Role of patient and family engagement in quality improvement for pediatric surgery.

In recent decades, the role of quality improvement (QI) in pediatric surgery has grown substantially. Patient and family engagement can help to maximize the impact of QI by enhancing safety and patient outcomes. Yet, broader, systematic efforts to actively involve patients and families in QI initiatives remain a persistent gap in pediatric surgery. To address this gap, we propose an agenda centered on three key goals for future quality improvement efforts: (1) building partnerships with patients and their families; (2) expanding the use of patient-reported outcomes (PROs) and novel, cross-disciplinary research methods; and (3) engaging patients and families consistently across all stages of pediatric surgical care. Fulfilling this agenda will be essential in shifting our mindset to view QI as a collective that involves patients, families, clinicians, and payers in continuous, system-wide opportunities to evaluate and improve care. Actively listening to and collaborating with patients and families may also help renew our focus on narrowing the gap between current practice and the best possible practice for children undergoing surgery.

Comparison of tetraploid blastocyst microinjection of outbred Crl:CD1(ICR), hybrid B6D2F1/Tac, and inbred C57BL/6NTac embryos for generation of mice derived from embryonic stem cells.

Embryo electrofusion and tetraploid blastocyst microinjection is a modification of the traditional embryonic stem cell (ES cell)-based method to generate targeted mutant mice. Viability of tetraploid embryos is reportedly lower than with diploid embryos, with considerable interstrain variation. Here we assessed fetus and pup viability after ES cell microinjection of tetraploid blastocysts derived from outbred, hybrid, and inbred mice. Two-cell mouse embryos (C57BL/6NTac [B6], n = 788; B6D2F1/Tac [BDF1], n = 1871; Crl:CD1(ICR) [CD1], n = 1308) were electrofused; most resultant tetraploid blastocysts were injected with ES cells and surgically transferred into pseudopregnant recipient mice. Reproductive tracts were examined at midgestation for embryologic studies using B6 and BDF1 blastocysts; implantation sites and viable fetuses were counted. Pregnancies were carried to term for studies of targeted mutant mice using BDF1 and CD1 blastocysts, and pup yield was evaluated. Electrofusion rates of 2-cell embryos did not differ among B6, BDF1, and CD1 mice (overall mean, 92.8% +/- 5.4%). For embryologic studies, 244 B6 blastocysts were surgically transferred and 1 fetus was viable (0.41%), compared with 644 BDF1 blastocysts surgically transferred and 88 viable fetuses (13.7%). For targeted mutant mouse studies, 259 BDF1 blastocysts were surgically transferred yielding 10 pups (3.9%); 569 CD1 blastocysts yielded 44 pups (7.7%).

Dating violence prevention in middle school and high school youth.

TOPIC: Dating violence and interpersonal abuse among middle school and high school students. PURPOSE: To review the current literature and evaluate the need of conducting further study in order to create early interventions for the prevention of relationship abuse. SOURCES USED: Case report and review of the literature. CONCLUSIONS: Dating violence among middle school and high school youth must be addressed by screening risk and offering anticipatory guidance during each health maintenance visit in order to prevent victimization of youth in dating and attraction relationships.

Homing defect of cultured human hematopoietic cells in the NOD/SCID mouse is mediated by Fas/CD95.

OBJECTIVE: To determine the bone marrow homing efficiency (20 hours) of cultured compared to noncultured umbilical cord blood (UCB)-derived human hematopoietic cells in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse, and to explain the difference in homing between these populations. METHODS: Human UCB CD34+ cells were cultured for up to 5 days, reselected, and used for transplantation, phenotype analysis, and functional studies, including adhesion and trans-endothelial migration assays. Seeding of CD34+ cells was measured after labeling of cells with 111-Indium, while homing of colony-forming cells (CFC) and SCID-repopulating (SRC) cells was determined using functional assays. RESULTS: Short-term culture was associated with a decrease in the 20-hour homing of CD34+ cells, CFC, and SRC to the BM. Although cultured compared to noncultured cells showed increased expression and function (adhesion/migration) of several cell adhesion molecules described to play a role in homing and engraftment, culture also induced expression of Fas/CD95 and rendered cells more susceptible to apoptosis. Finally, we demonstrate that the level of Fas/CD95 on cultured cells was inversely related to the ability of CFC to home to the BM, and that the homing of cultured CFC could be restored by incubating cells prior to transplantation with Fas/CD95-blocking mAb ZB4. CONCLUSION: These data implicate Fas/CD95 in the homing defect of cultured human hematopoietic cells in the NOD/SCID transplant model and suggest that prevention of apoptosis may be an important strategy to improve engraftment of ex vivo-manipulated HSC in a clinical setting.

Antigenically distinct conformations of CXCR4.

The major human immunodeficiency virus type 1 (HIV-1) coreceptors are the chemokine receptors CCR5 and CXCR4. The patterns of expression of the major coreceptors and their use by HIV-1 strains largely explain viral tropism at the level of entry. However, while virus infection is dependent upon the presence of CD4 and an appropriate coreceptor, it can be influenced by a number of factors, including receptor concentration, affinity between envelope gp120 and receptors, and potentially receptor conformation. Indeed, seven-transmembrane domain receptors, such as CCR5, can exhibit conformational heterogeneity, although the significance for virus infection is uncertain. Using a panel of monoclonal antibodies (MAbs) to CXCR4, we found that CXCR4 on both primary and transformed T cells as well as on primary B cells exhibited considerable conformational heterogeneity. The conformational heterogeneity of CXCR4 explains the cell-type-dependent ability of CXCR4 antibodies to block chemotaxis to stromal cell-derived factor 1 alpha and to inhibit HIV-1 infection. In addition, the MAb most commonly used to study CXCR4 expression, 12G5, recognizes only a subpopulation of CXCR4 molecules on all primary cell types analyzed. As a result, CXCR4 concentrations on these important cell types have been underestimated to date. Finally, while the factors responsible for altering CXCR4 conformation are not known, we found that they do not involve CXCR4 glycosylation, sulfation of the N-terminal domain of CXCR4, or pertussis toxin-sensitive G-protein coupling. The fact that this important HIV-1 coreceptor exists in multiple conformations could have implications for viral entry and for the development of receptor antagonists.

Relationships between CD4 independence, neutralization sensitivity, and exposure of a CD4-induced epitope in a human immunodeficiency virus type 1 envelope protein.

A CD4-independent version of the X4 human immunodeficiency virus type 1 (HIV-1) HXBc2 envelope (Env) protein, termed 8x, mediates infection of CD4-negative, CXCR4-positive cells, binds directly to CXCR4 in the absence of CD4 due to constitutive exposure of a conserved coreceptor binding site in the gp120 subunit, and is more sensitive to antibody-mediated neutralization. To study the relationships between CD4 independence, neutralization sensitivity, and exposure of CD4-induced epitopes associated with the coreceptor binding site, we generated a large panel of Env mutants and chimeras between 8x and its CD4-dependent parent, HXBc2. We found that a frameshift mutation just proximal to the gp41 cytoplasmic domain in 8x Env was necessary but not sufficient for CD4 independence and led to increased exposure of the coreceptor binding site. In the presence of this altered cytoplasmic domain, single amino acid changes in either the 8x V3 (V320I) or V4/C4 (N386K) regions imparted CD4 independence, with other changes playing a modulatory role. The N386K mutation resulted in loss of an N-linked glycosylation site, but additional mutagenesis showed that it was the presence of a lysine rather than loss of the glycosylation site that contributed to CD4 independence. However, loss of the glycosylation site alone was sufficient to render Env neutralization sensitive, providing additional evidence that carbohydrate structures shield important neutralization determinants. Exposure of the CD4-induced epitope recognized by monoclonal antibody 17b and which overlaps the coreceptor binding site was highly sensitive to an R298K mutation at the base of the V3 loop and was often but not always associated with CD4 independence. Finally, while not all neutralization-sensitive Envs were CD4 independent, all CD4-independent Envs exhibited enhanced sensitivity to neutralization by HIV-1-positive human sera, indicating that the humoral immune response can exert strong selective pressure against the CD4-independent phenotype in vivo. Whether this can be used to advantage in designing more effective immunogens remains to be seen.

Expression and coreceptor function of APJ for primate immunodeficiency viruses.

APJ is a seven transmembrane domain G-protein-coupled receptor that functions as a coreceptor for some primate immunodeficiency virus strains. The in vivo significance of APJ coreceptor function remains to be elucidated, however, due to the lack of an antibody that can be used to assess APJ expression, and because of the absence of an antibody or ligand that can block APJ coreceptor activity. Therefore, we produced a specific monoclonal antibody (MAb 856) to APJ and found that it detected this receptor in FACS, immunofluorescence, and immunohistochemistry studies. MAb 856 also recognized APJ by Western blot, enabling us to determine that APJ is N-glycosylated. Using this antibody, we correlated APJ expression with coreceptor activity and found that APJ had coreceptor function even at low levels of expression. However, we found that APJ could not be detected by FACS analysis on cell lines commonly used to propagate primate lentiviruses, nor was it expressed on human PBMC cultured under a variety of conditions. We also found that some viral envelope proteins could mediate fusion with APJ-positive, CD4-negative cells, provided that CD4 was added in trans. These findings indicate that in some situations APJ use could render primary cell types susceptible to virus infection, although we have not found any evidence that this occurs. Finally, the peptide ligand for APJ, apelin-13, efficiently blocked APJ coreceptor activity.

Multiple nonfunctional alleles of CCR5 are frequent in various human populations.

CCR5 is the major coreceptor for macrophage-tropic strains of the human immunodeficiency virus type I (HIV-1). Homozygotes for a 32-base pair (bp) deletion in the coding sequence of the receptor (CCR5Delta32) were found to be highly resistant to viral infection, and CCR5 became, therefore, one of the paradigms illustrating the influence of genetic variability onto individual susceptibility to infectious and other diseases. We investigated the functional consequences of 16 other natural CCR5 mutations described in various human populations. We found that 10 of these variants are efficiently expressed at the cell surface, bind [(125)I]-MIP-1beta with affinities similar to wtCCR5, respond functionally to chemokines, and act as HIV-1 coreceptors. In addition to Delta32, six mutations were characterized by major alterations in their functional response to chemokines, as a consequence of intracellular trapping and poor expression at the cell surface (C101X, FS299), general or specific alteration of ligand binding affinities (C20S, C178R, A29S), or relative inability to mediate receptor activation (L55Q). A29S displayed an unusual pharmacological profile, binding and responding to MCP-2 similarly to wtCCR5, but exhibiting severely impaired binding and functional responses to MIP-1alpha, MIP-1beta, and RANTES. In addition to Delta32, only C101X was totally unable to mediate entry of HIV-1. The fact that nonfunctional CCR5 alleles are relatively frequent in various human populations reinforces the hypothesis of a selective pressure favoring these alleles. (Blood. 2000;96:1638-1645)

Expression and coreceptor activity of STRL33/Bonzo on primary peripheral blood lymphocytes.

CCR5 and CXCR4 are the major coreceptors that mediate human immunodeficiency virus 1 (HIV-1) infection, while most simian immunodeficiency virus (SIV) isolates use CCR5. A number of alternative coreceptors can also mediate infection of some virus strains in vitro, although little is known about their in vivo relevance. Therefore, we characterized the expression pattern and coreceptor activity of one of these alternative coreceptors, STRL33/Bonzo, using a newly developed monoclonal antibody. In addition to being highly expressed (approximately 1000-7000 STRL33 ABS [antibody binding sites]) on specific subsets of natural killer cells (CD3(-)/CD16(-/low)/CD56(+) and CD3(-)/CD16(low)/CD56(-)) and CD19(+) B lymphocytes (approximately 300-5000 STRL33 ABS), STRL33 was expressed at levels sufficient to support virus infection on freshly isolated, truly naive CD4(+)/CD45RA(+)/CD62L(+) cells (6000-11 000 ABS). STRL33 expression on peripheral blood mononuclear cells (PBMCs) was increased by mitogenic stimulation (OKT3/IL-2 [interleukin-2] had a greater effect than phytohemaglutinin (PHA)/IL-2), but it was dramatically decreased upon Ficoll purification. Infection of CCR5(-) human peripheral blood lymphocytes (PBLs) showed that 2 different SIV envelope (Env) proteins mediated entry into STRL33(+) cells. More importantly, the preferential infection of STRL33(+) cells in CCR5(-) PBLs by an R5/X4/STRL33 HIV-1 maternal isolate in the presence of a potent CXCR4 antagonist (AMD3100) suggests that STRL33 can be used as a coreceptor by HIV-1 on primary cells. Rhesus macaque (rh) STRL33 was used less efficiently than human STRL33 by the majority of SIV Env proteins tested despite similar levels of expression, thereby making it less likely that STRL33 is a relevant coreceptor in the rhesus macaque system. In summary, the expression pattern and coreceptor activity of STRL33 suggest its involvement in trafficking of tumor-infiltrating lymphocytes and indicate that STRL33 may be a relevant coreceptor in vivo.

Expression of multiple functional chemokine receptors and monocyte chemoattractant protein-1 in human neurons.

Functional chemokine receptors and chemokines are expressed by glial cells within the CNS, though relatively little is known about the patterns of neuronal chemokine receptor expression and function. We developed monoclonal antibodies to the CCR1, CCR2, CCR3, CCR6, CXCR2, CXCR3 and CXCR4 chemokine receptors to study their expression in human fetal neurons cultured from brain tissue as well as the clonally derived NT2.N human neuronal cell line (NTera 2/cl.D1). Specific monoclonal antibody labeling demonstrated expression of CCR2, CXCR2, CXCR3 and CXCR4 on neurons from both sources. Co-labeling studies revealed strong expression of CXCR3 and CXCR4 on both dendritic and axonal processes, with a weaker expression of CXCR2 and CCR2. Reverse transcriptase-polymerase chain reaction analysis of pure NT2.N neurons confirmed RNA expression for CCR2, CXCR2, CXCR3 and CXCR4. No changes in the neuronal labeling pattern of chemokine receptor expression were noted when NT2.N neurons were grown on a supporting layer of astrocytes, again consistent with similar patterns seen in primary human fetal brain cultures. Analysis of single-cell calcium transients revealed a robust response to stromal derived factor-1alpha (CXCR4) and melanocyte growth-stimulating activity (CXCR2), and variable response to monocyte chemoattractant protein-1 (CCR2) or interferon-gamma inducible protein-10 (CXCR3). Finally, we detected the release of monocyte chemoattractant protein-1 from pure cultures of NT2.N neurons, but not undifferentiated NT2 cells. These data indicate that individual neurons may not only co-express multiple functional chemokine receptors, but also that neurons themselves produce chemokines which may influence cellular function within the central nervous system.

Extracellular cysteines of CCR5 are required for chemokine binding, but dispensable for HIV-1 coreceptor activity.

CCR5 is the major coreceptor for macrophage-tropic human immunodeficiency virus type I (HIV-1). For most G-protein-coupled receptors that have been tested so far, the disulfide bonds linking together the extracellular loops (ECL) are required for maintaining the structural integrity necessary for ligand binding and receptor activation. A natural mutation affecting Cys20, which is thought to form a disulfide bond with Cys269, has been described in various human populations, although the consequences of this mutation for CCR5 function are not known. Using site-directed mutagenesis, we mutated the four extracellular cysteines of CCR5 singly or in combination to investigate their role in maintaining the structural conformation of the receptor, its ligand binding and signal transduction properties, and its ability to function as a viral coreceptor. Alanine substitution of any single Cys residue reduced surface expression levels by 40-70%. However, mutation of Cys101 or Cys178, predicted to link ECL1 and ECL2 of the receptor, abolished recognition of CCR5 by a panel of conformation sensitive anti-CCR5 antibodies. The effects of the mutations on receptor expression and conformation were partially temperature-sensitive, with partial restoration of receptor expression and conformation achieved by incubating cells at 32 degrees C. All cysteine mutants were unable to bind detectable levels of MIP-1beta, and did not respond functionally to CCR5 agonists. Surprisingly, all cysteine mutants did support infection by R5 strains of HIV, though at reduced levels. These results indicate that both disulfide bonds of CCR5 are necessary for maintaining the structural integrity of the receptor necessary for ligand binding and signaling. Env binding and the mechanisms of HIV entry appear much less sensitive to alterations of CCR5 conformation.

Quantification of CD4, CCR5, and CXCR4 levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages.

CCR5 and CXCR4 are the major HIV-1 coreceptors for R5 and X4 HIV-1 strains, respectively, and a threshold number of CD4 and chemokine receptor molecules is required to support virus infection. Therefore, we used a quantitative fluorescence-activated cell sorting assay to determine the number of CD4, CCR5, and CXCR4 antibody-binding sites (ABS) on various T cell lines, T cell subsets, peripheral blood dendritic cells (PBDC), and monocyte-derived macrophages by using four-color fluorescence-activated cell sorting analysis on fresh whole blood. Receptor levels varied dramatically among the various subsets examined and typically varied from 2- to 5-fold between individuals. CCR5 was expressed at much higher levels in CD4+/CD45RO+/CD62L-true memory cells compared with CD4+/CD45RO+/CD62L+ cells. Fresh PBDC had the highest number of CCR5 ABS among the leukocyte subsets examined but had few CXCR4 ABS, affording a strategy for sort-purifying PBDC. In vitro maturation of PBDC resulted in median 3- and 41-fold increases in CCR5 and CXCR4 ABS, respectively. We found that macrophage colony-stimulating factor caused the greatest up-regulation of both CCR5 and CXCR4 on macrophage maturation (from approximately 5,000 to approximately 50, 000 ABS) whereas granulocyte-macrophage colony-stimulating factor caused a marked decrease of CXCR4 (from approximately 5,000 ABS to <500) while up-regulating CCR5 expression (from approximately 5,000 to approximately 20,000 ABS). Absolute ABS for CD4 and the major HIV-1 coreceptors serve as a more quantitative measure of cell surface expression, and we propose that this be used for future studies looking at the modulation of CD4 or chemokine receptor expression by cytokines, HIV-1 infection, or receptor polymorphisms.

Epitope mapping of CCR5 reveals multiple conformational states and distinct but overlapping structures involved in chemokine and coreceptor function.

The chemokine receptor CCR5 is the major coreceptor for R5 human immunodeficiency virus type-1 strains. We mapped the epitope specificities of 18 CCR5 monoclonal antibodies (mAbs) to identify domains of CCR5 required for chemokine binding, gp120 binding, and for inducing conformational changes in Env that lead to membrane fusion. We identified mAbs that bound to N-terminal epitopes, extracellular loop 2 (ECL2) epitopes, and multidomain (MD) epitopes composed of more than one single extracellular domain. N-terminal mAbs recognized specific residues that span the first 13 amino acids of CCR5, while nearly all ECL2 mAbs recognized residues Tyr-184 to Phe-189. In addition, all MD epitopes involved ECL2, including at least residues Lys-171 and Glu-172. We found that ECL2-specific mAbs were more efficient than NH2- or MD-antibodies in blocking RANTES or MIP-1beta binding. By contrast, N-terminal mAbs blocked gp120-CCR5 binding more effectively than ECL2 mAbs. Surprisingly, ECL2 mAbs were more potent inhibitors of viral infection than N-terminal mAbs. Thus, the ability to block virus infection did not correlate with the ability to block gp120 binding. Together, these results imply that chemokines and Env bind to distinct but overlapping sites in CCR5, and suggest that the N-terminal domain of CCR5 is more important for gp120 binding while the extracellular loops are more important for inducing conformational changes in Env that lead to membrane fusion and virus infection. Measurements of individual antibody affinities coupled with kinetic analysis of equilibrium binding states also suggested that there are multiple conformational states of CCR5. A previously described mAb, 2D7, was unique in its ability to effectively block both chemokine and Env binding as well as coreceptor activity. 2D7 bound to a unique antigenic determinant in the first half of ECL2 and recognized a far greater proportion of cell surface CCR5 molecules than the other mAbs examined. Thus, the epitope recognized by 2D7 may represent a particularly attractive target for CCR5 antagonists.

Use of GPR1, GPR15, and STRL33 as coreceptors by diverse human immunodeficiency virus type 1 and simian immunodeficiency virus envelope proteins.

Human and simian immunodeficiency viruses (HIV and SIV, respectively) use chemokine receptors as coreceptors along with CD4 to mediate viral entry. Several orphan receptors, including GPR1, GPR15, and STRL33, can also serve as coreceptors for a more limited number of HIV and SIV isolates. We investigated whether these orphan receptors could function as efficient coreceptors for a diverse group of HIV and SIV envelopes (Envs) in comparison with the principal coreceptors CCR5 and CXCR4. We found that a limited number of HIV-1 isolates could mediate inefficient cell-cell fusion with the orphan receptors relative to CCR5 and CXCR4; however, none of the orphan receptors tested could support pseudotype virus infection despite robust infection via CCR5 or CXCR4. All except one of the SIV Envs tested mediated some degree of cell-cell fusion and pseudotype infection, with target cells expressing at least one of these orphan receptors, although CCR5 proved to be the most efficient coreceptor for infection. Only one SIV Env protein, BK28, could mediate infection using GPR1 as a coreceptor, albeit much less efficiently than with CCR5. In addition, use of these coreceptors did not correlate with the published tropism of the SIV clones and was strictly CD4 dependent for both SIV and HIV. We also examined the expression of these molecules in cell lines and primary cells widely used for virus propagation and as targets for infection. All cells examined expressed STRL33, a more limited number expressed GPR15, and GPR1 was much more restricted in its expression pattern. Taken together, our results indicate that GPR15 and STRL33 are rarely used by HIV-1 but are more frequently used by SIV strains, although not in a manner that correlates with SIV tropism.

An orphan seven-transmembrane domain receptor expressed widely in the brain functions as a coreceptor for human immunodeficiency virus type 1 and simian immunodeficiency virus.

Both CD4 and an appropriate coreceptor are necessary for infection of cells by human immunodeficiency virus type 1 (HIV-1) and most strains of HIV-2. The chemokine receptors CCR5 and CXCR4 are the major HIV-1 coreceptors, although some virus strains can also utilize alternative coreceptors such as CCR3 to infect cells. In contrast, most if not all simian immunodeficiency virus (SIV) strains use CCR5 as a coreceptor, and many SIV strains can use CCR5 independently of CD4. In addition, several orphan seven-transmembrane receptors which can serve as HIV-1 and SIV coreceptors have been identified. Here we report that APJ, an orphan seven-transmembrane domain receptor with homology to the angiotensin receptor family, functions as a coreceptor for a number of HIV-1 and SIV strains. APJ was expressed widely in the human brain and in NT2N neurons. APJ transcripts were also detected by reverse transcription-PCR in the CD4-positive T-cell line C8166, but not in peripheral blood leukocytes, microglia, phytohemagglutinin (PHA)- or PHA/interleukin-2-stimulated peripheral blood mononuclear cells, monocytes, or monocyte-derived macrophages. The widespread distribution of APJ in the central nervous system coupled with its use as a coreceptor by some HIV-1 strains indicates that it may play a role in neuropathogenesis.

CXCR4 sequences involved in coreceptor determination of human immunodeficiency virus type-1 tropism. Unmasking of activity with M-tropic Env glycoproteins.

The interaction of human immunodeficiency virus type 1 (HIV-1) with CD4 and one of a cadre of chemokine receptors triggers conformational changes in the HIV-1 envelope (Env) glycoprotein that lead to membrane fusion. The coreceptor activity of the second extracellular loop of CXCR4, which is restricted to dual tropic and T-tropic strains, was insensitive to the removal of charged residues either singly or in combinations by alanine scanning mutagenesis or to the conversion of acidic residues to lysine. Conversion of Asp-187 to a neutral residue exclusively unmasked activity with M-tropic Env in fusion and infection experiments. Insertion of the D187V mutation into chimeras containing extracellular loop 2 of CXCR4 in a CXCR2 framework also resulted in the acquisition of M-tropic coreceptor activity. The independence of CXCR4 coreceptor activity from charged residues and the extension of its repertoire by removing Asp-187 suggest that this interaction is not electrostatic and that coreceptors have the potential to be utilized by a spectrum of Env, which may be masked by charged amino acids in extracellular domains. These findings indicate that the primary structural determinants of coreceptors that program reactivity with M-, dual, and T-tropic Env are surprisingly subtle and that relatively insignificant changes in CXCR4 can dramatically alter utilization by Env of varying tropism.

ChemR23, a putative chemoattractant receptor, is expressed in monocyte-derived dendritic cells and macrophages and is a coreceptor for SIV and some primary HIV-1 strains.

Leukocyte chemoattractants act through a rapidly growing subfamily of G protein-coupled receptors. We report the cloning of a novel human gene encoding an orphan receptor (ChemR23) related to the C3a, C5a and formyl Met-Leu-Phe receptors, and more distantly to the subfamilies of chemokine receptors. ChemR23 transcripts were found to be abundant in monocyte-derived dendritic cells and macrophages, treated or not with LPS. Low expression could also be detected by reverse transcription-PCR in CD4+ T lymphocytes. The gene encoding ChemR23 was assigned by radiation hybrid mapping to the q21.2-21.3 region of human chromosome 12, outside the gene clusters identified so far for chemoattractant receptors. Given the increasing number of chemoattractant receptors used by HIV-1, HIV-2 and SIV as coreceptors, ChemR23 was tested in fusion assays for potential coreceptor activity by a range of viral strains. None of the tested HIV-2 strains made use of ChemR23 as a coreceptor, but several SIV strains (SIVmac316, SIVmac239, SIVmacl7E-Fr and SIVsm62A), as well as a primary HIV-1 strain (92UG024-2) used it efficiently. ChemR23 therefore appears as a coreceptor for immunodeficiency viruses that does not belong to the chemokine receptor family. It is also a putative chemoattractant receptor relatively specific for antigen-presenting cells, and it could play an important role in the recruitment or trafficking of these cell populations. Future work will be required to identify the ligand(s) of this new G protein-coupled receptor and to define its precise role in the physiology of dendritic cells and macrophages.

Chemokine receptor utilization by human immunodeficiency virus type 1 isolates that replicate in microglia.

The role of human immunodeficiency virus (HIV) strain variability remains a key unanswered question in HIV dementia, a condition affecting around 20% of infected individuals. Several groups have shown that viruses within the central nervous system (CNS) of infected patients constitute an independently evolving subset of HIV strains. A potential explanation for the replication and sequestration of viruses within the CNS is the preferential use of certain chemokine receptors present in microglia. To determine the role of specific chemokine coreceptors in infection of adult microglial cells, we obtained a small panel of HIV type 1 brain isolates, as well as other HIV strains that replicate well in cultured microglial cells. These viruses and molecular clones of their envelopes were used in infections, in cell-to-cell fusion assays, and in the construction of pseudotypes. The results demonstrate the predominant use of CCR5, at least among the major coreceptors, with minor use of CCR3 and CXCR4 by some of the isolates or their envelope clones.

Utilization of chemokine receptors, orphan receptors, and herpesvirus-encoded receptors by diverse human and simian immunodeficiency viruses.

Human immunodeficiency virus type 1 (HIV-1) requires both CD4 and a coreceptor to infect cells. Macrophage-tropic (M-tropic) HIV-1 strains utilize the chemokine receptor CCR5 in conjunction with CD4 to infect cells, while T-cell-tropic (T-tropic) strains generally utilize CXCR4 as a coreceptor. Some viruses can use both CCR5 and CXCR4 for virus entry (i.e., are dual-tropic), while other chemokine receptors can be used by a subset of virus strains. Due to the genetic diversity of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) and the potential for chemokine receptors other than CCR5 or CXCR4 to influence viral pathogenesis, we tested a panel of 28 HIV-1, HIV-2, and SIV envelope (Env) proteins for the ability to utilize chemokine receptors, orphan receptors, and herpesvirus-encoded chemokine receptor homologs by membrane fusion and virus infection assays. While all Env proteins used either CCR5 or CXCR4 or both, several also used CCR3. Use of CCR3 was strongly dependent on its surface expression levels, with a larger number of viral Env proteins being able to utilize this coreceptor at the higher levels of surface expression. ChemR1, an orphan receptor recently shown to bind the CC chemokine I309 (and therefore renamed CCR8), was expressed in monocyte and lymphocyte cell populations and functioned as a coreceptor for diverse HIV-1, HIV-2, and SIV Env proteins. Use of ChemR1/CCR8 by SIV strains was dependent in part on V3 loop sequences. The orphan receptor V28 supported Env-mediated cell-cell fusion by four T- or dual-tropic HIV-1 and HIV-2 strains. Three additional orphan receptors failed to function for any of the 28 Env proteins tested. Likewise, five of six seven-transmembrane-domain receptors encoded by herpesviruses did not support Env-mediated membrane fusion. However, the chemokine receptor US28, encoded by cytomegalovirus, did support inefficient infection by two HIV-1 strains. These findings indicate that additional chemokine receptors can function as HIV and SIV coreceptors and that surface expression levels can strongly influence coreceptor use.

A small-molecule inhibitor directed against the chemokine receptor CXCR4 prevents its use as an HIV-1 coreceptor.

The chemokine receptor CXCR4 is the major coreceptor used for cellular entry by T cell- tropic human immunodeficiency virus (HIV)-1 strains, whereas CCR5 is used by macrophage (M)-tropic strains. Here we show that a small-molecule inhibitor, ALX40-4C, inhibits HIV-1 envelope (Env)-mediated membrane fusion and viral entry directly at the level of coreceptor use. ALX40-4C inhibited HIV-1 use of the coreceptor CXCR4 by T- and dual-tropic HIV-1 strains, whereas use of CCR5 by M- and dual-tropic strains was not inhibited. Dual-tropic viruses capable of using both CXCR4 and CCR5 were inhibited by ALX40-4C only when cells expressed CXCR4 alone. ALX40-4C blocked stromal-derived factor (SDF)-1alpha-mediated activation of CXCR4 and binding of the monoclonal antibody 12G5 to cells expressing CXCR4. Overlap of the ALX40-4C binding site with that of 12G5 and SDF implicates direct blocking of Env interactions, rather than downregulation of receptor, as the mechanism of inhibition. Thus, ALX40-4C represents a small-molecule inhibitor of HIV-1 infection that acts directly against a chemokine receptor at the level of Env-mediated membrane fusion.