Enzyme-linked Immunospot Method Research Articles

536 Comparing the Performance of Interferon Gamma Release Assays in Autoimmune Skin Diseases

Autoimmune skin disease patients are screened for tuberculosis (TB) before initiation of immunosuppressive drugs via interferon gamma release assays (IGRAs). The two available tests, the QuantiFERON-TB Gold Plus (QFT-Plus, Qiagen, Hilden, Germany) and the T-SPOT.TB test (T-SPOT, Oxford Immunotec, Abingdon, UK) measure IFN-γ levels produced in response to T-cell stimulation with TB antigens. The T-SPOT test uses an enzyme-linked immunospot method rather than the enzyme-linked immunosorbent assay, the basis of the QFT-Plus test.

Preparation of nanoliposomes linked to HER2/neu-derived (P5) peptide containing MPL adjuvant as vaccine against breast cancer.

The study was aimed at evaluating antitumor and immunomodulatory effects of liposomal vaccine composed of P5 human epidermal growth factor receptor 2 (HER2)/neu-derived peptide coupled to the surface of high-temperature nanoliposomes containing distearoylphosphocholine:distearoylphosphoglycerol:Chol:dioleoylphosphatidylethanolamine (DOPE) comprising monophosphoryl lipid A (MPL) adjuvant in HER2/neu overexpressing the breast cancer model. BALB/c mice bearing TUBO carcinoma were subcutaneously immunized with formulations containing 10 µg P5 peptide and 25 µg MPL three times with 2-week intervals. To determine immuno responses in immunized mice, the amount of released interferon-γ and IL-4 were measured by the enzyme-linked immunospot method and the flow cytometric analysis on the isolated splenocytes. The results demonstrated that tumor-bearing mice immunized with Lip/DOPE/MPL/P5 formulation had the most released interferon-γ and the highest cytotoxic T lymphocyte responses that led to the lowest tumor size and the longest survival time than those of other formulations. The results achieved by Lip/DOPE/MPL/P5 formulation could make it a suitable candidate to induce effective antigen-specific tumor immunity against breast cancer.

An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi

Lyme Borreliosis is an infectious disease caused by the spirochete Borrelia burgdorferi that is transmitted through the bite of infected ticks. Both B cell-mediated humoral immunity and T cell immunity develop during natural Borrelia infection. However, compared with humoral immunity, the T cell response to Borrelia infection has not been well elucidated. In this study, a novel T cell-based assay was developed and validated for the sensitive detection of antigen-specific T cell response to B. burgdorferi. Using interferon-γ as a biomarker, we developed a new enzyme-linked immunospot method (iSpot LymeTM) to detect Borrelia antigen-specific effector/memory T cells that were activated in vivo by exposing them to recombinant Borrelia antigens ex vivo. To test this new method as a potential laboratory diagnostic tool, we performed a clinical study with a cohort of Borrelia positive patients and healthy controls. We demonstrated that the iSpot Lyme assay has a significantly higher specificity and sensitivity compared with the Western Blot assay that is currently used as a diagnostic measure. A comprehensive evaluation of the T cell response to Borrelia infection should, therefore, provide new insights into the pathogenesis, diagnosis, treatment and monitoring of Lyme disease.

Clinical, Immunological, and Epidemiological Importance of Antituberculosis T Cell Responses in HIV-Infected Africans

Human immunodeficiency virus (HIV)-associated tuberculosis is a major cause of mortality in Africa. The assay of T cell interferon- gamma released in response to antigens of greater specificity than purified protein derivative is a useful improvement over the Mantoux tuberculin skin test, but few studies have evaluated interferon-gamma secretion in HIV-infected individuals. Mycobacterium tuberculosis antigen-specific interferon-gamma secretion was assessed by whole blood assay and enzyme-linked immunospot, which were compared with the Mantoux tuberculin skin test in HIV-infected and HIV-uninfected individuals without active tuberculosis and HIV-infected patients with pulmonary tuberculosis in Khayelitsha, South Africa. The skin test and whole blood assay responses to purified protein derivative in HIV-positive subjects were decreased, compared with responses in HIV-negative subjects (P < .001). By contrast, the responses to M. tuberculosis antigens (early secreted antigenic target 6, culture filtrate protein 10, TB10.3, and alpha-crystallin 2) were less affected, indicating a high prevalence of latent tuberculosis (approximately 80%) in both HIV-negative and HIV-positive subject groups. Whole blood assay responses did not differ between the HIV-positive subjects without tuberculosis and HIV-positive subjects with tuberculosis, but the enzyme-linked immunospot method response to early secreted antigenic target 6 and culture filtrate protein 10 was higher in the group of HIV-infected subjects with tuberculosis (P < or = .04), although this group had lower CD4+ cell counts. A ratio of the combined enzyme-linked immunospot method response divided by the CD4+ cell count of > 1.0 had 88% sensitivity and 80% specificity for active pulmonary tuberculosis in HIV-infected individuals. Interferon-gamma release appears to be less impaired than skin testing by HIV coinfection. The novel potential to relate the enzyme-linked immunospot method and CD4+ cell count to assist diagnosis of active tuberculosis in patients with HIV infection is important and deserves further evaluation.

Borrelia-specific interferon-gamma and interleukin-4 secretion in cerebrospinal fluid and blood during Lyme borreliosis in humans: association with clinical outcome.

The Borrelia-specific interferon (IFN)- gamma and interleukin (IL)-4 responses of 113 patients and control subjects were analyzed using the sensitive enzyme-linked immunospot method. Cerebrospinal fluid (CSF) and blood samples were obtained, during the course of disease, from patients with chronic or nonchronic neuroborreliosis (NB) and from control subjects without NB. Blood samples were obtained from patients with Lyme skin manifestations and from healthy blood donors. Early increased secretion of Borrelia-specific IFN- gamma (P<.05) and subsequent up-regulation of IL-4 (P<.05) were detected in the CSF cells of patients with nonchronic NB. In contrast, persistent Borrelia-specific IFN- gamma responses were observed in the CSF cells of patients with chronic NB (P<.05). In patients with erythema migrans, increased IFN- gamma (P<.001) was observed in blood samples obtained early during the course of disease, whereas increased IL-4 (P<.05) was observed after clearance. On the contrary, patients with acrodermatitis chronica atrophicans had Borrelia-specific IFN- gamma (P<.001), but not IL-4, detected in blood samples. The present data suggest that an initial IFN- gamma response, followed by up-regulation of IL-4, is associated with nonchronic manifestations, whereas a persistent IFN- gamma response may lead to chronic Lyme borreliosis.

Analysis of cellular immune responses in the peripheral blood of mice using real-time RT-PCR

Murine cancer models are commonly used in the evaluation of immunotherapeutic strategies. However, one of the major limitations in the monitoring of cellular immune responses induced by various vaccination approaches is that existing immunoassays require sacrifice of the animals for collection of the spleen or lymph nodes for analysis. We report here the development of an assay to quantitate antigen-specific T cell responses in murine blood, without euthanasia, using real-time RT-PCR for measurement of interferon-γ mRNA levels. C57BL/6 mice were immunized with an adenoviral vector encoding the melanoma antigen gp100 (Ad2/gp100) or were left untreated. Small samples of whole blood were collected by retro-orbital puncture for analysis of T cell reactivity. The mice were then euthanized and spleen cells were isolated for comparative analyses. Blood and spleen cells were restimulated with either a peptide containing the dominant gp100 MHC Class I-restricted epitope, gp100 25–33, or a negative control peptide containing an irrelevant Class I-restricted epitope from ovalbumin. IFN-γ mRNA was detected in gp100 peptide-pulsed whole blood as well as in spleen cells recovered from Ad2/gp100-treated mice, but not in untreated mice. In addition, there was a strong correlation in the magnitude of the gp100-specific response of spleen cells from an individual animal when measured by real-time RT-PCR with the more conventional enzyme-linked immunospot (ELISPOT) method ( P<0.001). Finally, the gp100-specific immune response measured in the peripheral blood of individual animals by real-time RT-PCR or ELISPOT showed a significant correlation with the response measured in the spleen ( P=0.001). We conclude that real-time RT-PCR measurement of IFN-γ mRNA induced by antigenic stimulation is an attractive method to measure an antigen-specific cellular immune response in small samples of whole blood as it does not require euthanasia, mirrors the response observed in the spleen and correlates with the response measured using the conventional ELISPOT method.

Exclusive Th2 Primary Effector Function in Spleens but Mixed Th1/Th2 Function in Lymph Nodes of Murine Neonates

Recent studies have shown that neonatal mice are competent to develop mature, Ag-specific Th1 function in situ. However, under many conditions, Th2 responses dominate in the neonate, while Th1 responses are more prevalent in adults. To compare further the immune responses of neonates and adults, we used the enzyme-linked immunospot method to measure the frequencies of primary Th1/Th2 effectors generated in situ in the spleens and lymph nodes. As assessed by the detection of IFN-gamma- or IL-4-producing cells, adults developed mixed Th1/Th2 responses in both organs. Neonatal lymph nodes contained mature frequencies of IFN-gamma- and IL-4-producing cells. In striking contrast, while mature frequencies of Th2 cells developed in neonatal spleens, virtually no IFN-gamma-secreting cells were detected. Exclusive Th2 function was observed in both BALB/c and C57BL/6 neonates, strains in which the Th2 and Th1 lineages, respectively, are favored in adults. Although Th1 effectors were virtually undetectable, the addition of rIL-12 boosted the frequency of IFN-gamma-secreting cells to adult levels. Therefore, Th1 effectors apparently developed in situ, but Th1 effector function either was not promoted or was inhibited upon subsequent exposure to the Ag in culture. Together, these results indicate that the quality of a primary Th response in neonates is strongly dependent on the site of initial Ag exposure; responses initiated in the lymph nodes are mixed Th1/Th2, whereas responses occurring in the spleen are heavily Th2 biased.

CAESAREAN SECTION DELIVERY IS ACCOMPANIED WITH DEFECTIVE CAPACITY TO GENERATE IGA IN NEONATES

63 AIM Microbial antigens preferentially expand T helper 1-type cells and thereby provide optimal conditions to redirect the immunologic memory away from the T helper 2-type phenotype of atopy. Caesarean section delivery interferes with the development of normal intestinal microflora which contribute to the development of mucosal immunity in the gut. This study aimed to evaluate the effect of caesarean delivery on IgA generation. METHODS Blood samples were obtained during 12 vaginal deliveries (VD) and 16 elective caesarean sections (ECS), and 2 and 6 months later. The number of immunoglobulin secreting cells (ICS)/ 106 cord and peripheral blood mononuclear cells were determined by the enzyme-linked immunospot method, the immunophenotypes by a flow cytometer and the cytokine concentrations in plasma/serum samples by ELISA. RESULTS The number of ISC/106 IgA-secreting cells expressed in median (interquartile ranges) in cord blood samples of VD vs. ECS were 19.5 (5.0-25.7) vs. 4.9 (0.0-8.4) respectively, p=0.03. The percentage of NK cells in ECS samples tended to be lower than that in VD; 12.2 (14.6-9.7) in ECS and 18.3 (25.1-11.3) in VD. The concentration of IL6 of ECS samples was lower than that of VD. These differences at birth were no longer significant when the infants were 2 and 6 months old. CONCLUSIONS These results indicate that ECS predisposes neonates to low capacity to generate IgA concomitantly with low circulating levels of IL6 and NK cells. Caesarean section delivery can hinder the first-line nonspecific immunity to enteral antigens of microbial or dietary origin at a critical period of life when the immune memory is yet to be established. This may explain the observation that the incidence of atopic diseases increases concurrently with the frequency of elective caesarean section deliveries in the developed countries.

Diltiazem modulates monokine production in human mixed lymphocyte culture.

Calcium channel blockers are widely used in transplantation. Their immunosuppressive activity is well known and has been demonstrated both in vitro and in vivo. Nevertheless, their effect on cytokine production has never been reported. One-way mixed lymphocyte cultures (MLCs) have been obtained from healthy human subjects. Cytokine production has been assessed by three different methods: by enzyme-linked immunosorbent assay on supernatants of MLC, by enzyme-linked immunospot method on MLC cells for measuring cytokine-producing cells, and by the reverse transcription polymerase chain reaction technique on MLC cells for measuring cytokine mRNAs. An interesting effect on proinflammatory monokines was observed: in this study, we demonstrate that the calcium antagonist diltiazem enhances interleukin-1beta and slightly reduces interleukin-6 production in MLC, but it has no effect on tumor necrosis factor-alpha levels. For the first time, a modulation of monokine production by diltiazem can be demonstrated. This evidence suggests that calcium antagonist drugs may exert effects on monocytes and possibly on other antigen-presenting cells.

Enhancement of the antibody response to protein antigens by specific IgG under different experimental conditions.

Specific stimulation of the antibody response to protein antigens by IgG antibodies was studied in vivo. The response to TNP-coupled keyhole limpet haemocyanin (KLH-TNP) was enhanced by a TNP-specific IgG monoclonal antibody, 7B4, as measured both in ELISA (enzyme-linked immunosorbent assay) and by ELISPOT (enzyme-linked immunospot) method. Enhancement was seen when using both high and low doses of antigen and antibody. The antibody response to TNP-coupled bovine serum albumin (BSA-TNP), but not to TNP-coupled ovalbumin (OA-TNP), tetanus toxoid (TT-TNP) or diphtheria toxoid (DT-TNP), was also efficiently augmented by 7B4. Enhancement was seen when using both high and low coupling ratio of TNP to KLH. The fact that IgG-mediated enhancement is seen under many different experimental conditions suggests that it is a physiologically relevant phenomenon. In order to enhance the response to KLH-TNP and BSA-TNP, 7B4 had to be injected while antigen was circulating in blood. This supports that IgG-mediated stimulation acts by concentrating circulating antigen into lymphoid centres.