Analytical and biological considerations in the measurement of cell-associated CCR5 and CXCR4 mRNA and protein.

The accurate measurement of T cell-associated CC chemokine receptor type 5 (CCR5) and CXC chemokine receptor type 4 (CXCR4) expression, including expression of CCR5 and CXCR4 mRNA as an immune measure of immunologic response to highly active antiretroviral therapy (HAART) and newer agents, including entry inhibitors, is essential. Previous studies have reported alterations in lymphocyte cell membrane CCR5 expression that were related to blood collection and cell separation media. Clinical trials often require the transport of specimens to central laboratories for evaluation, resulting in significant time delays between specimen procurement and analysis. This study shows that CCR5 expression on naïve and memory T cells is influenced by blood collection media and specimen age. Peripheral blood collected in Streck Vacutainer tubes containing a cell stabilizer and fixative was found to improve detection of CCR5 expression compared to specimens collected in K2 EDTA anticoagulant. The selection of flow cytometry gating strategies for the identification of naïve and memory T-helper cells can also significantly influence the sensitivity of detection of CCR5 expression. Procedural methods are described that allow for the optimal measurement of naïve and memory T-helper cell CCR5 and CXCR4 expression as well as the quantitation of CCR5 and CXCR4 mRNA.

Cryopreservation decreases receptor PD-1 and ligand PD-L1 coinhibitory expression on peripheral blood mononuclear cell-derived T cells and monocytes.

The B7-CD28 immunoglobulin superfamily of costimulatory and coinhibitory ligands and their cell receptors play a critical role in modulating immune responses. Imbalances in these immune regulatory signals occur in pathological conditions characterized by chronic antigenic stimulation. Clinical studies often rely on the use of cryopreserved peripheral blood mononuclear cells (PBMC) to evaluate cellular immune responses. The impact of cryopreservation on these coinhibitory ligands and their cell receptors is unknown. In our studies, cryopreservation significantly reduced the expression of both PD-1 and PD-L1 on PBMC-derived CD3+/CD8+ T cells and CD45+/CD14+ monocytes obtained from adult control subjects. Blockade of PD-1, PD-L1, and PD-L2 using both freshly isolated and cryopreserved PBMC led to higher levels of phytohemagglutinin (PHA) and Candida-induced gamma interferon (IFN-gamma), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-alpha) with no effect on IL-10 production. Coinhibitory signaling blockade of freshly isolated, PHA-stimulated PBMC from normal adult controls and human immunodeficiency virus (HIV)-infected subjects led to increased production of IL-4 and IL-5. Candida-stimulated PBMC preferentially induced IFN-gamma and TNF-alpha production, with reduced production of IL-2 and IL-10. This is in contrast to high levels of IFN-gamma, IL-2, and TNF-alpha production with PHA-stimulated cells. The effects of coinhibitory blockade on PHA and Candida-induced lymphoproliferation were varied, with freshly isolated PBMC from adult control subjects and HIV-infected patients yielding higher levels of lymphoproliferation in response to PD-1/PD-L1 blockade. Immune function studies employing cryopreserved cells may lead to increased T-cell effector cytolytic and regulatory immune responses.

Full-length and truncated neurokinin-1 receptor expression and function during monocyte/macrophage differentiation.

The substance P (SP)-preferring receptor neurokinin-1 receptor (NK-1R) has two forms: a full-length receptor consisting of 407 aa and a truncated receptor consisting of 311 aa. These two receptors differ in the length of the C terminus of NK-1R. We studied the undifferentiated and phorbol myristate acetate (PMA)-differentiated human monocyte/macrophage cell line THP-1 to investigate the expression and function of NK-1R. The expression of full-length and truncated NK-1R in this cell line was determined by using real-time PCR and immunofluorescence staining. Undifferentiated THP-1 cells expressed only truncated NK-1R. The differentiation of THP-1 cells with PMA to a macrophage-like phenotype resulted in the expression of full-length NK-1R, which was functionally accompanied by an SP (10(-6) M)-induced Ca2+ increase. In contrast, the addition of SP (10(-6) M) did not trigger Ca2+ response in undifferentiated THP-1 cells; however, SP did enhance the CCR5-preferring ligand RANTES (CCL5)-mediated Ca2+ increase. When a plasmid containing the full-length NK-1R was introduced into undifferentiated THP-1 cells, exposure to SP triggered Ca2+ increase, demonstrating that the full-length NK-1R is required for SP-induced Ca2+ increase. The NK-1R antagonist aprepitant (Emend, Merck) inhibited both the SP-induced Ca2+ increase in PMA-differentiated THP-1 cells and the SP priming effect on the CCL5-mediated Ca2+ increase, indicating that these effects are mediated through the full-length and truncated NK-1R, respectively. Taken together, these observations demonstrate that there are unique characteristics of NK-1R expression and NK-1R-mediated signaling between undifferentiated THP-1 cells and THP-1 cells differentiated to the macrophage phenotype.

Substance P enhances HIV-1 replication in latently infected human immune cells.

Substance P (SP) is a potent modulator of neuroimmunoregulation. SP receptors are present on human monocytes and T lymphocytes, and SP alters the function of these immune cells. We investigated the effects of SP on HIV-1 replication in latently infected human immune cells. SP significantly enhanced HIV-1 replication in the latently infected promonocytic cell line (U1) and T lymphocyte line (ACH-2) stimulated with tumor necrosis factor (TNF-alpha). When added to these cells in combination with TNF-alpha, SP also enhanced HIV-1 gag gene expression in U1 and ACH-2 cells. This stimulatory effect of SP was associated with the activation of HIV-LTR (long terminal repeat) driven chloramphenicol acetyltransferase (CAT) gene expression, and could be blocked by pretreatment of U1 and ACH-2 cells with an SP receptor antagonist RP-67,580, indicating specific SP receptor-mediated regulation. Furthermore, the addition of SP to the cultures of latently infected peripheral blood mononuclear cells isolated from HIV-1-infected patients enhanced HIV-1 gag gene expression. Thus, SP may play a potentially important role as a positive regulator of HIV-1 replication in latently infected monocytes and lymphocytes. These observations may have significant implications toward understanding the role of neuropeptide SP in the immunopathogenesis of HIV-1 infection and AIDS.

Elevated substance P levels in HIV-infected men.

The neuropeptide, substance P, is a potent modulator of neuroimmunoregulation. Substance P and its receptor modulate HIV infection. HIV-seropositive men had significantly higher plasma substance P levels compared with uninfected controls, which were associated with decreased CD16 and CD56 natural killer (NK) cell populations. The changes in plasma substance P levels and decreases in NK subsets did not correlate with CD4 cell levels, but a diurnal pattern was suggested for substance P. The balance between substance P expression and functions of immune cells may be important in the immunopathogenesis of HIV infection.

Interactions between the nervous and immune systems.

Substantial morphologic and functional evidence exists that supports the reciprocal interactions that occur between the nervous and immune systems. The nervous and immune systems have been increasingly found to use a common chemical language in the form of neuropeptides, cytokines, and hormones. Sophisticated immunologic techniques such as the identification and detection of immune cell surface markers enable researchers to determine the origin and activity of diverse cells in the blood and central nervous system. These techniques have elucidated the activity of immune cells in the central nervous system (CNS) that was previously thought to be privileged from immune surveillance in the presence of an intact blood brain barrier. Immune cells in the CNS play a central role in several degenerative diseases such as Alzheimer's disease, Huntington's disease, Multiple sclerosis, AIDS dementia complex, and nerve destruction associated with trauma. Immune cells also play a role in demyelinating peripheral nerve disorders. Cytokines and neuropeptides secreted by peripheral immune cells have profound effects on behavior that is mediated by the CNS. The close integration between immune and nervous system responses is being increasingly recognized in physiologic and pathologic conditions.

HIV-1 infection of placental cord blood monocyte-derived dendritic cells.

Dendritic cells (DC), the most potent antigen-presenting cells (APC), have been implicated as the initial targets of HIV infection in skin and mucosal surfaces. DC can be generated in vitro from blood-isolated CD14(+) monocytes or CD34(+) hematopoietic progenitor cells in the presence of various cytokines. In this study, we investigated whether monocytes obtained from placental cord blood are capable of differentiation into dendritic cells when cultured with a combination of cytokines - granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and tumor necrosis factor-alpha (TNF-alpha). We then examined HIV infection, HIV receptor (CD4, CCR5) expression, and beta-chemokine [macrophage inflammatory protein-1alpha and -1beta (MIP-1alpha, MIP-1beta)] production by placental cord monocyte-derived dendritic cells (MDDC) as compared to that of autologous cord monocyte-derived macrophages (MDM). Monocytes isolated from placental cord blood differentiate into DC after 7 days in culture with the mixture of cytokines, as demonstrated by development of characteristic DC morphology, loss of CD14 expression, and gain of CD83, a marker for mature DC. Mature cord MDDC had significantly lower susceptibility to M-tropic ADA (CCR5-dependent) envelope-pseudotyped HIV infection in comparison to autologous placental cord MDM, whereas there was no significant difference in virus replication in cord MDDC and MDM infected with murine leukemia virus envelope-pseudotyped HIV (HIV receptor-independent). This limited susceptibility of cord MDDC to M-tropic HIV infection may be due to lower expression of CD4 and CCR5 on the cell membrane and higher production of MIP-1alpha and MIP-1beta. These data provide important information toward our understanding of the biological properties of cord MDDC in relation to HIV infection.

Immunology and the REACH study: HIV immunology and preliminary findings. Reaching for Excellence in Adolescent Care and Health.

This review paper presents the immunology findings in human immunodeficiency virus (HIV) infected and uninfected youth in the Reaching for Excellence in Adolescent Care and Health (REACH) Project within the context of basic and HIV immunology concepts. Methods employed in the study for specimen collection, management, and laboratory analysis are presented. This paper reviews published analyses of cross-sectional data; longitudinal analyses are underway. These preliminary data extend the work of others in demonstrating the potential for substantial thymic reserve in youth. This finding in HIV infected adolescents has implications for a fuller response to antiretroviral or immune-based therapies compared to that seen in adults. Dysregulation in mucosal immunity may appear before systemic HIV effects are seen and requires attention particularly to screening and treatment of genital co-infections. REACH has demonstrated gender differences in immunologic measures irrespective of HIV infection status.

Methadone may promote HIV replication: study.

Investigators at the University of Pennsylvania School of Medicine have found that HIV replication in vitro is promoted by the opiate methadone. Although this research is a long way from concluding that injection-drug-using HIV patients should avoid methadone treatment, it does raise questions that will need to be answered through further studies.

Natural killer cell enumeration and function in HIV-infected and high-risk uninfected adolescents.

This is the first report of natural killer cell enumeration and function in HIV-infected and high-risk uninfected adolescents. We examined the association of demographic characteristics of this cohort with three outcomes: CD16+ cell absolute count, lytic units per peripheral blood mononuclear cell (PBMC), and lytic units per natural killer (NK) cell. We also examined the association of CD4, CD38, and antiretroviral therapy (ART) use with these outcomes in the subset of HIV-infected adolescents. Adolescents participating in an on-going longitudinal study (the REACH study) were sampled for CD16+ cell count and NK function. This cross-sectional analysis was performed on 412 subjects with NK cell data available. HIV-positive males had higher numbers of CD3-/CD16+/CD56+ NK cells than HIV-positive females. However, for the HIV-negative subjects, we did not observe a gender-related effect for absolute NK cell numbers. Gender, however, was a significant covariate for the analysis, using lytic units per PBMC as the unit of measurement, with males showing higher values than females. Age was not a predictive covariate for any of the three assessments of NK cell number and function examined. Our observations concerning the HIV-positive individuals indicate that reduced CD4+ T cell counts were associated with decreased circulating CD3-/CD16+/CD56+ NK cells. We also observed an association between elevation of CD8+/CD38+/DR+ lymphocytes and lower NK lytic units per PBMC. The results of our multivariate models indicate that there is a reduced number of NK cells and reduced lytic units per PBMC in patients receiving single or multidrug antiretroviral therapy. There are changes in circulating NK cell number and function in HIV-infected adolescents, in comparison with high-risk HIV-negative adolescents. The data suggest that these changes may occur early in the course of HIV disease but that quantitative changes continue to occur with advancing depletion of the CD4+ T cell pool.

Postpartum smoking behaviors and immune response in mothers of term and preterm infants.

Infants exposed to secondhand smoke, especially preterm infants with a very low birth weight (VLBW), have an increased risk for developing health problems. Smoking has been associated with numerous health problems in mothers and may reduce immune functioning as well. The purposes of this study were to examine smoking in postpartum mothers of term and preterm infants and to examine the relationship between smoking and immune status. Peripheral blood was drawn on 142 women at four data-collection points and tested for cotinine, immune cell phenotypes, and immune functioning. Overall, 39% of the participants smoked in the postpartum period, but 49% of mothers who delivered preterm infants smoked compared to only 28% of mothers who delivered term infants. There was no difference in cotinine levels between the smokers in both groups of postpartum mothers, nor was smoking related to immune phenotypes or immune function. Given the documented health risks to the mother and infant and the significant number of women who continue to smoke in the postpartum period, it is imperative that health care providers continue to assess smoking status and provide smoking-cessation counseling at every encounter.

Substance P antagonist (CP-96,345) inhibits HIV-1 replication in human mononuclear phagocytes.

Substance P (SP) is a potent modulator of neuroimmunoregulation. We recently reported that human immune cells express SP and its receptor. We have now investigated the possible role that SP and its receptor plays in HIV infection of human mononuclear phagocytes. SP enhanced HIV replication in human blood-isolated mononuclear phagocytes, whereas the nonpeptide SP antagonist (CP-96,345) potently inhibited HIV infectivity of these cells in a concentration-dependent fashion. CP-96,345 prevented the formation of typical giant syncytia induced by HIV Bal strain replication in these cells. This inhibitory effect of CP-96,345 was because of the antagonism of neurokinin-1 receptor, a primary SP receptor. Both CP-96,345 and anti-SP antibody inhibited SP-enhanced HIV replication in monocyte-derived macrophages (MDM). Among HIV strains tested (both prototype and primary isolates), only the R5 strains (Bal, ADA, BL-6, and CSF-6) that use the CCR5 coreceptor for entry into MDM were significantly inhibited by CP-96,345; in contrast, the X4 strain (UG024), which uses CXCR4 as its coreceptor, was not inhibited. In addition, the M-tropic ADA (CCR5-dependent)-pseudotyped HIV infection of MDM was markedly inhibited by CP-96,345, whereas murine leukemia virus-pseudotyped HIV was not affected, indicating that the major effect of CP-96,345 is regulated by Env-determined early events in HIV infection of MDM. CP-96,345 significantly down-regulated CCR5 expression in MDM at both protein and mRNA levels. Thus, SP-neurokinin-1 receptor interaction may play an important role in the regulation of CCR5 expression in MDM, affecting the R5 HIV strain infection of MDM.

Performance of antigens used in detecting delayed-type hypersensitivity in adolescents infected with the human immunodeficiency virus.

We examined the performance of delayed-type hypersensitivity (DTH) antigens employing a new Candida albicans product in a human immunodeficiency virus (HIV)-infected and nonanergic adolescent population. Diameters of induration (in millimeters) for three intradermally applied antigens (C. albicans, tetanus toxoid, and mumps) were compared in a population of HIV-infected 12 to 18 year olds at study entry in a national multicenter study of HIV disease progression. CD4+ T-cell counts were measured in quality-controlled laboratories. The influence of past immunization, gender, and clinical status on antigen reactivity was evaluated with contingency table comparisons and relative risk estimation. Nearly one-half of the 123 eligible subjects were untreated, and almost three-quarters were early in HIV disease by clinical indicators. There was no statistically significant difference in reactivity by past immunization status. Candida antigen (CASTA; Greer Laboratories) evoked DTH response in a significantly higher number of males and females at every level of induration (largest P value, 0.049 for male comparisons; all P values, <0.001 for females) and in subjects with early and intermediate HIV disease at every level of induration (all P values, <0.0001) than either tetanus or mumps antigens. No two-antigen combination was as useful as all three antigens across either gender or clinical categories, although candida and tetanus was the most useful two-antigen combination at indurations of <3 mm. The superior performance of a new C. albicans antigen may extend the utility of DTH assessment in monitoring immune function.

Recombinant human granulocyte colony-stimulating factor therapy for patients with neutropenia and/or neutrophil dysfunction secondary to glycogen storage disease type 1b.

The purpose of this study was to evaluate the efficacy and toxicity of recombinant human granulocyte colony-stimulating factor (rhG-CSF) therapy in patients with neutropenia and/or neutrophil dysfunction secondary to glycogen storage disease (GSD) type 1b. Thirteen patients with neutropenia and/or neutrophil dysfunction secondary to GSD type 1b were treated with rhG-CSF. The effects of therapy on neutrophil numbers and in vitro neutrophil function and on bone marrow cellularity and morphology were studied. The clinical status of the patients and the occurrence of adverse events associated with rhG-CSF use were monitored. Use of rhG-CSF therapy was associated with a significant increase in circulating neutrophil numbers (P <. 01) and an improvement in neutrophil function as assessed in vitro. In addition, rhG-CSF therapy produced a significant increase in marrow cellularity and an increase in myeloid:erythroid (M:E) ratio, indicating stimulation of granulopoeisis. No adverse effects on marrow function were noted; in particular, no myelodysplasia or marrow exhaustion was seen. Use of rhG-CSF therapy was associated with objective and subjective improvements in infection-related morbidity. The therapy was well tolerated, although all patients developed splenomegaly, and 5 patients developed mild hypersplenism that did not require any specific treatment. rhG-CSF therapy is efficacious in the management of neutropenia and neutrophil dysfunction associated with GSD type 1b. Patients on this therapy need to be monitored for hypersplenism. Continued follow-up will be necessary to confirm long-term safety; however, no significant short-term toxicity was noted.

Morphine Up-regulates expression of substance P and its receptor in human blood mononuclear phagocytes and lymphocytes.

In vitro and in vivo studies have indicated that there is an important relationship between morphine and neuropeptide substance P (SP). We therefore investigated the interaction of morphine and cultured human immune cells on the expression of SP, a neuropeptide which we have recently demonstrated to be produced by human monocytes and lymphocytes. Morphine up-regulated SP production in human mononuclear phagocytes and lymphocytes at both the mRNA and the protein level. In addition, morphine induced SP receptor (NK-1R) expression in human lymphocytes. The specific morphine receptor antagonist (naltrexone) blocked morphine-induced SP expression in human mononuclear phagocytes, supporting the concept of authentic morphine receptor-mediated regulation. Since SP modulates neurogenic inflammation and immunologic events, these data suggest that morphine-induced SP expression in cells of the immune system may be of importance in the pathogenesis of immune-mediated diseases, including neuroimmunologic diseases and AIDS.

Detection of substance P and its receptor in human fetal microglia.

Substance P, the most abundant neurokinin in the CNS, is a major modulator of the immune system. We have examined the gene expression of substance P and its receptor in human fetal brain microglia. Using reverse transcription-polymerase chain reaction and Southern blotting assay, the four isoforms of preprotachykinin-A gene transcripts (alpha, beta, gamma and delta) were detected in the microglia. The human fetal microglia produced significantly higher levels of endogenous substance P protein (640-850 pg/10(6) cells) than did human peripheral blood monocyte-derived macrophages (25-50 pg/10(6) cells), as determined by an enzyme immunoassay. Using immunohistochemical staining with an anti-substance P antibody, cell membrane substance P immunoreactivity was observed. In addition, we identified the presence of messenger RNA for neurokinin-1 receptor, a primary receptor for substance P in human fetal microglia.From these data, we propose that substance P and its receptor are biologically involved in regulating the functions of microglia, and potentially play an important role in host defense of the central nervous system.

The prevalence of anergy in human immunodeficiency virus-infected adolescents and the association of delayed-type hypersensitivity with subject characteristics.

PURPOSE: To examine the prevalence of anergy in HIV-infected adolescents and factors associated with its occurrence. METHODS: Anergy was defined as less than 2mm induration to each of three intradermally applied antigens (Candida albicans, tetanus toxoid, and mumps) between 24 and 96 hours in a population of HIV-infected adolescents aged 12-18 at entry in a national multicenter study of HIV disease progression. CD4(+) T-cell counts and plasma HIV-1 RNA were measured in quality controlled laboratories. Factors associated with the probability of anergy were examined with contingency table comparisons, tree-structured classification, and logistic regression analyses. RESULTS: Overall prevalence of anergy in this clinic-based population of 167 was 11% [7% in males and 12% in females (p = 0.57)]. The sole significant predictor of anergy was decreased CD4(+) T-cell count (p = 0.005). CONCLUSION: The prevalence of anergy is low in this HIV-infected population compared to older infected cohorts. The occurrence of differential rates of anergy in particular age and sex groupings that may be related to intrinsic immunologic differences requires further study.

Measurement of cytokine secretion, intracellular protein expression, and mRNA in resting and stimulated peripheral blood mononuclear cells.

Quantitation of cytokine production is a valuable adjunct to standard immunologic assays in defining several pathologic processes. Nevertheless, there is little agreement about which tissues should be assayed, which type of assay should be performed, and which stimulation protocol should be used. As these types of assays enter the clinical arena, there is need for standardization. There is also a need to maximize the amount of information which may be derived from a single sample. We compared secreted interleukin 4 (IL-4), IL-2, IL-6, tumor necrosis factor alpha (TNF-alpha), and gamma interferon proteins as measured by enzyme-linked immunosorbent assay with intracellular cytokine production (IL-2 and gamma interferon) as detected by flow cytometry and quantitative competitive PCR for IL-2, IL-4, TNF-alpha, and gamma interferon mRNA and cDNA. Results from unstimulated cells and cells stimulated with phorbol myristate acetate, phytohemagglutinin, and phorbol myristate acetate plus phytohemagglutin were compared. All three methodologies detected significant stimulation of cytokine production. The combination of phytohemagglutinin and phorbol myristate acetate was overall the most-potent stimulus.

Human stem cells express substance P gene and its receptor.

We studied the expression of Substance P (SP) and its receptor in an established human stem cell line (TF-1) and primary stem cells derived from human placental cord blood (HPCB). Using reverse transcriptase polymerase chain reaction (RT-PCR) assay, SP mRNA is detected in both TF-1 cells and HPCB stem cells. Among the alpha, beta, gamma, and delta transcripts of the SP gene, only the beta, gamma, and delta transcripts are detectable in these cells. These RT-PCR-amplified transcripts are confirmed by Southern blot assay using a specific SP probe. Sequence analysis of the RT-PCR-amplified products transcribed from mRNA extracted from the HPCB stem cells also confirmed that these transcripts are identical to those found in human neurons. At the protein level, TF-1 cells produced endogenous SP as determined by an enzyme-linked immunosorbent assay (EIA). Capsaicin, a vanillyl fatty acid amide (ingredient of hot pepper), released SP from TF-1 cells. In addition, using RT nested-PCR analysis, we identified the presence of mRNA for neurokinin-1 receptor (NK-1R, the receptor for SP) in both TF-1 cells and HPCB stem cells, which was confirmed by Southern blot and DNA sequencing analysis. The demonstration that human stem cells express SP and its receptor support the notion that SP is biologically involved in the hematopoietic regulating network.

Immunologic response to combination nucleoside analogue plus protease inhibitor therapy in stable antiretroviral therapy-experienced human immunodeficiency virus-infected children.

The response of 40 immunologic parameters was studied for 147 clinically stable, protease inhibitor-naive, human immunodeficiency virus (HIV)-infected children aged 2-17 years when antiretroviral therapy was changed to either a dual nucleoside analogue regimen or a protease inhibitor-containing regimen. Immunologic response to therapy, as measured by lymphocyte subsets, 3-color flow cytometric measures, and lymphoproliferative assays, were investigated for changes in weeks 44 and 48. The most significant changes after baseline that were associated with the administration of a protease inhibitor-containing regimen were seen for percentages of CD8(+)/CD38(+)/HLA-DR(+), CD8(+)/CD95(+)/CD28(-), and CD8. The percentages of CD8(+)/CD38(+)/HLA-DR(+) and CD8(+)/CD95(+)/CD28(-) decreased from baseline medians of 33% and 46% to medians of 18% and 30% at week 44 (P<.0001 for both). Median CD4 cell count increased 168 cells/microL (from 694 cells/microL to 862 cells/microL; P=.02) by week 48 in this clinically stable population. Changes in lymphoproliferative responses to HIV antigens and recall antigens did not increase over time and between groups.