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Bullous pemphigoid (BP) is a prototypic autoimmune disorder of the elderly, characterized by serum IgG autoantibodies, namely anti-BP180 and anti-BP230, directed against components of the basal membrane zone that lead to sub-epidermal loss of adhesion. Pruritus may be indicative of a pre-clinical stage of BP, since a subset of these patients shows serum IgG autoantibodies against BP230 and/or BP180 while chronic pruritus is increasingly common in the elderly population and is associated with a variety of dermatoses. Clinical and experimental evidence further suggests that pruritus of the elderly may be linked to autoimmunity with loss of self-tolerance against cutaneous autoantigens. Thus, the objective of this study was to determine autoreactive T cell responses against BP180 in elderly patients in comparison to patients with BP. A total of 22 elderly patients with pruritic disorders, 34 patients with bullous or non-bullous BP and 34 age-matched healthy controls were included in this study. The level of anti-BP180 and anti-BP230 IgG serum autoantibodies, Bullous Pemphigoid Disease Area Index (BPDAI), and pruritus severity were assessed for all patients and controls. For characterization of the autoreactive T cell response, peripheral blood mononuclear cells were stimulated ex vivo with recombinant BP180 proteins (NH2- and COOH-terminal domains) and the frequencies of BP180-specific T cells producing interferon-γ, interleukin (IL)-5 or IL-17 were subsequently determined by ELISpot assay. Patients with BP showed a mixed Th1/Th2 response against BP180 while autoreactive Th1 cells were identified in a minor subset of elderly patients with pruritic disorders. Furthermore, our T cell characterization revealed that therapeutic application of topical clobetasol propionate ointment in BP patients significantly reduced peripheral blood BP180-specific T cells, along with clinically improved symptoms, strongly suggesting a systemic immunosuppressive effect of this treatment.

Adeno-associated virus (AAV)-based gene therapies have recently shown promise as a novel treatment for hereditary diseases. Due to the viral origin of the vector capsid, however, cellular immune response may be elicited that could eliminate transduced target cells. To monitor cellular immune responses in clinical trials, we optimized and bioanalytically validated a sensitive, robust, and reliable interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) assay. For method performance validation, human peripheral blood mononuclear cells (PBMCs) were stimulated with peptides derived from AAV5 capsid proteins and the encoded transgene product, human blood clotting factor VIII (FVIII), in addition to positive controls, such as peptides from the 65-kDa phosphoprotein of cytomegalovirus. We statistically assessed the limit of detection and confirmatory cutpoint, evaluated precision and linearity, and confirmed specificity using HIV peptides. Robustness parameter ranges and sample stability periods were established. The validated IFN-γ ELISpot assay was then implemented in an AAV5-FVIII gene therapy clinical trial. Cellular immune responses against the AAV5 capsid were observed in most participants as soon as 2 weeks following dose administration; only limited responses against the transgene product were detected. These data underscore the value of using validated methods for monitoring cellular immunity in AAV gene therapy trials., Graphical abstract, This article describes experimental and statistical approaches for the bioanalytical validation of an interferon-γ ELISpot assay to measure cellular immune responses against AAV5 gene therapy. The validated method was subsequently used with clinical trial PBMC samples following AAV5-FVIII administration and detected anti-AAV5 capsid responses in the majority of participants tested.

Evaluation of antigen-specific T-cell responses to viral antigens is frequently performed on IFN-γ secreting cells. However, T-cells are capable of producing many more functions than just IFN-γ, some of which, like Perforin, are associated with immune protection in HIV-1 disease elite controllers. We evaluated the extent of missed T-cell functions when IFN-γ secretion is used as a surrogate marker for further evaluation of T-cell functions. Intracellular cytokine staining assay and flow cytometry were used to assess peripheral blood mononuclear cells (PBMCs) from 31 HIV-infected ART-naive individuals for the extent to which gated CD4+ and CD8+ IFN-γ producing and non-producing T-cells also secreted IL-2, Perforin, and TNF-α functions. Similarly, the extent of missed virus-specific responses in IFN-γ ELISpot assay negative T-cells from 5 HIV-1 uninfected individuals was evaluated. Cells from HIV-infected individuals were stimulated with pooled consensus group M (Con M) peptides; and those from healthy individuals were stimulated with pooled adenovirus (Ad) peptides. Overall, frequencies of virus-specific IFN-γ secreting CD4+ and CD8+ cells were low. Proportions of IFN-γ negative CD4+ expressing IL-2, Perforin, or TNF-α to Con M were significantly higher (5 of 7 functional profiles) than the corresponding IFN-γ positive CD4+ (0 of 7) T-cell phenotype, p = 0.02; Fisher's Exact test. Likewise, proportions of CD8+ T-cells expressing other functions were significantly higher in 4 of the 7 IFN-γ negative CD8+ T-cells. Notably, newly stimulated Perforin, identified as Perforin co-expression with IL-2 or TNF-α, was significantly higher in IFN-γ negative CD8+ T-cell than in the positive CD8+ T-cells. Using SEB, lower responses in IFN-γ positive cells were most associated with CD4+ than CD8+ T-cells. These findings suggest that studies evaluating immunogenicity in response to HIV and Adenovirus viral antigens should not only evaluate T-cell responsiveness among IFN-γ producing cells but also among those T-cells that do not express IFN-γ.

Background Protection against the influenza virus by a specific antibody is relatively strain specific; meanwhile broader immunity may be conferred by cell-mediated immune response to the conserved epitopes across influenza virus subtypes. A universal broad-spectrum influenza vaccine which confronts not only seasonal influenza virus, but also avian influenza H5N1 virus is promising. Methods This study determined the specific and cross-reactive T cell responses against the highly pathogenic avian influenza A (H5N1) virus in four survivors and 33 non-H5N1 subjects including 10 H3N2 patients and 23 healthy individuals. Ex vivo IFN-γ ELISpot assay using overlapping peptides spanning the entire nucleoprotein (NP), matrix (M) and hemagglutinin (HA) derived from A/Thailand/1(KAN-1)/2004 (H5N1) virus was employed in adjunct with flow cytometry for determining T cell functions. Microneutralization (microNT) assay was performed to determine the status of previous H5N1 virus infection. Results IFN-γ ELISpot assay demonstrated that survivors nos. 1 and 2 had markedly higher T cell responses against H5N1 NP, M and HA epitopes than survivors nos. 3 and 4; and the magnitude of T cell responses against NP were higher than that of M and HA. Durability of the immunoreactivity persisted for as long as four years after disease onset. Upon stimulation by NP in IFN-γ ELISpot assay, 60% of H3N2 patients and 39% of healthy subjects exhibited a cross-reactive T cell response. The higher frequency and magnitude of responses in H3N2 patients may be due to blood collection at the convalescent phase of the patients. In H5N1 survivors, the effector peptide-specific T cells generated from bulk culture PBMCs by in vitro stimulation displayed a polyfunction by simultaneously producing IFN-γ and TNF-α, together with upregulation of CD107a in recognition of the target cells pulsed with peptide or infected with rVac-NP virus as investigated by flow cytometry. Conclusions This study provides an insight into the better understanding on the homosubtypic and heterosubtypic T cell-mediated immune responses in H5N1 survivors and non-H5N1 subjects. NP is an immunodominant target of cross-recognition owing to its high conservancy. Therefore, the development of vaccine targeting the conserved NP may be a novel strategy for influenza vaccine design.

Summary Objective This study aimed to assess the performance of a laboratory-developed recombinant CFP-10/ESAT-6 fusion protein (rCFP-10/ESAT-6)-based enzyme-linked immunospot (ELISPOT) assay for the diagnosis of spinal tuberculosis (TB) in China, and to evaluate the value of the ELISPOT assay for monitoring the efficacy of surgical treatment. Methods In the first part of the study, a total of 78 participants were consecutively recruited for ELISPOT using rCFP-10/ESAT-6 as a stimulus. The cutoff value for ELISPOT positivity was based on the results of receiver operating characteristic curve analysis. In the second part, this approach was evaluated in a prospective study including 102 patients with suspected spinal TB. Data on clinical characteristics of the patients and conventional laboratory results were collected, and blood samples were obtained for ELISPOT using rCFP-10/ESAT-6 as a stimulus. Results Among the 102 patients with suspected spinal TB, 11 were excluded from the study. Twenty-three patients (25.2%) had culture-confirmed TB and 29 (31.9%) patients had probable TB. Among the spinal TB patients, the ELISPOT had a sensitivity of 82.7%, compared to a sensitivity of 61.5% for the purified protein derivative (PPD) skin test. The specificity was 87.2% for ELISPOT and 46.2% for the PPD skin test among 39 subjects with non-TB disease. The number of spot-forming cells and/or the positive rate of the ELISPOT assay were associated with aging, emaciation, and paravertebral abscess. The number of subjects with responses to rCFP-10/ESAT-6 slightly decreased after surgical treatment in spinal TB patients. Conclusions A laboratory-developed rCFP-10/ESAT-6 ELISPOT assay is a useful adjunct to current tests for the diagnosis of spinal TB.

International audience; Hypersensitivity pneumonitis (HP) is an immunoallergic disease due to chronic exposure to high quantities of different microorganisms such as Mycobacterium immunogenum (Mi), a mycobacterium, and Lichtheimia corymbifera (Lc), a filamentous fungus. It has recently been demonstrated that the protein DLDH (dihydrolipoyl dehydrogenase), is common to these microorganisms. This study aimed to investigate the immune potential of overlapping peptide pools covering the MiDLDH and LcDLDH.; A selection of 34 peptides, from the MiDLDH and LcDLDH, able to interact with Major Histocompatibility Complex (MHC) 1 and MHC 2, was obtained using three different epitope prediction websites. By means of ELISPOT assays, we compared the frequency of Interferon gamma (IFNγ) secreting peripheral blood mononuclear cells (PBMC) after stimulation with overlapping peptide pools. Tests were performed using cells from 35 healthy blood donors.; One peptide pool containing five peptides from MiDLDH and able to interact with MHC 2 induced a marked IFNγ specific immune response (Pool F, p

Die Entwicklung von Allergien ist auf das komplexe Zusammenspiel zwischen genetischen Faktoren und bestimmten Umwelteinflüssen zurückzuführen. Bei etwa 25% der Menschen entwickeln sich überschießende Immunantworten, die durch harmlose Umweltstoffe wie Pollen oder Nahrungsmittel aber auch Arzneimittel ausgelöst werden können. Die Arzneimittelallergie hat in den letzten Jahren stetig an Bedeutung zugenommen. Hierbei kommt es zu einer immunologisch vermittelten Überempfindlichkeit auf ein Medikament, die sich nach den zugrunde liegenden Pathomechanismen als allergische Soforttypreaktion oder als Spättypreaktion nach Coombs und Gell äußern kann. Die Betalactmantibiotika gehören zu den häufigsten Auslösern allergischer Medikamentenreaktionen. Der Pathomechanismus einer Soforttypreaktion auf Penicilline entspricht einer klassischen IgE-vermittelten Reaktion. Demgegenüber steht die Spättypreaktion, bei der es zur Aktivierung spezifischer T-Lymphozyten und zur Sekretion proinflammatorischer Zytokine kommt, durch die schließlich die entsprechenden Hautreaktion in Form eines Exanthems hervorgerufen wird. Die vorliegende Studie wurde mit vier unterschiedlichen Probandengruppen durchgeführt, bei denen Untersuchungen mit verschiedenen Betalactamantibiotika (Penicillin G, Amoxicillin, Ampicillin und Cefuroxim) durchgeführt wurden. Im Fokus der Studie standen dabei Allergiker vom Spättyp. Diese wurden klinisch und immunologisch mit Kollektiven von Soforttypallergikern sowie von nicht-allergischen Individuen mit und ohne Betalactamexposition als Kontrollgruppen verglichen. Zu diesem Zweck wurden allergologische In-vivo-Testverfahren wie Hauttests (Prick-, Intrakutan-, Epikutantest) und die orale Provokationstestung durchgeführt. Auf Basis der hierbei gewonnenen Ergebnisse erfolgte neben der Anamnese die klinische Charakterisierung und Einteilung in die vorab festgelegten Probandengruppen. Durch die anschließenden Verfahren der In-vitro-Diagnostik wurden sowohl allergenspezifische Antikörper im Serum als auch die Frequenzen allergenspezifischer T-Zellpopulationen im peripheren Blut anhand Zell-spezifischer Markerzytokine (IFN-γ, IL-5 und IL-10) bestimmt. Zusätzlich wurde die Frequenz regulatorischer T(reg)-Zellen anhand des Oberflächenmarkerprofils CD4+CD25+CD127low untersucht. Die hierbei ermittelten immunologischen Parameter der Spättypallergiker wurden mit denen der anderen Studienkollektive verglichen. Die Studienergebnisse zeigten eine unauffällige humorale Antwort im Vergleich der vier verschiedenen Probandengruppen. Auf zellulärer Ebene konnte das Kollektiv der Spättypallergiker nicht über die häufig postulierte vermehrte IFN-γ-Sekretion von den anderen drei Patientengruppen abgegrenzt werden, jedoch fand sich eine gesteigerte Th2-Zellantwort bei den Soforttypallergikern. Es ließen sich keine Unterschiede der Anzahl IL-10-sezernierender Tr1-Zellen nach In-vitro-Betalactamstimulation in allen vier Gruppen detektieren, allerdings zeigte sich in der mit Betalactamantibiotika behandelten Kontrollgruppe ein tendenzieller Anstieg von IFN-γ+ und IL-10+ T-Zellen. Dies könnte ein Hinweis für die Induktion eines protektiven Mechanismus bei gesunden Probanden nach Antibiotikastimulation sein. Andererseits fand sich eine tendenzielle Abnahme der CD4+CD25+CD127low Treg-Population in dieser Kontrollgruppe während der dreitägigen systemischen Betalactamtherapie, was die Komplexität möglicher immunologischer Reaktionsmechanismen auf Betalaktamantibiotika unterstreicht. Insofern liefern die Ergebnisse der vorliegenden Arbeit einige interessante Ansatzpunkte für weitere wissenschaftliche Untersuchungen zu dem insgesamt noch sehr wenig erforschten Gebiet der Arzneimittelallergie., The development of allergies is a complex interaction between genetic factors and environ-mental influences. About 25% of the population acquires an immune-mediated hypersensitivi-ty reaction to harmless environmental antigens like pollen, food or drugs during lifetime. In the last years the impact of drug allergies has increased gradually. The immune-mediated hypersensitivity response to drugs can be classified into an immediate and a non-immediate type according to Coombs and Gell. The pathomechanism of the immediate hypersensitivity reaction is mediated by specific IgE antibodies. In case of delayed-type hypersensitivity reac-tions, drug-specific T cells are activated and secretion of proinflammatory cytokines leads to the respective skin responses like occurrence of exanthema. Among drug-allergies beta-lactam antibiotics are one of the most common triggers. In the present study different antibiotics (penicillin G, amoxicillin, ampicillin and cefuroxime) were tested on four distinct subject cohorts. Since this study mainly focused on the delayed-type of drug reactions, patients with non-immediate type reactions were compared to a cohort of patients with immediate-type hypersensitivity and to two cohorts of non-allergic individu-als, one of them was exposed to beta-lactams. For this purpose different in-vivo-skin tests like intracutaneous, skin prick and epicutaneous tests as well as oral provocation tests were per-formed. On the basis of these test results and the anamneses, subjects were clinically charac-terized and categorized into the four different cohorts. Subsequently, in-vitro-diagnostic methods were performed to analyze the allergen-specific serum antibodies and the frequencies of allergen-specific T cell populations depending on their effector cytokines (IFN-γ, IL-5 and IL-10) in the peripheral blood of the patients. In addition, regulatory T (Treg) cells were quantified according to the surface marker profile CD4+CD25+CD127low. Finally, all of the immunological parameters obtained for the patients with delayed-type drug reactions were compared to the other three cohorts. The results of the study showed no substantial differences in the humoral response between all four tested cohorts. Although an increase of drug-specific IFN-γ+ T cells could not be de-tected in delayed-type, there was an increase of IL-5+ Th2 cells in the cohort of patients with immediate-type drug allergy. Additionally, there were no differences in IL-10-secreting type-1-Treg (Tr1) cells detectable after beta-lactam stimulation of peripheral blood mononuclear cells (PBMC) comparing the four cohorts. However, the cohort of healthy individuals showed an increase of drug-specific IFN-γ+ and IL-10+ T cells by trend. This could indicate a protec-tive immune mechanism in healthy individuals after exposure to beta-lactam antibiotics. On the other hand, a decrease of the CD4+CD25+CD127low Treg population could be observed in the non-allergic control cohort during the three days of systemic beta-lactam treatment. These findings highlight the complexity of immunological mechanisms after drug exposure. Thus, the results of this study might provide both novel insights and new starting points for future research in the field of drug allergy.

In this Phase I/II trial, the patient's peripheral blood dendritic cells were pulsed with an autologous tumour lysate of the glioma. Seven patients with glioblastoma and three patients with anaplastic glioma, ranging in age from 20 to 69 years, participated in this study. The mean numbers of vaccinations of tumour lysate-pulsed dendritic cells were 3.7 times intradermally close to a cervical lymph node, and 3.2 times intratumorally via an Ommaya reservoir. The percentage of CD56-positive cells in the peripheral blood lymphocytes increased after immunisation. There were two minor responses and four no-change cases evaluated by radiological findings. Dendritic cell vaccination elicited T-cell-mediated antitumour activity, as evaluated by the ELISPOT assay after vaccination in two of five tested patients. Three patients showed delayed-type hypersensitivity reactivity to the autologous tumour lysate, two of these had a minor clinical response, and two had an increased ELISPOT result. Intratumoral CD4+ and CD8+ T-cell infiltration was detected in two patients who underwent reoperation after vaccination. This study demonstrated the safety and antitumour effects of autologous tumour lysate-pulsed dendritic cell therapy for patients with malignant glioma.

F4 enterotoxigenic Escherichia coli (ETEC) cause diarrhoea and mortality in piglets leading to severe economic losses. Oral immunization of piglets with F4 fimbriae induces a protective intestinal immune response evidenced by an F4-specific serum and intestinal IgA response. However, successful oral immunization of pigs with F4 fimbriae in the presence of maternal immunity has not been demonstrated yet. In the present study we aimed to evaluate the effect of maternal immunity on the induction of a systemic immune response upon oral immunization of piglets. Whereas F4-specific IgG and IgA could be induced by oral immunization of pigs without maternal antibodies and by intramuscular immunization of pigs with maternal antibodies, no such response was seen in the orally immunized animals with maternal antibodies. Since maternal antibodies can mask an antibody response, we also looked by ELIspot assays for circulating F4-specific antibody secreting cells (ASCs). Enumerating the F4-specific ASCs within the circulating peripheral blood mononuclear cells, and the number of F4-specific IgA ASCs within the circulating IgA(+) B-cells revealed an F4-specific immune response in the orally immunized animals with maternal antibodies. Interestingly, results suggest a more robust IgA booster response by oral immunization of pigs with than without maternal antibodies. These results demonstrate that oral immunization of piglets with F4-specific maternal antibodies is feasible and that these maternal antibodies seem to enhance the secondary systemic immune response. Furthermore, our ELIspot assay on enriched IgA(+) B-cells could be used as a screening procedure to optimize mucosal immunization protocols in pigs with maternal immunity. ispartof: Veterinary Research vol:46 issue:1 ispartof: location:England status: published

Immunological monitoring for CMV can be useful in transplant patients; however, few centers perform it on a routine basis.In this study, CMV-specific cellular response was evaluated in a population of kidney transplant recipients and related to viral infection/reactivation and other demographic and clinical features.Three hundred and twenty-eight patients were studied by EliSPOT assay: 201 prospectively monitored in the first year posttransplantation, 127 with a single determination at1 year. Clinical features, including occurrence of CMV-DNAemia, CMV serostatus, anti-viral strategies and immunosuppressive protocols, were evaluated.Overall, 66.5% of patients were CMV-responders at EliSPOT assay. No episode of infection occurred at follow-up (mean 24.5 months) in 73.4% responders versus 55.5% non-responders (p0.005); CMV-free period was significantly longer in responders (p0.001). Although no significant difference of peak viral load was found, prevalence of CMV-DNAemia values10(5)copies/mL was significantly higher in non-responders versus responders (8.2% and 2.3%, p0.05). Non-responder status was significantly associated to CMV-seronegativity (p0.0001), anti-viral prophylaxis use (p0.0001), and immunosuppression induction with basiliximab (p0.005). No significant association was found for other clinical features and immunosuppressive protocols.Immunological data for CMV could be used in the clinical evaluation and decision-making process, in combination with virological monitoring, in kidney transplant recipients.

Tuberculous pericarditis is a rare disease in developed countries. The diagnosis is difficult to set since there are no robust rapid tests, and culture of pericardial fluid forMycobacterium tuberculosisis often negative. T-SPOT.TB, an enzyme-linked immunospot (ELISPOT) test, measures the gamma interferon response of lymphocytes against tuberculosis antigens and can be performed on blood and body fluids. We describe a patient with tuberculous pericarditis for which the diagnosis was rapidly set by positive T-SPOT.TBresults, which were confirmed by isolation ofMycobacterium tuberculosisin pericardial fluid culture. We performed a literature search to assess the diagnostic potential of ELISPOT testing in tuberculous pericarditis. The limited data on this subject indicate that T-SPOT.TBaids in diagnosing active tuberculosis (TB) infection and results in a more rapid decision to start antituberculosis treatment. Enumerating TB-specific lymphocytes and testing blood/compartmental fluid simultaneously can provide useful information on active tuberculous pericarditis.

No consensus has been reached on how to determine if an immune response has been detected based on raw data from an ELISPOT assay. The goal of this paper is to enable investigators to understand and readily implement currently available methods for response determination. We describe empirical and statistical approaches, identifying the strengths and limitations of each approach to allow readers to rationally select and apply a scientifically sound method appropriate to their specific laboratory setting. Five representative approaches were applied to data sets from the CIMT Immunoguiding Program and the response detection and false positive rates were compared. Simulation studies were also performed to compare empirical and statistical approaches. Based on these, we recommend the use of a non-parametric statistical test. Further, we recommend that six medium control wells or four wells each for both medium control and experimental conditions be performed to increase the sensitivity in detecting a response, that replicates with large variation in spot counts be filtered out, and that positive responses arising from experimental spot counts below the estimated limit of detection be interpreted with caution. Moreover, a web-based user interface was developed to allow easy access to the recommended statistical methods. This interface allows the user to upload data from an ELISPOT assay and obtain an output file of the binary responses. Electronic supplementary material The online version of this article (doi:10.1007/s00262-010-0875-4) contains supplementary material, which is available to authorized users.

An HIV-1 reservoir comprised primarily of latently infected resting CD4(+) T lymphocytes that can be stimulated in vivo to produce virus may play a critical role in mother-to-child postnatal transmission of HIV-1 by breastfeeding. Here, we describe an experimental protocol for the detection of resting CD4(+) T cell HIV-1 reservoir from breast milk. We adapted a method for the purification of resting CD4(+) T lymphocytes in blood to isolate resting CD4 T+ cells in breast milk from HIV-1-infected-lactating women (n = 18) and from controls (n = 3). Purified resting CD4 T+ cells from blood and breast milk samples of HIV-1-infected-lactating women were polyclonally stimulated to characterize and enumerate HIV-1-antigen-secreting cells (HIV-1-Ag-SCs) by an enzyme-linked immunospot (ELISpot) assay. Resting CD4 T+ cells represented more than 90% of purified viable breast milk cells. CD4(+) T cell polyclonal stimulation combined with the ELISpot assay led to the characterization of a breast milk T cell HIV-1 reservoir greater than the blood reservoir (median 400 and 57.14 HIV-1-Ag-SCs/10(6) resting CD4(+) T cells, respectively,p < 0.001). our strategy could be adapted to other body fluids and be useful for characterizing new HIV-1 reservoirs.