Ambrosia Garcia

Optimization of storage and shipment of cryopreserved peripheral blood mononuclear cells from HIV-infected and uninfected individuals for ELISPOT assays

Functional immunologic assays using cryopreserved peripheral blood mononuclear cells (PBMC) are influenced by blood processing, storage and shipment. The objective of this study was to compare the viability, recovery and ELISPOT results of PBMC stored and shipped in liquid nitrogen (LN/LN) or stored in LN and shipped on dry ice (LN/DI) or stored at -70°C for 3 to 12 weeks and shipped on DI (70/DI 3 to 12); and to assess the effect of donor HIV infection status on the interaction between storage/shipment and the outcome measures. PBMC from 12 HIV-infected and 12 uninfected donors showed that LN/LN conferred higher viability and recovery than LN/DI or 70/DI 3, 6, 9 or 12. LN/DI PBMC had higher viability than any 70/DI PBMC. The PBMC viability and recovery linearly decreased with the duration of storage at -70°C from 3 to 12 weeks. This effect was more pronounced in samples from HIV-infected than uninfected donors. Results of ELISPOT assays using CMV pp65, CEF and Candida albicans antigens were qualitatively and quantitatively similar across LN/LN, LN/DI and 70/DI 3. However, ELISPOT values significantly decreased with the duration of storage at -70°C both in HIV-infected and uninfected donors. ELISPOT results also decreased with PBMC viability <70%.

Diminished mononuclear cell function is associated with chronic venous insufficiency

PURPOSE With clinical progression of chronic venous insufficiency (CVI), dermal infiltration of mononuclear cells increases. Because these cells regulate chronic inflammatory responses and modulate wound healing, cellular dysfunction could explain alterations in wound healing with CVI. The purpose of this study was to determine whether monocytes in patients with CVI are dysfunctional. METHODS Mononuclear cell function was measured as the degree of proliferation in response to a mitogenic challenge. Fifty patients were separated into four groups: group 1, 14 patients with normal limbs; group 2, 10 patients with class 2 CVI; group 3, 15 patients with active venous ulcers; group 4, 11 patients with healed venous ulcers and current evidence of lipodermatosclerosis. Duplex scanning and air plethysmography correlated with the clinical classification of CVI. Systemically circulating monocytes and lymphocytes were obtained by antecubital venipuncture from groups 1 to 4. Cells were cultured in the presence of staphylococcal enterotoxins A, B, C1, D, and E (mitogens) at 1, 8, 31, and 125 micrograms/well on the basis of previous dose-response experiments. Phytohemagglutinin (PHA), 5 micrograms/well, served as a control mitogen. The dose-response curves indicated that 8 micrograms/well elicited the greatest degree of cell proliferation. Proliferative responses at 8 micrograms/well were analyzed for statistical significance among groups 1 to 4. Comparisons among groups were performed by use of the nonparametric Mann Whitney U post tests and a one-tailed unpaired t test. Results were considered significant at p < or = 0.05. RESULTS Proliferative responses to PHA indicate that lymphocytes and monocytes from patients with CVI are not globally depressed. However, patients in group 2 did not exhibit the same degree of proliferation to PHA as did groups 1, 3 and 4. Proliferative responses between groups 2 and 1 (44.38 +/- 43.9 vs 118.87 +/- 27.1, p < or = 0.05) and groups 2 and 3 (44.38 +/- 43.9 vs 105.95 +/- 60.99, p < or = 0.05) were significant. Challenges with staphylococcal enterotoxin A and B reveal significant diminution of proliferative responses in groups 2 (42.73 +/- 11.55, p < or = 0.05) and 3 (45.57 +/- 9.1, p < or = 0.05) and groups 3 (36.81 +/- 6.9, p < or = 0.05) and 4 (35.04 +/- 7.5, p < or = 0.05), compared with staphylococcal enterotoxin A controls (68.68 +/- 9.9) and staphylococcal enterotoxin B controls (66.25 +/- 13.56), respectively. A trend of diminished mononuclear cell function with progression of CVI was observed with staphylococcal enterotoxins B, C1, D, and E, strongly suggesting biologic significance. Furthermore, patients with lipodermatosclerosis uniformly exhibited the poorest proliferative responses. CONCLUSIONS Deterioration of mononuclear cell function is associated with CVI. A trend of diminishing proliferative responses with clinical disease progression is observed and suggests biologic significance. The decreased capacity for mononuclear cell proliferation in response to various challenges may manifest itself clinically as poor and prolonged wound healing.

Implementation of Good Clinical Laboratory Practice (GCLP) guidelines within the External Quality Assurance Program Oversight Laboratory (EQAPOL)

The EQAPOL contract was awarded to Duke University to develop and manage global proficiency testing programs for flow cytometry-, ELISpot-, and Luminex bead-based assays (cytokine analytes), as well as create a genetically diverse panel of HIV-1 viral cultures to be made available to National Institutes of Health (NIH) researchers. As a part of this contract, EQAPOL was required to operate under Good Clinical Laboratory Practices (GCLP) that are traditionally used for laboratories conducting endpoint assays for human clinical trials. EQAPOL adapted these guidelines to the management of proficiency testing programs while simultaneously incorporating aspects of ISO/IEC 17043 which are specifically designed for external proficiency management. Over the first two years of the contract, the EQAPOL Oversight Laboratories received training, developed standard operating procedures and quality management practices, implemented strict quality control procedures for equipment, reagents, and documentation, and received audits from the EQAPOL Central Quality Assurance Unit. GCLP programs, such as EQAPOL, strengthen a laboratorys ability to perform critical assays and provide quality assessments of future potential vaccines.

Establishment and maintenance of a PBMC repository for functional cellular studies in support of clinical vaccine trials.

A large repository of cryopreserved peripheral blood mononuclear cells (PBMCs) samples was created to provide laboratories testing the specimens from human immunodeficiency virus-1 (HIV-1) vaccine clinical trials the material for assay development, optimization, and validation. One hundred thirty-one PBMC samples were collected using leukapheresis procedure between 2007 and 2013 by the Comprehensive T cell Vaccine Immune Monitoring Consortium core repository. The donors included 83 human immunodeficiency virus-1 (HIV-1) seronegative and 32 HIV-1 seropositive subjects. The samples were extensively characterized for the ability of T cell subsets to respond to recall viral antigens including cytomegalovirus, Epstein-Barr virus, influenza virus, and HIV-1 using Interferon-gamma (IFN-γ) enzyme linked immunospot (ELISpot) and IFN-γ/interleukin 2 (IL-2) intracellular cytokine staining (ICS) assays. A subset of samples was evaluated over time to determine the integrity of the cryopreserved samples in relation to recovery, viability, and functionality. The principal results of our study demonstrate that viable and functional cells were consistently recovered from the cryopreserved samples. Therefore, we determined that this repository of large size cryopreserved cellular samples constitutes a unique resource for laboratories that are involved in optimization and validation of assays to evaluate T, B, and NK cellular functions in the context of clinical trials.

The External Quality Assurance Oversight Laboratory (EQAPOL) proficiency program for IFN-gamma enzyme-linked immunospot (IFN-γ ELISpot) assay.

The interferon-gamma enzyme-linked immunospot (IFN-γ ELISpot) assay has been developed and used as an end-point assay in clinical trials for infectious diseases and cancer to detect the magnitude of antigen-specific immune responses. The ability to compare data generated by different laboratories across organizations is pivotal to understand the relative potency of different therapeutic and vaccine strategies. We developed an external proficiency program for the IFN-γ ELISpot assay that evaluates laboratory performance based on five parameters: timeliness for data reporting; ability to handle cellular samples; detection of background (non-specific) responses; accuracy to consensus of the results; and precision of the measurements. Points are awarded for each criterion, and the sum of the points is used to determine a numeric and adjectival performance rating. Importantly, the evaluation of the accuracy to the consensus mean for the detection of antigen-specific responses using laboratory-specific procedures informs each laboratory and its sponsor on the degree of concordance of its results with those obtained by other laboratories. This study will ultimately provide the scientific community with information on how to organize and implement an external proficiency program to evaluate longitudinally the performance of the participating laboratories and, therefore, fulfill the requirements of the GCLP guidelines for laboratories performing end-point IFN-γ ELISpot assay for clinical trials.

The Immunology Quality Assessment Proficiency Testing Program for CD3⁺4⁺ and CD3⁺8⁺ lymphocyte subsets: a ten year review via longitudinal mixed effects modeling.

Since 1999, the National Institute of Allergy and Infectious Diseases Division of AIDS (NIAID DAIDS) has funded the Immunology Quality Assessment (IQA) Program with the goal of assessing proficiency in basic lymphocyte subset immunophenotyping for each North American laboratory supporting the NIAID DAIDS HIV clinical trial networks. Further, the purpose of this program is to facilitate an increase in the consistency of interlaboratory T-cell subset measurement (CD3(+)4(+)/CD3(+)8(+) percentages and absolute counts) and likewise, a decrease in intralaboratory variability. IQA T-cell subset measurement proficiency testing was performed over a ten-year period (January 2003-July 2012), and the results were analyzed via longitudinal analysis using mixed effects models. The goal of this analysis was to describe how a typical laboratory (a statistical modeling construct) participating in the IQA Program performed over time. Specifically, these models were utilized to examine trends in interlaboratory agreement, as well as successful passing of proficiency testing. Intralaboratory variability (i.e., precision) was determined by the repeated measures variance, while fixed and random effects were taken into account for changes in interlaboratory agreement (i.e., accuracy) over time. A flow cytometer (single-platform technology, SPT) or a flow cytometer/hematology analyzer (dual-platform technology, DPT) was also examined as a factor for accuracy and precision. The principal finding of this analysis was a significant (p<0.001) increase in accuracy of T-cell subset measurements over time, regardless of technology type (SPT or DPT). Greater precision was found in SPT measurements of all T-cell subset measurements (p<0.001), as well as greater accuracy of SPT on CD3(+)4(+)% and CD3(+)8(+)% assessments (p<0.05 and p<0.001, respectively). However, the interlaboratory random effects variance in DPT results indicates that for some cases DPT can have increased accuracy compared to SPT. Overall, these findings demonstrate that proficiency in and among IQA laboratories have, in general, improved over time and that platform type differences in performance do exist.

Leukopak PBMC sample processing for preparing quality control material to support proficiency testing programs

External proficiency testing programs designed to evaluate the performance of end-point laboratories involved in vaccine and therapeutic clinical trials form an important part of clinical trial quality assurance. Good clinical laboratory practice (GCLP) guidelines recommend both assay validation and proficiency testing for assays being used in clinical trials, and such testing is facilitated by the availability of large numbers of well-characterized test samples. These samples can be distributed to laboratories participating in these programs and allow monitoring of laboratory performance over time and among participating sites when results are obtained with samples derived from a large master set. The leukapheresis procedure provides an ideal way to collect samples from participants that can meet the required number of cells to support these activities. The collection and processing of leukapheresis samples require tight coordination between the clinical and laboratory teams to collect, process, and cryopreserve large number of samples within the established ideal time of ≤8 hours. Here, we describe our experience with a leukapheresis cryopreseration program that has been able to preserve the functionality of cellular subsets and that provides the sample numbers necessary to run an external proficiency testing program.

Comparison of Human Immunodeficiency Virus Antigens as Stimulants for Lymphocyte Proliferation Assays

ABSTRACT CD4 proliferative responses to the human immunodeficiency virus (HIV) type 1 (HIV-1) p24 (gag) antigen inversely correlate with the plasma viral load in HIV-infected subjects who control viral replication without antiretroviral therapy. Use of a single HIV-1 protein to assess CD4 proliferative responses may not reflect the global response to this pathogen. We compared the abilities of HIV p24 and gp120 antigens from two different vendors, an inactivated whole HIV-1 MN virion preparation and an HIV-1E culture supernatant antigen, to elicit proliferative responses in HIV-seropositive and HIV-seronegative donors. Peripheral blood mononuclear cells from 12 HIV-seropositive donors (each with HIV-1 loads <4,000 copies/ml of plasma, >350 CD4 T lymphocytes/mm3, and no antiretroviral therapy) and 15 HIV-seronegative donors were assessed with multiple concentrations of each stimulant by standard lymphocyte proliferation assays. Wide variations in response rates were found, with zero, three, five, and eight individuals demonstrating stimulation indices of >3 for the HIV culture antigen supernatant, gp120, p24, and inactivated whole-virus preparations, respectively. These results suggest that the use of the inactivated whole virus resulted in a more sensitive assay for detection of CD4 T-lymphocyte function in HIV-infected subjects.

Lymphocyte Immunoregulatory Cells Present in Semen From Human Immunodeficiency Virus (HIV)-Infected Individuals: A Report From the HIV Heterosexual Transmission Study

The purpose of this study was to determine the types and distribution of immune subsets present in semen from human immunodeficiency virus (HIV)-infected (HIV+) individuals and to compare these values with those measures in semen from HIV-negative (HIV-) individuals. To accomplish this, a direct three-color monoclonal antibody labeling technique was employed to identify immune cells in fresh ejaculates. Once labeled, the percent of each immune subset present in the ejaculate was determined by flow cytometric analysis. The percent of CD3+ cells present in the semen of the HIV+ group showed no significant difference when compared with semen from the HIV- group. Analysis of the CD4+ subset yielded a significantly lower percent in the HIV+ group than in the HIV- group. The analysis of the CD8+ subset yielded a higher percent of cells present in semen from HIV+ individuals. The CD8 higher value along with lower CD4 value results in a lower CD4/CD8 ratio in the HIV+ group. Further subset studies showed that the percent of cells expressing naive (CD4+ CD45RA+) and memory (CD4+ CD45RO+) markers was lower in the HIV+ group. This study provides additional data supporting the utility of flow cytometry and monoclonal antibodies to immunophenotypic cells present in semen ejaculates. It is also the first reported application of the technique to a disease-based model and may be useful to better understand issues of mucosal immunity and transmission of sexually transmitted diseases such as HIV.

Effect of pregnancy and human immunodeficiency virus infection on intracellular interleukin-2 production patterns.

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infection decreases the production of interleukin-2 (IL-2) from CD4+ and CD8+ T cells. Recombinant IL-2 (rIl-2) has been given to HIV-infected individuals to generate significant increases in CD4+ T-cell counts. There are limited data regarding the effects of pregnancy and HIV infection on IL-2 production in humans. To investigate the effects of human pregnancy, HIV infection, and HIV therapy on IL-2 production, we evaluated 61 women. Intracellular IL-2 production by CD4+ T cells from nonpregnant HIV-infected women was significantly lower than in that in uninfected women (45% ± 8% versus 52% ± 8%, P = 0.04). In contrast, there was no difference in levels of intracellular IL-2 production between HIV-infected and uninfected pregnant women. These observations suggest that pregnancy may down-regulate IL-2 production regardless of HIV infection status. Future studies should evaluate IL-2 production patterns in larger cohorts of women so that the physiological significance of IL-2 down-regulation in pregnancy can be further evaluated. This information is essential to assess the possible use of IL-2 supplementation therapy as a means of enhancing immune responses among HIV-infected pregnant women.