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Biomedical subjects

M Connors

Publications and source records attributed to M Connors.

At least 19 recordsLinked to original sources
PubMed2004

Islet isolation under cGMP conditions: replacing the coil.

INTRODUCTION: Clinical islet transplantation is increasingly regulated, with isolation standards defined under current good manufacturing practices (cGMPs). cGMP requires validation of equipment cleaning and sterilization. The automated process for islet isolation requires rapid thermal exchange during processing, manipulating a metal coil containing cellular product into and out of chilled/heated waterbaths. We recognize challenges of validating cleaning and sterilization of this coil and propose replacement with a disposable blood warming system. Our specific aims were to assess the system's ability to accommodate flow rates utilized during various phases of pancreatic digestion and to assess its efficiency of heat exchange and temperature control. METHODS: In a pancreas digestion circuit, heat exchange occurred via the coil in a digital water bath, or Warmflo blood warming bag in a digital warming unit. Temperature within the digestion chamber was assessed using an inserted thermocouple. Time to achieve 37 degrees C was measured for set heating element temperatures 38.5 degrees C to 41 degrees C. Circuit temperature maintenance characteristics were also recorded. RESULTS: The Warmflo bloodbag easily accommodated flows of 150 and 300 mL/min. At all set temperatures, the bag resulted in shorter or equivalent time to 37 degrees C than the coil. Maintenance of 37 degrees C was also equivalent. We have utilized this system for four human islet isolations with mean time to 37 degrees C of 5.5 minutes, without difference in digestion quality. CONCLUSIONS: Use of a disposable blood warming system in place of the coil during islet isolation provides adequate flow characteristics, heat exchange, and temperature control and may facilitate evolution of islet isolation toward cGMP compliance.

Cell Culture Techniques↗
PubMed2001

High-level HIV-1 viremia suppresses viral antigen-specific CD4(+) T cell proliferation.

In chronic viral infections of humans and experimental animals, virus-specific CD4(+) T cell function is believed to be critical for induction and maintenance of host immunity that mediates effective restriction of viral replication. Because in vitro proliferation of HIV-specific memory CD4(+) T cells is only rarely demonstrable in HIV-infected individuals, it is presumed that HIV-specific CD4(+) T cells are killed upon encountering the virus, and maintenance of CD4(+) T cell responses in some patients causes the restriction of virus replication. In this study, proliferative responses were absent in patients with poorly restricted virus replication although HIV-specific CD4(+) T cells capable of producing IFN-gamma were detected. In a separate cohort, interruption of antiretroviral therapy resulted in the rapid and complete abrogation of virus-specific proliferation although HIV-1-specific CD4(+) T cells were present. HIV-specific proliferation returned when therapy was resumed and virus replication was controlled. Further, HIV-specific CD4(+) T cells of viremic patients could be induced to proliferate in response to HIV antigens when costimulation was provided by anti-CD28 antibody in vitro. Thus, HIV-1-specific CD4(+) T cells persist but remain poorly responsive (produce IFN-gamma but do not proliferate) in viremic patients. Unrestricted virus replication causes diminished proliferation of virus-specific CD4(+) T cells. Suppression of proliferation of HIV-specific CD4(+) T cells in the context of high levels of antigen may be a mechanism by which HIV or other persistently replicating viruses limit the precursor frequency of virus-specific CD4(+) T cells and disrupt the development of effective virus-specific immune responses.

CD4-Positive T-Lymphocytes↗
PubMed2001

Frequency and function of HIV-specific CD8(+) T cells.

The virus-specific CD8(+) T cell responses of 27 HIV-infected patients were studied, including a unique cohort of long term nonprogressors (LTNP) with normal CD4(+) T cell counts, low levels of plasma viral RNA, strong proliferative responses to HIV antigens and an over-representation of the HLA B*5701 class I allele. The frequencies of CD8(+) T cells specific to the majority of HIV gene products were measured by flow cytometric detection of intracellular interferon-gamma (IFN-gamma) in response to HIV-vaccinia recombinant infected autologous B cells. Very high frequencies (1.4-22%) of circulating CD8(+) T cells were found to be HIV-specific and were not only found in LTNP with reduced plasma virus. No correlation was evident between the frequency of HIV-specific CD8(+) T cells and levels of plasma viremia. In each case, the vast majority of cells (up to 17.2%) responded to Gag-Pol gene products. Although similar frequencies of Gag peptide-specific CD8(+) T cells were found in LTNP and progressors by either intracellular IFN-gamma or MHC class I tetramer staining, the breadth of these responses was greater in patients with progressive HIV infection compared with the LTNP group. The frequency of CD8(+) T cells specific for a single peptide was not representative of an individual patient's total HIV-specific CD8(+) T cell response. These data demonstrate that high numbers of HIV-specific CD8(+) T cells exist even in patients with high level viremia and progressive disease. Further, they suggest that other qualitative parameters of the CD8(+) T cell response may differentiate some patients with very low levels of plasma virus and nonprogressive infection.

Adult↗
PubMed2001

Managing values in personal food systems.

People in post-industrial societies are faced with many food products and diverse eating situations that can make food-choice decisions complex. This study examined the ways that people managed values in making food choices in various contexts. An analysis of 86 semi-structured, in-depth qualitative interviews from a diverse population of urban adults living in upstate New York revealed that all participants used a personal food system, which was a dynamic set of processes constructed to enact food choices. Within these personal food systems people managed the five main food-related values of taste, health, cost, time and social relationships, and other less prominent values of symbolism, ethics, variety, safety, waste and quality. The salience of these values varied among the participants as well as across the eating situations that confronted each participant. Participants used three main processes in their personal food systems: (i) categorizing foods and eating situations; (ii) prioritizing conflicting values for specific eating situations; and (iii) balancing prioritizations across personally defined time frames. Understanding the personal food systems people use to help them make food choices can be useful for developing theories about eating behavior and communicating health messages related to food and eating.

Adolescent↗
PubMed2001

Managing healthy eating: definitions, classifications, and strategies.

This study sought to enhance understanding of how people conceptualize and manage healthy eating. An interpretivist approach employed the constant comparative method to analyze 79 open-ended interviews with individuals about food choices and eating behaviors for health-related themes. Participant reports depicted cognitive systems for defining healthy eating, where personal meanings evolved through ongoing exposure to a variety of experiential and informational sources. Participants' definitions of healthy eating clustered around seven themes forrelating food and eating to their personal health. Healthy eating definitions shaped how participants categorized food and eating situations as healthy and unhealthy. Participants described healthy eating strategies that were differentially associated with various healthy eating themes. These findings provide an emic perspective of how a diverse sample of adults conceptualize and manage healthy eating. Exposing the implicit and multiplistic nature of healthy eating conceptions provides information useful to health educators promoting behavior changes.

Adolescent↗
PubMed2000

Longitudinal changes in CD4+ T cell antigen receptor diversity and naive/memory cell phenotype during 9 to 26 months of antiretroviral therapy of HIV-infected patients.

Although skewing of the CD4+ TCR repertoire in advanced HIV infection is well documented, increases in polyclonality during antiretroviral therapy have been less consistently observed. Ten patients, each with documented abnormalities within the CD4+ TCR repertoire, were studied by CDR3 spectratyping, semiquantitative PCR, and SSCP during 9-26 months of therapy. Naive and memory cell phenotypes were analyzed by flow cytometry. Six of 10 patients showed increased polyclonality of their TCR repertoires, 1 showed no change, and 3 showed increased TCR skewing, despite suppressed viral replication. Overall, there was no significant change in the percentage of abnormal BV subfamilies (from a mean of 25.5 to 17.1%) or the percentage of naive CD4+ T cells (from a mean of 18 to 25%). Further, progression of TCR repertoire disruptions was observed in some patients even with suppression of plasma viral RNA below 500 copies/ml. Although a spectrum of changes may be seen within the CD4+ TCR repertoire in the setting of antiretroviral therapy, increases in polyclonality are observed in some patients.

Anti-HIV Agents↗
PubMed2000

Maintenance of large numbers of virus-specific CD8+ T cells in HIV-infected progressors and long-term nonprogressors.

The virus-specific CD8+ T cell responses of 21 HIV-infected patients were studied including a unique cohort of long-term nonprogressors with low levels of plasma viral RNA and strong proliferative responses to HIV Ags. HIV-specific CD8+ T cell responses were studied by a combination of standard cytotoxic T cell (CTL) assays, MHC tetramers, and TCR repertoire analysis. The frequencies of CD8+ T cells specific to the majority of HIV gene products were measured by flow cytometric detection of intracellular IFN-gamma in response to HIV-vaccinia recombinant-infected autologous B cells. Very high frequencies (0.8-18.0%) of circulating CD8+ T cells were found to be HIV specific. High frequencies of HIV-specific CD8+ T cells were not limited to long-term nonprogressors with restriction of plasma virus. No correlation was found between the frequency of HIV-specific CD8+ T cells and levels of plasma viremia. In each case, the vast majority of cells (up to 17.2%) responded to gag-pol. Repertoire analysis showed these large numbers of Ag-specific cells were scattered throughout the repertoire and in the majority of cases not contained within large monoclonal expansions. These data demonstrate that high numbers of HIV-specific CD8+ T cells exist even in patients with high-level viremia and progressive disease. Further, they suggest that other qualitative parameters of the CD8+ T cell response may differentiate some patients with very low levels of plasma virus and nonprogressive disease.

Adult↗
PubMed2000

HLA B*5701 is highly associated with restriction of virus replication in a subgroup of HIV-infected long term nonprogressors.

A unique cohort of HIV-1-infected long term nonprogressors (LTNP) with normal CD4(+) T cell counts and <50 copies/ml of plasma were prospectively recruited for study. HLA typing revealed a dramatic association between the HLA B*5701 class I allele and nonprogressive infection [85% (11 of 13) vs. 9.5% (19 of 200) in progressors; P < 0. 001]. Antigen-specific CD8(+) T cells were enumerated by flow cytometric detection of intracellular IFN-gamma in response to HIV antigens and HLA B*57-gag tetramer staining. No quantitative differences in the total HIV-specific CD8(+) T cell responses were observed between B*57(+) LTNP and five B*57(+) progressors (P = 0.4). Although similar frequencies of peptide specific CD8(+) T cells were also found, the gag-specific CD8(+) T cell response in the LTNP group was highly focused on peptides previously shown to be B*57-restricted. These findings indicate that, within this phenotypically and genotypically distinct cohort, a host immune factor is highly associated with restriction of virus replication and nonprogressive disease. They also strongly suggest a mechanism of virus specific immunity that directly operates through the B*5701 molecule. Further characterization of qualitative differences in the virus-specific responses that distinguish HLA B*57(+) LTNP from progressors may ultimately define mechanisms of effective immune mediated restriction of virus replication.

Alleles↗
PubMed2000

Resistance to replication of human immunodeficiency virus challenge in SCID-Hu mice engrafted with peripheral blood mononuclear cells of nonprogressors is mediated by CD8(+) T cells and associated with a proliferative response to p24 antigen.

High levels of resistance to challenge with human immunodeficiency virus type 1 SF162 were observed in animals engrafted with peripheral blood mononuclear cells of four long-term nonprogressors (LTNPs). Resistance was abrogated by depletion of CD8(+) T cells in vivo and was observed only in LTNPs with proliferative responses to p24. In a subgroup of nonprogressors, CD8(+) T cells mediated restriction of challenge viruses, and this response was associated with strong proliferative responses to p24 antigen.

Animals↗
PubMed1999

Increased vaccine-specific T cell frequency after peptide-based vaccination correlates with increased susceptibility to in vitro stimulation but does not lead to tumor regression.

Although in vitro sensitization assays have shown increased melanoma Ag (MA)-specific CTL reactivity after vaccination with MA peptides, clinical responses have been uncommon. This paradox questions whether data obtained from the in vitro stimulation and expansion of T cells lead to an overestimation of the immune response to vaccines. Using HLA/peptide tetramer (tHLA), we enumerated MA-specific T cell precursor frequency (TCPF) directly in PBMC from 23 melanoma patients vaccinated with gp100:209-217(210M) (g209-2M) peptide. Vaccine-specific TCPF was higher in postvaccination PBMC from seven of seven patients treated with peptide alone and four of five patients treated with peptide plus IL-12 (range of postvaccination TCPF, 0.2-2.4% and 0.2-2.5%, respectively). The increased TCPF correlated with enhanced susceptibility to in vitro stimulation with the relevant epitope. Paradoxically, no increase in postvaccination TCPF was observed in most patients who had been concomitantly treated with IL-2 (1 of 11 patients; range of postvaccination TCPF, 0.02-1.0%), a combination associated with enhanced rates of tumor regression. The lack of increase in TCPF seen in these patients corresponded to inability to elicit expansion of vaccine-specific T cells in culture. This study shows that a peptide-based vaccine can effectively generate a quantifiable T cell-specific immune response in the PBMC of cancer patients, though such a response does not associate with a clinically evident regression of metastatic melanoma.

CD8-Positive T-Lymphocytes↗
PubMed1999

HIV-1-specific CD4+ T cells are detectable in most individuals with active HIV-1 infection, but decline with prolonged viral suppression.

The role of HIV-1-specific CD4+ T-cell responses in controlling HIV-1 infection remains unclear. Previous work has suggested that such cells are eliminated in the early stages of infection in most subjects, and thus cannot substantially contribute to host defense against HIV-1. Here, using flow cytometric detection of antigen-induced intracellular cytokines, we show that significant frequencies of gag specific, T-helper-1 CD4+ memory T cells are detectable in most subjects with active/progressive HIV-1 infection (median frequency, 0.12% of memory subset; range, 0-0.66%). Median frequencies of these cells were considerably higher in nonprogressive HIV-1 disease (0.40%), but there was substantial overlap between the two groups (range of nonprogressors, 0.10-1.7%). Continuous HIV-1 suppression with anti-retroviral therapy was associated with a time-dependent reduction in median frequencies of gag-specific CD4+ memory T cells: 0.08% in subjects treated for 4-24 weeks, and 0.03% in subjects treated for 47-112 weeks. Thus, functional HIV-1-specific CD4+ T cells are commonly available for support of anti-HIV-1 effector responses in active disease, but their decline with anti-retroviral therapy indicates that immunologic participation in long-term HIV-1 control will probably require effective vaccination strategies.

Adult↗
PubMed1998

Progression of human immunodeficiency virus disease is associated with increasing disruptions within the CD4+ T cell receptor repertoire.

The immunodeficiency caused by human immunodeficiency virus (HIV) infection may be related to loss of diversity in the T cell receptor (TCR) repertoire. A cross-sectional study of the CD4 TCR repertoire was done for patients at various stages of HIV infection. Semiquantitative polymerase chain reaction was used to study the relative usage of the variable chain beta (BV) subfamilies and the size distributions of transcripts (CDR3 size analysis) within these subfamilies. The relative usage of the TCRBV subfamilies of patients and controls was not significantly different. The proportion of subfamilies with abnormal CDR3 size patterns was higher in the HIV-infected patients (25%, 95% confidence interval [CI], 17%-33%) than in the controls (7.2%, 95% CI, 2.3%-12.1%; P < .001), with a significant negative correlation between the number of CD4 cells and the percentage of abnormal TCRBV subfamilies. These results indicate that progressive loss of CD4 T cells is accompanied by increasing disruptions within the T cell receptor repertoire.

CD4 Lymphocyte Count↗
PubMed1998

Administration of an anti-CD8 monoclonal antibody interferes with the clearance of chimeric simian/human immunodeficiency virus during primary infections of rhesus macaques.

Parenteral administration of a mouse anti-human CD8 monoclonal antibody (MAb) to rhesus macaques resulted in a transient depletion of CD8+ cells in both the peripheral blood and lymphoid tissues. When administered during primary chimeric simian/human immunodeficiency virus infections, the CD8 MAb caused marked elevations of plasma and cell-associated virus levels in both the peripheral blood and lymphoid tissues and led to prolonged depletion of CD4 cells. Taken together, these results directly demonstrate that CD8+ T lymphocytes are actively involved in controlling the acute phase of primate lentivirus infections.

Animals↗
PubMed1997

Effects of CD28 costimulation on long-term proliferation of CD4+ T cells in the absence of exogenous feeder cells.

In this report, conditions for prolonged in vitro proliferation of polyclonal adult CD4+ T cells via stimulation with immobilized anti-CD3 plus anti-CD28 have been established. CD4+ cells maintained exponential growth for more than 60 days during which a total 10(9)- to 10(11)-fold expansion occurred. Cell cultures exhibited cyclical changes in cell volume, indicating that, in terms of proliferative rate, cells do not have to rest before restimulation. Indeed, electronic cell size analysis was the most reliable method to determine when to restimulate with additional immobilized mAb. The initial approximately 10(5)-fold expansion was autocrine, occurring in the absence of exogenous cytokines or feeder cells. Addition of recombinant human IL-2 after the initial autocrine expansion resulted in continued exponential proliferation. Phorbol ester plus ionomycin also induced long-term growth when combined with anti-CD28 stimulation. Analysis of the T cell repertoire after prolonged expansion revealed a diverse repertoire as assessed by anti-TCR Vbeta Abs or a PCR-based assay. Cytokines produced were consistent with maintenance of both Th1 and Th2 phenotypes; however, the mode of CD3 and CD28 stimulation could influence the cytokine secretion pattern. When anti-CD3 and anti-CD28 were immobilized on the same surface, ELISAs on culture supernatants revealed a pattern consistent with Th1 secretion. Northern analysis revealed that cytokine gene expression remained inducible. Spontaneous growth or cell transformation was not observed in more than 100 experiments. Together, these observations may have implications for gene therapy and adoptive immunotherapy. Furthermore, these culture conditions establish a model to study the finite lifespan of mature T lymphocytes.

Antibodies, Monoclonal↗
PubMed1997

HIV infection induces changes in CD4+ T-cell phenotype and depletions within the CD4+ T-cell repertoire that are not immediately restored by antiviral or immune-based therapies.

Changes in CD4+ T-cell surface marker phenotype and antigen receptor (TCR) repertoire were examined during the course of HIV infection and following therapy. A preferential decline in naive CD4+ T cells was noted as disease progressed. Following protease inhibitor therapy, naive CD4+ T cells increased only if they were present before initiation of therapy. Disruptions of the CD4+ TCR repertoire were most prevalent in patients with the lowest CD4+ T-cell counts. Antiviral or IL-12 therapy-induced increases in CD4+ T-cell counts led to only minor changes in previously disrupted repertoires. Thus, CD4+ T-cell death mediated by HIV-1 infection may result in a preferential decline in the number of naive CD4+ T cells and disruptions of the CD4+ T-cell repertoire that are not immediately corrected by antiviral or immune-based therapies.

CD4-Positive T-Lymphocytes↗
PubMed1997

The relationship between overnight GH levels and insulin concentrations in adolescents with insulin-dependent diabetes mellitus (IDDM) and the impact of recombinant human insulin-like growth factor I (rhIGF-I).

OBJECTIVE: We examined the relationship between GH concentrations and free insulin concentrations, used as an index of insulin sensitivity, before and after recombinant human insulin-like growth factor I (RhIGF-I) administration in adolescents with insulin-dependent diabetes mellitus (IDDM). DESIGN AND PATIENTS: Growth hormone concentrations were assessed by a peak detection programme (Pulsar) on a control night (2000 h-0800 h) and a night when rhIGF-I administered in a subcutaneous dose of 40 micrograms/kg at 1800 h to 16 adolescent subjects. Stable euglycaemia was maintained by a continuous intravenous insulin infusion and changes in free insulin levels on the two nights were compared with growth hormone data. RESULTS: Mean overnight GH concentrations (2000 h-0800 h) on the control night were positively related to glycated haemoglobin (Hba1) concentrations (r = 0.63; P < 0.01) and were reduced following rhIGF-I administration (24.9 +/- 3.6 mU/I on the control night versus 17.4 +/- 2.2 mU/I after administration, P = 0.01). The mean GH pulse amplitude on the control night was related to the change in GH levels after rhIGF-I (rs = -0.66, P < 0.001). Multiple regression analysis revealed that mean GH pulse amplitude was the only determinant of free insulin concentrations (0500 h-0700 h on both study nights (P < 0.01). The percentage change in mean growth hormone pulse amplitude between the two nights was related to the percentage reduction in free insulin concentrations (r = 0.53, P = 0.03). CONCLUSIONS: Growth hormone pulse amplitude is related to early morning insulin sensitivity in adolescents with IDDM on control nights and after rhIGF-I administration. The reduction in insulin levels following rhIGF-I may be linked to the change in GH pulse amplitude and not just to direct insulin-like actions. Individuals with the higher GH (and thus HbA1 levels) were most sensitive to the GH-suppressive effects of rhIGF-I.

Adolescent↗
PubMed1997

Body composition and health-related quality of life in patients with obstructive airways disease.

This study evaluated the effects of body weight and lean mass abnormalities on health-related quality of life (HRQL) in obstructive airways disease. Body weight, lean mass (using dual-energy X-ray absorptiometry), and HRQL (using the St George's Respiratory Questionnaire (SGRQ)) were measured in 50 patients. Low lean mass was defined as a lean mass index (lean mass/height2) below the fifth percentile of a control population. Dyspnoea was measured by the baseline dyspnoea index. The mean (SD) age was 69+/-9 yrs; the forced expiratory volume in one second (FEV1) was 39+/-19% of predicted. Patients had 2.4+/-4.1 kg less lean mass than predicted. Increased dyspnoea was the most influential predictor of poor HRQL. Compared to normal-weight patients, those who were underweight had significantly greater impairment in activity, impact, and total SGRQ scores, while those who were overweight had greater impairment in impact and total SGRQ scores. Low lean mass was associated with greater impairment in symptoms, activity and impact subscores and the total SGRQ score. When dyspnoea was added to the model as a covariate, neither weight nor lean mass remained significantly related to HRQL. Thus, although body weight and lean mass abnormalities influence health-related quality of life, their effects appear to be mediated through increased levels of dyspnoea.

Absorptiometry, Photon↗