Search results for "ELISPOT"
showing 10 items of 48 documents
Differences in Kaposi sarcoma-associated herpesvirus-specific and herpesvirus-non-specific immune responses in classic Kaposi sarcoma cases and match…
2011
Kaposi sarcoma (KS) might develop because of incompetent immune responses, both non-specifically and specifically against the KS-associated herpesvirus (KSHV). Peripheral blood mononuclear cells from 15 classic (non-AIDS) KS cases, 13 KSHV seropositives (without KS) and 15 KSHV-seronegative controls were tested for interferon-γ T-cell (enzyme-linked immunospot [Elispot]) responses to KSHV-latency-associated nuclear antigen (LANA), KSHV-K8.1 and CMV/Epstein-Barr virus (EBV) peptide pools. The forearm and thigh of each participant was also tested for delayed-type hypersensitivity (DTH) against common recall antigens. Groups were compared with Fisher exact test and multinomial logistic regress…
The Impact of Harmonization on ELISPOT Assay Performance
2011
During more than 25 years of application in immunological sciences, ELISPOT has been established as a routine, robust, versatile, and reliable assay. From basic research to clinical immune monitoring, ELISPOT is being used to address the quantification and (to a lesser extent) functional characterization of immune cells secreting different molecules in the context of health and disease, immune intervention, and therapy in humans and other species [Kalyuzhny (Ed.) (2005) Handbook of Elispot: methods and protocols, Vol. 302, Humana Press Inc., Totowa, NJ]. Over the last decade, ELISPOT assays have been increasingly implemented as an immune-monitoring tool in clinical trials [Schmittel et al. …
Response Determination Criteria for ELISPOT: Toward a Standard that Can Be Applied Across Laboratories
2011
ELISPOT assay readout is often dichomized as positive or negative responses according to prespecified criteria. However, these criteria can vary widely across institutions. The adoption of a common response criterion is a key step toward cross-laboratory comparability. This chapter describes the two main approaches to response determination, identifying the strengths and limitations of each. Nonparametric statistical tests and consideration of data quality are recommended and instructions provided for their ready implementation by nonstatisticians and statisticians alike.
The use of HLA-A*0201-transfected K562 as standard antigen-presenting cells for CD8+ T lymphocytes in IFN-γ ELISPOT assays
2001
ELISPOT assays are increasingly used for a direct detection and quantification of single antigen-specific T cells in freshly isolated peripheral blood mononuclear cells (PBMC). They are particularly attractive for the monitoring of specific T lymphocyte responses in clinical trials assessing antigen-specific immunizations in patients with cancer or chronic viral infections. However, one major limitation for the broad clinical implementation of ELISPOT assays is the lack of an inexhaustible source of suitable HLA-matched antigen-presenting cells (APC). Currently available allogeneic or xenogeneic APC (such as the human lymphoid hybrid T2 or HLA-transfected insect cells) can either lead to st…
Rapid Expansion of Acute Myeloid Leukemia-Reactive Cytotoxic T Cells from CD8+CD62L+ Blood Lymphocytes of HLA-Matched Healthy Donors In Vitro
2006
Abstract Allogeneic cytotoxic T-lymphocyte (CTL) therapy in acute myeloid leukemia (AML) is hampered by the poor efficiency of growing leukemia-reactive CTLs from healthy donors in vitro. We established an allogeneic mini-mixed lymphocyte-leukemia culture (MLLC) approach by stimulating comparably small numbers (104/well) of CD8+ T cells isolated from healthy donors against irradiated primary AML blasts in 96-well plates. Prior to use, CD8+ T cells were immunomagnetically separated into a CD62L(high)+ subset enriched for naive precursors and central memory cells as well as a CD62L(low)+/negative subset containing effector memory cells. The culture medium contained IL-7, IL-12, and IL-15. Aft…
Evolution of viral quasispecies in four dominant HlA-A2 restricted T cell epitopes is not a major reason for viral persistence in interferon-treated …
2002
In most patients, chronic hepatitis C virus (HCV) infection persists despite antiviral treatment with interferon-alpha (IFN-alpha) and ribavirin. The aim of the study was to determine whether HCV could evade cellular immune responses through mutations within T cell epitopes. Viral sequences flanking four major CTL epitopes within the HCV core and envelope regions were analyzed by PCR amplification, cloning and sequencing in seven HLA-A2 positive HCV patients before, during and after antiviral therapy. In addition, cytotoxic T lymphocyte precursor (CTLp) frequencies specific to these epitopes were quantitated by ELISPOT. A total of 13 coding mutations were observed among 650 cloned and seque…
Quantification of CD8+ T lymphocytes responsive to human immunodeficiency virus (HIV) peptide antigens in HIV-infected patients and seronegative pers…
1998
/ T cells responding to HIV-1 peptides were observed in none of 11 HIV- seronegative donors without a history of HIV exposure. ELISPOT assays are relatively fast and easy to perform and appear to reliably detect T cell reactivity due to previous exposure to HIV. These findings support the use of the ELISPOT assay for monitoring T cell responsiveness to HIV peptides. In acute infection with the human immunodeficiency virus We described recently an enzyme-linked immunospot (ELISPOT) assay to detect and quantitate single blood-de- type 1 (HIV-1), initial reduction in virus load is associated with the appearance of a high frequency of antiviral cytotoxic T rived CD8 / T lymphocytes forming tumo…
Cellular and humoral immune responses induced by intradermal or intramuscular vaccination with the major hepatitis B surface antigen
2000
The vaccination route may influence the success of immunization against pathogens. The conventional intramuscular (i.m.) application of a vaccine containing the hepatitis B virus (HBV) surface antigen (HBsAg) led to protective anti-HBs antibody levels in the majority of vaccine recipients. In this study, we vaccinated healthy volunteers and a group of i.m. vaccine nonresponders via the intradermal (i.d.) route and analyzed the HBV-specific B-cell response as well as class-II- and class-I-restricted T-cell responses by 3 H-thymidine uptake, enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISPOT). The results were then compared with i.m. vaccinated controls. I.d…
Clinical and immunological efficacy of intradermal vaccine plus lamivudine with or without interleukin-2 in patients with chronic hepatitis B
2002
To evaluate therapeutic immunostimulation nine chronic hepatitis B patients received six monthly intradermal vaccinations with HBsAg in combination with daily lamivudine. Another five patients received six doses of the vaccine and daily lamivudine together with daily Interleukin-2 (IL-2) s.c. within 3 months in an open-labeled trial. Clinical efficacy was assessed by alanine transaminase levels and HBV serology. The induction of specific T and B cell responses was analyzed serially by 3H-thymidine uptake, ELISA and ELISPOT assays. After the therapy was stopped, seven of nine vaccine/lamivudine and two of five vaccine/lamivudine/IL-2 recipients did not have detectable HBV DNA. Four complete …
Identification of a Conserved HLA-A2-Restricted Decapeptide from the IE1 Protein (pUL123) of Human Cytomegalovirus
2002
Abstract Control of human cytomegalovirus (HCMV) infection is predominantly mediated by cytolytic CD8 + T lymphocytes (CTL). Among the roughly 200 HCMV-encoded polypeptides, the tegument protein pp65 (ppUL83) and the nonstructural IE1 protein are considered to be dominant CTL targets. Yet the importance of CTL against IE1 for protective immunity against HCMV reactivation and disease has remained elusive. Analyses have been difficult, as all MHC class I presented peptides of IE1 defined so far are located in parts of the protein that are variable between viral strains. In this study a conserved decameric peptide from IE1 (P6, IE1 354–363 ) that bound to HLA-A2 was identified. Using peptide-p…