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S D Douglas

Publications and source records attributed to S D Douglas.

At least 37 records · Page 2Linked to original sources
PubMed2000

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.

Brain↗
PubMed2000

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.

Adolescent↗
PubMed2000

Cytokine profile in genital tract secretions from female adolescents: impact of human immunodeficiency virus, human papillomavirus, and other sexually transmitted pathogens.

Quantitative enzyme-linked immunosorbent assays were used to measure interleukin (IL)-2, IL-10, and IL-12 in cervical secretions from female adolescents with and without sexually transmitted infections. Compared with human immunodeficiency virus [HIV]-negative patients, HIV-positive patients had higher concentrations of IL-10 (118.2 pg/mL vs. 34.5 pg/mL; P=.002) and IL-12 (175.5 pg/mL vs. 85.1; P=.03). IL-2 concentrations were not statistically different. Furthermore, genital tract infections were predictors of IL-10 and IL-12 concentrations. Coinfection with HIV and human papillomavirus predicted the highest IL-10 concentrations; coinfection with HIV, human papillomavirus, and other sexually transmitted pathogens predicted the highest IL-12 concentrations. The data indicate that concomitant infection of the genital tract with HIV and other viral, bacterial, or protozoan pathogens influences the local concentrations of some immunoregulatory cytokines.

Adolescent↗
PubMed2000

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.

Capsaicin↗
PubMed2000

Differential expression of neurokinin-1 receptor by human mucosal and peripheral lymphoid cells.

Substance P (SP) has been implicated in peripheral and mucosal neuroimmunoregulation. However, confusion remains regarding immunocyte expression of the receptor for SP, neurokinin-1 receptor (NK-1R), and whether there is differential NK-1R expression in the mucosal versus the peripheral immune system. In the same assay systems, we examined the expression of NK-1R in human lamina propria mononuclear cells (LPMC), peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL), monocytes, and monocyte-derived macrophages (MDM). Using standard reverse transcription (RT)-PCR, mRNA expression of both the long and the short isoforms of the NK-1R was evident in LPMC but not in PBMC, PBL, monocytes, or MDM. However, by using nested RT-PCR NK-1R mRNA expression was detected in PBMC, PBL, monocytes, and MDM. This level of expression was found to represent one NK-1R mRNA transcript in >1,000 cells. In contrast, by using competitive RT-PCR we demonstrate that LPMC express a more biologically significant level of eight NK-1R mRNA transcripts per cell. Flow cytometric detection of NK-1R expression at the protein level was evident in LPMC but not in PBMC. These findings illustrate the extreme sensitivity of nested RT-PCR and the advantages of competitive RT-PCR in comparative studies of receptor expression in different cell populations. This study suggests that, under normal conditions, readily detectable expression of NK-1R in human mononuclear cells occurs at the mucosal level rather than in the peripheral circulation.

Adenocarcinoma↗
PubMed2000

Need for an external proficiency testing program for cytokines, chemokines, and plasma markers of immune activation.

An external evaluation program for measuring the performance of laboratories testing for cytokines and immune activation markers in biological fluids was developed. Cytokines, chemokines, soluble cytokine receptors, and other soluble markers of immune activation (CSM) were measured in plasma from a healthy human immunodeficiency virus (HIV)-seronegative reference population and from HIV-seropositive individuals as well as in supernatant fluids from in vitro-stimulated human immune cells. The 14 components measured were tumor necrosis factor (TNF) alpha, gamma interferon, interleukin-1 (IL-1), IL-2, IL-4, IL-6, IL-10, Rantes, MIP-Ia, MIP-Ibeta, soluble TNF receptor II, soluble IL-2 receptor alpha, beta(2)-microglobulin, and neopterin. Twelve laboratories associated with the Adult and Pediatric AIDS Clinical Trial Groups participated in the study. The performance features that were evaluated included intralaboratory variability, interlaboratory variability, comparison of reagent sources, and ability to detect CSM in the plasma of normal subjects as well as the changes occurring in disease. The principal findings were as follows: (i) on initial testing, i.e., before participating in the program, laboratories frequently differed markedly in their analytic results; (ii) the quality of testing of a CSM in individual participating laboratories could be assessed; (iii) most commercial kits allowed distinction between normal and abnormal plasma CSM levels and between supernatants of stimulated and unstimulated cells; (iv) different sources of reagents and reference standards frequently provided different absolute values; (v) inexperienced laboratories can benefit from participating in the program; (vi) laboratory performance improved during active participation in the program; and (vii) comparability between analyses conducted at different sites can be ensured by an external proficiency testing program.

Adult↗
PubMed2000

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.

Adult↗
PubMed1999

Human immunodeficiency virus infection of human placental cord blood CD34+AC133+ stem cells and their progeny.

The AC133 is a novel antigen selectively expressed on primitive CD34bright stem cells and is a valuable marker for the selection of long-term culture-initiating cells (LTC-ICs) and severe combined immunodeficiency (SCID)-repopulating cells. Human placental cord blood (HPCB) is a rich source of CD34+AC133+ cells. Since AC133 antibody is likely to be used as an alternative to CD34 for the selection of stem cells in transplant and gene therapy situations, we examined the susceptibility of HPCB-isolated CD34+AC133+ stem cells to infection with free and cell-associated HIV-1 in vitro. Freshly isolated HPCB CD34+AC133+ stem cells were not susceptible to HIV-1 infection as determined by PCR and reverse transcriptase assays. Inoculation with HIV-1 did not affect the viability and clonogenic ability of HPCB CD34+AC133+ cells. Although the highly purified HPCB CD34+AC133+ stem cells contained mRNA for CD4 and CXCR4 receptors, CD4 and CXCR4 proteins were not expressed on these cells. Similarly, CCR5 protein, the major macrophage-tropic HIV-1 coreceptor, was not expressed in freshly isolated HPCB CD34+AC133+ stem cells, although the transcript for CCR5 was identified in these cells. Expression of CD4, CXCR4, and CCR5 receptor proteins on the progeny derived from HPCB CD34+AC133+ stem cells was detected and correlated with susceptibility to HIV-1 infection in vitro. These findings suggest that freshly isolated HPCB CD34+AC133+ stem cells are not susceptible to HIV-1 infection and may not be a viral reservoir. These data have important implications for the use of AC133 antibody as a means of enriching for primitive hematopoietic stem cells from placental cord blood and in the design of stem cell or progenitor cell-based gene therapeutic strategies for perinatal HIV-1 infection.

AC133 Antigen↗
PubMed1999

Quantification of substance P mRNA in human mononuclear phagocytes and lymphocytes using a mimic-based RT-PCR.

We have recently demonstrated that human monocytes and lymphocytes express the substance P (SP) gene at both the mRNA and protein level [Ho, W.Z., Lai, J.P., Zhu, X.H., Uvaydova, M., Douglas S.D., 1997. Human monocytes and macrophages express substance P and neurokinin-1 receptor. Journal of Immunology, 159, p. 5654; Lai, J.P., Douglas, S. D., Ho, W.Z., 1998. Human lymphocytes express substance P and its receptor. Journal of Neuroimmunology, 86, p. 80; Lai, J.-P., Douglas, S.D., Rappaport, E., Wu, J., Ho, W.-Z., 1998. Identification of a delta isoform of preprotachykinin mRNA in human mononuclear phagocytes and lymphocytes. Journal of Neuroimmunology, 91, p. 121]. Using RT-PCR assay with several specific human SP primer pairs, we were able to differentiate four isoforms of preprotachykinin (PPT-A, the SP precursor) mRNA transcripts on ethidium bromide-stained agarose gels and clone the PCR amplified cDNA of the four isoforms (alpha, beta, gamma, and delta) of the PPT-A gene. In an effort to quantitatively measure PPT-A mRNA levels, we have developed a mimic-based RT-PCR assay to analyze total PPT-A mRNA levels in human monocytes and lymphocytes. We designed a specific human SP primer pair (HSP4/HSP3) to amplify a single fragment of cDNA derived from all four isoforms of PPT-A mRNA transcripts, with a sensitivity of 120 molecules per reaction. Thus the PPT-A mRNA transcripts in an unknown sample can be quantitatively analyzed using the mimic-based RT-PCR. The accuracy and reproducibility of this assay were confirmed by the plasmids containing alpha, beta, gamma and delta cDNA inserts and by in vitro synthesized mRNA from a plasmid containing beta isoform cDNA insert. Our data indicate that the SP mimic-based RT-PCR assay has potential advantages in studies of SP levels in a variety of human cells as well as in clinical specimens.

Adult↗
PubMed1999

Peripheral blood mononuclear cell markers in antiretroviral therapy-naive HIV-infected and high risk seronegative adolescents. Adolescent Medicine HIV/AIDS Research Network.

OBJECTIVE: To examine potential hematologic and immunologic markers for healthy adolescents and for adolescents infected with HIV. DESIGN: The REACH Project (Reaching for Excellence in Adolescent Care and Health) of the Adolescent Medicine HIV/AIDS Research Network (AMHARN) recruits HIV-infected and high-risk HIV-uninfected adolescents, aged at least 13 but less than 19 years. The study evaluates biomedical and behavioral features of HIV infection as observed while under medical care for HIV infection and adolescent health. METHODS: Blood samples were collected from HIV-infected and HIV-uninfected subjects at 16 clinical sites. Cell phenotypes were determined using standard single, dual or three-color flow cytometry. RESULTS: This report includes data at enrollment for 94 HIV-positive adolescents who had never received antiretroviral therapy (ART) (mean age, 17.4 +/- 1.0 years for males and 16.5 +/- 1.3 years for females) and 149 HIV-negative adolescents (mean age, 16.7 +/- 1.2 years for males and 16.6 +/- 1.2 years for females); this is the antiretroviral therapy-naive subset drawn from 294 HIV-positive and 149 HIV-negative adolescents enrolled in the REACH Cohort. The total leukocyte count was significantly reduced in the HIV-positive females in comparison with the HIV-negative females (P < 0.001). There was a reduction in natural killer cells (P < 0.05) in HIV-positive females (mean, 140.6 +/- 104.2 x 10(6) cells/l) in comparison with HIV-negative females (184.3 +/- 142.5 x 10(6) cells/l), whereas no differences were found between the two groups of males. The reduction in the total CD4 cell count in HIV-positive males and females in comparison with the HIV-negative subjects was the consequence of a decrease in both the naive CD4 and memory CD4 components. There was a striking increase in the mean number of CD8 memory cells in HIV-positive compared with HIV-negative adolescents, and a corresponding increase in the percentage of these cells. In contrast, naive CD8 cells were present in increased numbers but their percentage was decreased. CONCLUSIONS: These studies of adolescents provide normative data for high-risk healthy adolescents as well as baseline immunologic data for a cohort of ART-naive HIV-positive adolescents. This comparison suggests that this untreated, recently infected group had relatively intact immunologic parameters.

Adolescent↗
PubMed1999

Protective role of beta-chemokines associated with HIV-specific Th responses against perinatal HIV transmission.

To examine the protective role of cellular immunity in the vertical transmission of HIV, we analyzed HIV-specific IL-2 and CTL responses, as well as beta-chemokine expression in HIV-infected and uninfected infants of HIV+ mothers. Our results showed that HIV envelope (env) peptide-specific IL-2 responses associated with beta-chemokine production were detectable at birth in the majority of uninfected infants of HIV+ mothers. The responses falling to background before the infants were 1 yr old were rarely associated with HIV-specific CTL activity. Conversely, HIV-specific Th and CTL cellular responses were absent at birth in HIV-infected infants. Infants with AIDS-related symptoms exhibited undetectable or very low levels of HIV-specific cellular immunity during the first year of life, whereas those with a slowly progressive disease showed evidence of such immunity between their second and ninth month. The latter group of infected infants tested negative for plasma HIV RNA levels shortly after birth, suggesting lack of intrauterine exposure to HIV. The presence of HIV-specific Th responses at birth in uninfected newborns of HIV+ mothers, but absence of such activities in HIV-infected infants without evidence of intrauterine HIV infection, suggests that in utero development of HIV-specific Th responses associated with beta-chemokines could mediate nonlytic inhibition of infection during vertical transmission of HIV.

Alleles↗
PubMed1999

Interferon-gamma upregulates CCR5 expression in cord and adult blood mononuclear phagocytes.

The C-C chemokine receptors CCR5 and CCR3 are fusion coreceptors for human immunodeficiency virus (HIV) entry into macrophages. The regulation of their expression influences infectivity by HIV. We report here that interferon-gamma (IFN-gamma) a cytokine that has bidirectional effects on HIV infection of macrophages, significantly upregulated CCR5 and CCR3 cell surface expression in human mononuclear phagocytes isolated from placental cord blood and adult peripheral blood. Monocytes treated with IFN-gamma showed increased chemotaxis to the CCR5 ligands macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta, confirming the functional relevance of IFN-gamma-induced CCR5 expression. However, IFN-gamma suppressed HIV entry into macrophages. Interestingly, we demonstrated that IFN-gamma inhibited cell surface expression of CD4, the major receptor for HIV. This finding may explain the suppressive effect of IFN-gamma on HIV entry into macrophages, despite its enhancing effect on the expression of CCR5 and CCR3 by these cells. In addition, IFN-gamma-induced secretion of C-C chemokines (RANTES, MIP-1alpha, and MIP-1beta) by mononuclear phagocytes may also suppress HIV entry into macrophages. These data provide further evidence for cytokine-mediated regulation of CCR5 expression and are consistent with a novel paradigm in which cytokines regulate HIV infection and leukocyte migration by reciprocal and opposing effects on the expression of CD4 and chemokine receptors.

Adult↗
PubMed1999

Recombinant human gamma interferon in human immunodeficiency virus-infected children: safety, CD4(+)-lymphocyte count, viral load, and neutrophil function (AIDS Clinical Trials Group Protocol 211).

Nineteen children with human immunodeficiency virus (HIV) infection were treated with recombinant human gamma interferon (rIFN-gamma) (50 microg/m2 subcutaneously three times each week during weeks 1 through 12 and 100 microg/m2 subcutaneously three times each week during weeks 13 through 24) in a phase I/II clinical trial. All children continued to receive previously prescribed therapy with oral zidovudine or didanosine. Children were assessed clinically and with laboratory studies during 24 weeks of study treatment and for 12 weeks after completion of rIFN-gamma therapy. In general, rIFN-gamma therapy was well tolerated. There were two clinical or laboratory adverse events thought to be possibly or probably study drug associated. One child developed acute pancreatitis; another child developed granulocytopenia. Median CD4(+)-lymphocyte counts and plasma HIV RNA concentrations did not change significantly during therapy. In vitro neutrophil bactericidal activity against Staphylococcus aureus and superoxide production were not significantly affected by rIFN-gamma therapy. We conclude that rIFN-gamma therapy in HIV-infected children receiving single-agent antiretroviral therapy is safe and does not produce consistent changes in CD4(+)-lymphocyte count, plasma HIV RNA concentration, or in vitro neutrophil function.

Anti-HIV Agents↗
PubMed1999

Decreased superoxide production, degranulation, tumor necrosis factor alpha secretion, and CD11b/CD18 receptor expression by adherent monocytes from preterm infants.

Preterm infants have an increased incidence of infection, which is principally due to deficiencies in neonatal host defense mechanisms. Monocyte adherence is important in localizing cells at sites of infection and is associated with enhanced antimicrobial functions. We isolated cord blood monocytes from preterm and full-term infants to study their adhesion and immune functions, including superoxide (O2-) generation, degranulation, and cytokine secretion and their adhesion receptors. O2- production and degranulation were significantly diminished, by 28 and 37%, respectively, in adherent monocytes from preterm infants compared to full-term infants (P < 0. 05); however, these differences were not seen in freshly isolated cells. We also observed a significant decrease of 35% in tumor necrosis factor alpha secretion by lipopolysaccharide-stimulated adherent monocytes from preterm infants compared to full-term infants (P < 0.05); however, this difference was not observed in interleukin-1beta or interleukin-6 production by the monocytes. The cell surface expression of the CD11b/CD18 adhesion receptor subunits was significantly decreased (by 60 and 52%, respectively) in monocytes from preterm infants compared to full-term infants (P < 0. 01). The cascade of the immune response to infection involves monocyte upregulation and adherence via CD11b/CD18 receptors followed by cell activation and the release of cytokines and bactericidal products. We speculate that monocyte adherence factors may be important in the modulation of immune responses in preterm infants.

Birth Weight↗
PubMed1998

Identification of a delta isoform of preprotachykinin mRNA in human mononuclear phagocytes and lymphocytes.

We have characterized preprotachykinin (PPT-A) gene transcript splicing products and identified a fourth isoform of PPT-A mRNA transcript in human peripheral blood-isolated monocytes and PBL. Using RT-PCR, Southern blot analysis and nucleotide sequencing analysis, we have identified the four isoforms of PPT-A transcripts (alpha, beta, gamma and delta) in human peripheral blood-isolated monocytes and PBL. The delta-PPT transcript present in the immune cells lacks exons 4 and 6. The sequences of exons 3, 5 and 7 of delta-PPT transcript completely match those of beta-PPT transcript. The alpha-PPT and beta-PPT sequences in these cells are identical to those obtained by Tan and Too (GenBank accession number U37539) and Harmar et al. (Genbank accession number X54469), but differ by a single nucleotide from another entry by Chiwakata et al. (Genbank accession number M68906). In comparison to this latter sequence, there was a C-->T change at amino acid position 87 (CCT-->CTT) which may result in a Pro to Leu change. Identification of the new SP mRNA transcript in both human CNS and immune cells supports the concept of an important biological link between CNS and immune system.

Adult↗
PubMed1998

Serum levels of substance P are elevated in patients with sickle cell disease and increase further during vaso-occlusive crisis.

As a mediator of neurogenic inflammation and pain, we hypothesized that levels of the neuropeptide Substance P (SP) would be elevated in patients with sickle cell disease (SCD) with vaso-occlusive pain crisis. SP is a known stimulator of tumor necrosis factor-alpha (TNF-alpha) release and a promoter of interleukin-8 (IL-8), which are reported to be increased in SCD. These cytokines enhance adhesion of leukocytes to endothelium and may play a role in vaso-occlusive events. Serum levels of IL-8, TNFalpha, and SP were studied in three groups of children aged 2 to 18 years: 30 well children with SCD, 21 with SCD in pain crisis, and 20 healthy age-matched controls. Serum levels of SP were elevated in all SCD patients and were highest in patients in pain crisis. The percentage of sera with detectable levels of IL-8 (>5.0 pmol/L) was increased in SCD patients as compared with the control group. IL-8 levels were similar for well SCD patients and those with pain. TNFalpha levels were not significantly different among the three groups. In three children with SCD, SP was measured at baseline and again during pain crisis. In each case, serum levels during pain crisis were higher than they were when the patient was well. We conclude that levels of SP are high in patients with SCD and increase during pain crisis. These results imply that SP plays a prominent role in the pain and inflammation of SCD and may be a measurable laboratory marker of vaso-occlusive crisis. We speculate that neurokinin receptor antagonists may have a therapeutic potential in the treatment of crisis pain.

Acute Disease↗
PubMed1998

Human lymphocytes express substance P and its receptor.

We present data demonstrating the gene expression of substance P (SP) and its receptor in human peripheral blood-isolated lymphocytes. Using reverse transcribed polymerase chain reaction (RT-PCR) assay, preprotachykinin-A (substance-P) mRNA is detected in human peripheral blood-isolated lymphocytes. Among the alpha, beta, and gamma transcripts of the SP gene, only the beta and gamma transcripts are detectable in these cells. These RT-PCR amplified transcripts are recognized by Southern blot assay using a specific SP probe. Direct DNA sequence analysis of the RT-PCR products from lymphocytes also confirmed the structure of these transcripts which are identical to those found in human neuronal cells. At the protein level, human lymphocytes produced endogenous SP as determined by an enzyme immunoassay. Capsaicin, a vanillyl fatty acid amide (ingredient of hot pepper), released preformed SP from lymphocytes. In addition, using RT/nested-PCR analysis, we identified the presence of mRNA for neurokinin-1 receptor (the receptor for SP) in human peripheral blood-isolated lymphocytes, which was confirmed by Southern blot and DNA sequencing analysis. The demonstration that human lymphocytes express SP and its receptor support the notion that SP is biologically involved in regulating the functions of these cells in an autocrine fashion.

Adult↗