compared with controls (P < 0.05). The plasma concentration and cell surface expression of CTLA-4 were found to exhibit positive and significant correlations with those of CD28 (both P < 0.05). Serum total IgE concentration correlated positively and significantly with sCTLA-4 and sCD28 concentrations in allergic asthmatic patients (both P < 0.05). The increased expression of these soluble co-stimulatory molecules may reflect the dysregulation of T cell activation, thereby contributing to the immunopathogenesis of allergic asthma.

A Novel Caspase-1/Toll-like Receptor 4-independent Pathway of Cell Death Induced by Cytosolic Shigella in Infected Macrophages

Shigella-induced macrophage cell death is an important step in the induction of acute inflammatory responses that ultimately lead to bacillary dysentery. Cell death was previously reported to be dependent upon the activation of caspase-1 via interaction with IpaB secreted by intracellular Shigella, but in this study, we show that Shigella infection of macrophages can also induce cell death independent of caspase-1 or IpaB activity. Time-lapse imaging and electron microscopic analyses indicated that caspase-1-dependent and -independent cell death is morphologically indistinguishable and that both resemble necrosis. Analyses of Shigella mutants or Escherichia coli using co-infection with Listeria suggested that a component common to Gram-negative bacteria is involved in inducing caspase-1-independent cell death. Further studies revealed that translocation of bacterial lipid A into the cytosol of macrophages potentially mediates cell death. Notably, cell death induced by cytosolic bacteria was TLR4-independent. These results identify a novel cell death pathway induced by intracellular Gram-negative bacteria that may play a role in microbial-host interactions and inflammatory responses.

Vpu-mediated CD4 down-regulation and degradation is conserved among highly divergent SIV cpz strains

Human immunodeficiency virus type 1 (HIV-1) along with simian immunodeficiency viruses from chimpanzees (SIV cpz) and three species of Old World monkeys from the genus Cercopithecus have been shown to encode a Vpu protein. To date, the functional characterization of Vpu has been limited to a small number of subtype B and more recently subtype C Vpu proteins. Using a recently developed VpuEGFP reporter system, we have shown that the subtype B and C Vpus are capable of preventing CD4 from being expressed on the cell surface. Using the same reporter system, we report here on the expression and functional analysis of Vpu protein from four SIV cpz isolates (CAM13, ANT, TAN1, and GAB1). All four SIV Vpu fusion proteins were efficiently expressed and prevented CD4 expression on the cell surface and induced CD4 degradation. This was surprising as three of the SIV cpz Vpu fusion proteins had only one canonical casein kinase II (CK-II) site (CAM13, ANT, TAN1) while previous studies with laboratory adapted HXB2 had indicated that both CK-II sites were required for CD4 degradation. Both ANT and TAN1 Vpu sequences encoded five consecutive negatively charged amino acids residues following the only CKII site ( SAIEEDEE for ANT; SGVEEDEE for TAN1). We thus explored the possibility that this stretch of negatively charged amino acids might substitute for the lack of second CK-II site. Substitution of the aspartic acid at position 61 and glutamic acid at position 63 in the SIV cpz ANT Vpu within with lysine residues abolished the ability of this protein to down-modulate cell surface expression of CD4. Similarly, change of a serine to an alanine residue following the single consensus CK-II site of the CAM13 Vpu ( SGNESDGGEEE) abolished CD4-down-regulation, suggesting that this serine was phosphorylated in the absence of a canonical CK-II site. Our results indicate that the serine was required, suggesting that this serine was phosphorylated by CK-II or possibly another cellular kinase. Taken together, these results show for the first time that Vpu proteins from SIV cpz isolates, although quite diverse in sequence and predicted secondary structure from the HIV-1 subtype B protein, are capable of down-regulating CD4, which is one of the major functions of the HIV-1 protein.

CD4 Down-regulation by HIV-1 and Simian Immunodeficiency Virus (SIV) Nef Proteins Involves Both Internalization and Intracellular Retention Mechanisms

Among the pleiotropic effects of Nef proteins of HIV and simian immunodeficiency virus (SIV), down-modulation of cell surface expression of CD4 is a prominent phenotype. It has been presumed that Nef proteins accelerate endocytosis of CD4 by linking the receptor to the AP-2 clathrin adaptor. However, the related AP-1 and AP-3 adaptors have also been shown to interact with Nef, hinting at role(s) for these complexes in the intracellular retention of CD4. By using genetic inhibitors of endocytosis and small interfering RNA-induced knockdown of AP-2, we show that accelerated CD4 endocytosis is not a dominant mechanism of HIV-1 (NL4-3 strain) Nef in epithelial cells, T lymphocyte cell lines, or peripheral blood lymphocytes. Furthermore, we show that both the CD4 recycling from the plasma membrane and the nascent CD4 in transit to the plasma membrane are susceptible to intracellular retention in HIV-1 Nef-expressing cells. In contrast, AP-2-mediated enhanced endocytosis constitutes the predominant mechanism for SIV (MAC-239 strain) Nef-induced down-regulation of human CD4 in human cells.

CD44 Potentiates the Adherence of Metastatic Prostate and Breast Cancer Cells to Bone Marrow Endothelial Cells

The aim of this current study was to examine the significance of CD44 expression in mediating cancer cell adhesion to human bone marrow endothelial cell(s) (hBMEC). Differential CD44 expression on two metastatic prostate cancer cell lines, PC3 (CD44 ve) and DU145 (CD44 ve) and four breast cancer cell lines was confirmed by immunoblotting and immunocytochemistry. In cell adhesion assays, PC3 but not DU145 cells demonstrated a rapid adhesion to hBMECs. Treatment of PC3 cells with a neutralizing antibody against CD44 standard (CD44s) and CD44 splice variants decreased PC3 cell adhesion to hBMECs. Similarly, depletion of CD44 expression using RNA interference decreased the ability of PC3 cells and two CD44 ve breast cancer cell lines (MDA-MB-231 and MDA-MB-157) to bind FITC-conjugated hyaluronan (FITC-HA) and to adhere to hBMECs. In contrast, transfection of DU145 cells or the T47D and MCF-7 breast cancer cell lines to express CD44s increased cell surface binding of FITC-HA and cell adherence to hBMECs. Treatment of PC3 and MDA-MD-231 cells but not hBMECs with hyaluronidase attenuated cell adhesion, suggesting that cell surface expression of CD44 on prostate and breast cancer cells may promote the retention of a HA coat that facilitates their initial arrest on bone marrow endothelium.

Cross‐linking of neutrophil CD11b results in rapid cell surface expression of molecules required for antigen presentation and T‐cell activation

Recent studies suggest that neutrophils may play a role in antigen presentation. In support of this hypothesis it has been shown that these cells appear to contain cytoplasmic stores of molecules required for this function, i.e. major histocompatibility complex class II (DR) antigen, CD80 and CD86. In this study we have considered a mechanism for the translocation of these preformed molecules onto the cell surface which does not require active synthesis. Cross-linking of the Mac-1 molecule (CD18 + CD11b) was shown to result in rapid cell surface expression of CD80, CD86 and DR antigen on the surface of normal human peripheral blood neutrophils. A distinct subpopulation (approximately 20%) of neutrophils appeared to be enlarged and were found to express significantly elevated levels of these molecules on the cell surface following cross-linking of CD11b when compared with control cells. The level of expression of CD80, CD86 and DR antigen on these large cells was comparable to, and in some cases greater than, the levels found expressed on the surface of monocytes obtained from the same donors. In addition, these cytoplasmic molecules were shown by confocal laser microscopy and by immunoelectron microscopy to be located within secretory vesicles. Following rapid translocation onto the cell surface, CD80 and CD86 appeared to be colocalized within large clusters reminiscent of the supramolecular antigen clusters previously found on conventional antigen-presenting cells. These findings therefore lend further support for the hypothesis that neutrophils may have a role to play in antigen presentation and/or T-cell activation.

A2A Adenosine Receptor Induction Inhibits IFN-γ Production in Murine CD4+ T Cells

Incubation of purified C57BL/6 murine CD4(+) T lymphocytes with anti-CD3 mAb serves as a model of TCR-mediated activation and results in increased IFN-gamma production and cell surface expression of CD25 and CD69. We demonstrate here that signaling through the TCR causes a rapid (4-h) 5-fold increase in A(2A) adenosine receptor (AR) mRNA, which is correlated with a significant increase in the efficacy of A(2A)AR-mediated cAMP accumulation in these cells. A(2A)AR activation reduces TCR-mediated production of IFN-gamma by 98% with a potency order of 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl]prop-2-ynyl}cyclohexanecarboxylic acid methyl ester (ATL146e; EC(50) = 0.19 +/- 0.03 nM) > 4-{3-[6-amino-9-(5-cyclopropyl-carbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl]prop-2-ynyl}piperidine-1-carboxylic acid methyl ester (ATL313; 0.43 +/- 0.06 nM) > 5'-N-ethylcarboxamidoadenosine (3.5 +/- 0.77 nM) > 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680; 7.2 +/- 1.4 nM) >> N(6)-cyclohexyladenosine (110 +/- 33 nM) > 2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarboxamide (390 +/- 160 nM), similar to the potency order to compete for radioligand binding to the recombinant murine A(2A)AR but not the A(3)AR. The selective A(2A)AR antagonist, 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM241385), inhibits the effect of ATL146e with a pA(2) of 0.34 nM and also inhibits the effects of N(6)-cyclohexyl-adenosine and 2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarboxamide. In CD4(+) T cells derived from A(2A)AR(-/-) and A(2A)AR(+/-) mice, the IFN-gamma release response to ATL146e is reduced by 100 and 50%, respectively, indicative of a gene dose effect. The response of T cells to the phosphodiesterase inhibitor, 4-(3'-cyclopentyloxy-4'-methoxyphenyl)-2-pyrrolidone (rolipram), is not affected by A(2A)AR deletion. We conclude that the rapid induction of the A(2A)AR mRNA in T cells provides a mechanism for limiting T cell activation and secondary macrophage activation in inflamed tissues.

An aqueous stem bark extract ofMangifera indica (Vimang®) inhibits T cell proliferation and TNF-induced activation of nuclear transcription factor NF-κB

A commercial aqueous stem bark extract of Mangifera indica L. (Vimang) has been reported to have antiinflammatory, immunomodulatory and antioxidant activities. The molecular basis for these diverse properties is still unknown. This study shows that a stem bark extract of M. indica inhibits early and late events in T cell activation, including CD25 cell surface expression, progression to the S-phase of the cell cycle and proliferation in response to T cell receptor (TCR) stimulation. Moreover, the extract prevented TNFalpha-induced IkappaBalpha degradation and the binding of NF-kappaB to the DNA. This study may help to explain at the molecular level some of the biological activities attributed to the aqueous stem bark extract of M. indica (Vimang).