Lymphocyte Activation Test Research Articles

Clinical characteristics and immune profiles of patients with immune-mediated alopecia associated with COVID-19 vaccinations

BackgroundThe clinical characteristics and pathomechanism for immune-mediated alopecia following COVID-19 vaccinations are not clearly characterized. ObjectiveWe investigated the causality and immune mechanism of COVID-19 vaccines-related alopecia areata (AA). Study design27 new-onset of AA patients after COVID-19 vaccinations and 106 vaccines-tolerant individuals were enrolled from multiple medical centers for analysis. ResultsThe antinuclear antibody, total IgE, granulysin, and PARC/CCL18 as well as peripheral eosinophil count were significantly elevated in the patients with COVID-19 vaccines-related AA compared with those in the tolerant individuals (P = 2.03 × 10−5-0.039). In vitro lymphocyte activation test revealed that granulysin, granzyme B, and IFN-γ released from the T cells of COVID-19 vaccines-related AA patients could be significantly increased by COVID-19 vaccine excipients (polyethylene glycol 2000 and polysorbate 80) or spike protein (P = 0.002–0.04). ConclusionsSpike protein and excipients of COVID-19 vaccines could trigger T cell-mediated cytotoxicity, which contributes to the pathogenesis of immune-mediated alopecia associated with COVID-19 vaccines.

Associations of HLA-A and HLA-B with vancomycin-induced drug reaction with eosinophilia and systemic symptoms in the Han-Chinese population.

Vancomycin is a commonly used antibiotic; however, it can cause life-threatening severe cutaneous adverse reactions, such as drug reaction with eosinophilia and systemic symptoms (DRESS). A previous study has reported a strong association between HLA-A*32:01 and vancomycin-induced DRESS in European ethnicity. Herein, we aim to investigate the genetic predisposition of vancomycin-induced DRESS in the Han-Chinese population. In this study, we enrolled a total of 26 patients with vancomycin-induced DRESS, 1,616 general population controls, and 51 subjects tolerant to vancomycin. In vitro granulysin-based lymphocyte activation tests (LAT) were conducted among 6 vancomycin-induced DRESS patients who were concomitantly receiving other medicines. HLA-A and HLA-B genotypes were determined by sequencing-based typing. Our results found that vancomycin-induced DRESS was associated with HLA-A*32:01 [odds ratio (OR) = 7.8, 95% confidence interval (CI) = 1.7-35.8; p-value = 0.035], HLA-B*07:05 (OR = 32.3, 95% CI = 2.8-367.7; p-value = 0.047), HLA-B*40:06 (OR = 4.7, 95% CI = 1.3-16.1; p-value = 0.036) and HLA-B*67:01 (OR = 44.8, 95% CI = 7.2-280.4; p-value = 0.002) when comparing the vancomycin-induced DRESS patients with the general population controls. LAT results showed that granulysin significantly increased in the vancomycin-induced DRESS patients upon vancomycin stimulation (4.7 ± 3.7 fold increased), but not upon other co-medicines. This study identified that, in addition to HLA-A*32:01, HLA-B*07:05, HLA-B*40:06, and HLA-B*67:01 were also genetic markers for vancomycin-induced DRESS in the Han-Chinese population. Associations of ethnic variances in HLA with vancomycin-DRESS were observed.

The risk of anti-osteoporotic agent-induced severe cutaneous adverse drug reactions and their association with HLA.

There is increasing use of anti-osteoporotic agents (AOA) worldwide for prevention or management of patients with osteoporosis. However, there have been reports of severe cutaneous adverse reactions (SCAR) induced by AOA. A recent study showed weak association between HLA and strontium ranelate (SR)-SCAR. To characterize patients with AOA-SCAR and investigate the HLA association and utility of in vitro diagnostic methods. We enrolled 16 cases with AOA-cutaneous adverse drug reactions (cADR), including SCAR (n=10: 8 with Stevens-Johnson syndrome [SJS] and 2 with drug rash with eosinophilia and systemic symptoms [DRESS]) and maculopapular exanthema (MPE) (n=6) from Taiwan and Hong Kong. We analysed the clinical characteristics, outcomes, HLA alleles and in vitro testing of AOA-SCAR, and tolerability to alternative drugs. We further performed literature review and meta-analysis on the HLA association of AOA-SCAR. Our data showed strontium ranelate is the most common causality of AOA-SCAR in Asian populations. There was no cross-hypersensitivity of SR-SCAR with other AOA. HLA genotyping showed that SR-SJS was most significantly associated with HLA-A*33:03 (Pc=5.17×10-3 , OR: 25.97, 95% CI: 3.08-219.33). Meta-analysis showed that HLA-A*33:03 was associated with SR-SJS (P=5.01×10-5 ; sensitivity: 85.7%) in Asians. The sensitivity of lymphocyte activation test (LAT) for identifying the culprit drug of SR-SJS was 83.3%. Strontium ranelate is identified as the most notorious AOA associated with SCAR. The HLA-A*33:03 genetic allele and LAT testing may add benefits to the diagnosis of SR-SCAR in patients whose reaction developed while taking multiple drugs.

Granulysin-Based Lymphocyte Activation Test for Evaluating Drug Causality in Antiepileptics-Induced Severe Cutaneous Adverse Reactions

Aromatic antiepileptic drugs (AEDs) are common causes of cutaneous adverse drug reactions, which range from morbilliform drug eruption to life-threatening severe cutaneous adverse reactions, including drug reaction with eosinophilia and systemic symptoms, Stevens‒Johnson syndrome, and toxic epidermal necrolysis. Different invitro methods for identifying the culprit drugs have been developed; however, it is particularly challenging for Stevens‒Johnson syndrome-toxic epidermal necrolysis. In this study, we enrolled 63 patients (39 with Stevens‒Johnson syndrome-toxic epidermal necrolysis, 13 with drug reaction with eosinophilia and systemic symptoms, and 11 with morbilliform drug eruption) and 30 tolerant controls to examine the performance of lymphocyte activation tests by measuring the expression of granulysin, granzyme B, and IFN-γ. Granulysin-based lymphocyte activation tests displayed the best sensitivity and specificity to identify the causality: 73.9% sensitivity and 96.7% specificity for carbamazepine and 68.2% sensitivity and 96.7% specificity for phenytoin. Oxcarbazepine and lamotrigine show weak antigenicity. Granulysin-based lymphocyte activation tests expanded predominantly memory cytotoxic T lymphocytes with characteristics of drug-specific T-cell receptor, major histocompatibility complex I dependence, and cross reactivity to different aromatic AEDs. Among 29 follow-up patients, 28 alternatively used nonaromatic AEDs, and none developed cutaneous adverse drug reactions. Our data suggest that granulysin-based lymphocyte activation tests represent invitro cytotoxic T-lymphocyte memory response to offending drugs and are useful to confirm drug causality of AED-induced severe cutaneous adverse reactions. Implementing these tests will improve the AED-induced severe cutaneous adverse reactions prevention and clinical care.

Evaluation of Cellular Responses for the Diagnosis of Allergic Bronchopulmonary Mycosis: A Preliminary Study in Cystic Fibrosis Patients.

Background: Allergic bronchopulmonary mycosis (ABPM) is an underestimated allergic disease due to fungi. Most reported cases are caused by Aspergillus fumigatus (Af) and are referred to as allergic bronchopulmonary aspergillosis (ABPA). The main risk factor of ABPA is a history of lung disease, such as cystic fibrosis, asthma, or chronic obstructive pulmonary disease. The main diagnostic criteria for ABPA rely on the evaluation of humoral IgE and IgG responses to Af extracts, although these cannot discriminate Af sensitization and ABPA. Moreover, fungi other than Af have been incriminated. Flow cytometric evaluation of functional responses of basophils and lymphocytes in the context of allergic diseases is gaining momentum.Objectives: We hypothesized that the detection of functional responses through basophil and lymphocyte activation tests might be useful for ABPM diagnosis. We present here the results of a pilot study comparing the performance of these cellular assays vs. usual diagnostic criteria in a cystic fibrosis (CF) cohort.Methods: Ex vivo basophil activation test (BAT) is a diagnostic tool highlighting an immediate hypersensitivity mechanism against an allergen, e.g., through CD63 upregulation as an indirect measure of degranulation. Lymphocyte stimulation test (LST) relies on the upregulation of activation markers, such as CD69, after incubation with allergen(s), to explain delayed hypersensitivity. These assays were performed with Af, Penicillium, and Alternaria extracts in 29 adult CF patients.Results: BAT responses of ABPA patients were higher than those of sensitized or control CF patients. The highest LST result was for a woman who developed ABPA 3 months after the tests, despite the absence of specific IgG and IgE to Af at the time of the initial investigation.Conclusion: We conclude that basophil and lymphocyte activation tests could enhance the diagnosis of allergic mycosis, compared to usual humoral markers. Further studies with larger cohorts and addressing both mold extracts and mold relevant molecules are needed in order to confirm and extend the application of this personalized medicine approach.

Lyme Borreliosis: Serological and Microbiological Diagnostics andDifferential Diagnosis

Lyme borreliosis caused by at least six Borrelia burgdorferi species, is the most important tick-borne disease in the northern hemisphere. With a wide spectrum of possible symptoms, the microbiological diagnosis of this disease isassociated with a wide variety of ideas, divergent approaches and much uncertainty. The aim of the article is to introduce the treating physicians to a meaningful microbiological diagnostic procedure. Most important messages include that (I) the suspected diagnosis of Lyme borreliosis is first based on anamnestic data and clinical symptoms (compare "case definitions") which is substantiated by microbiological examinations; (II) microbiological diagnostics - primarily antibody detection, downstream PCR and culture - are only indicated if there are indicative symptoms, with typical erythema migrans not requiring microbiological diagnostics; (III) keep in mind: The more unspecific the symptoms are the lower the positive and the higher the negative predictive value of microbiological testing; (IV) serological diagnosis should follow a two-step procedure: a sensitive ELISA as first step, if reactive followed by immunoblot (IgM and IgG). Detection rates are ca. 50 % in localized, 70- > 90 % in disseminated early and nearly 100 % (only IgG relevant) in late disease; (V) in the immunoblot early forms of disease show a narrow band spectrum, late forms show a broad spectrum; (VI) methods that are not recommended for diagnostic purposes include lymphocyte activation or transformation tests (LTT, MELISA, ELISPOT), PCR or antigen detection from urine or blood, lymphocyte subpopulations, or direct detection of borreliae from patient material using dark field- or focus floating microscopy.

Development of a Peptide Derived from Platelet-Derived Growth Factor (PDGF-BB) into a Potential Drug Candidate for the Treatment of Wounds.

Objective: This study evaluated the use of novel peptides derived from platelet-derived growth factor (PDGF-BB) as potential wound healing stimulants. One of the compounds (named PDGF2) was subjected for further research after cytotoxicity and proliferation assays on human skin cells. Further investigation included evaluation of: migration and chemotaxis of skin cells, immunological and allergic safety, the transcriptional analyses of adipose-derived stem cells (ASCs) and dermal fibroblasts stimulated with PDGF2, and the use of dorsal skin wound injury model to evaluate the effect of wound healing in mice. Approach: Colorimetric lactate dehydrogenase and tetrazolium assays were used to evaluate the cytotoxicity and the effect on proliferation. PDGF2 effect on migration and chemotaxis was also checked. Immunological safety and allergic potential were evaluated with a lymphocyte activation and basophil activation test. Transcriptional profiles of ASCs and primary fibroblasts were assessed after stimulation with PDGF2. Eight-week-old BALB/c female mice were used for dorsal skin wound injury model. Results: PDGF2 showed low cytotoxicity, pro-proliferative effects on human skin cells, high immunological safety, and accelerated wound healing in mouse model. Furthermore, transcriptomic analysis of ASCs and fibroblasts revealed the activation of processes involved in wound healing and indicated its safety. Innovation: A novel peptide derived from PDGF-BB was proved to be safe drug candidate in wound healing. We also present a multifaceted in vitro model for the initial screening of new compounds that may be potentially useful in wound healing stimulation. Conclusion: The results show that peptide derived from PDGF-BB is a promising drug candidate for wound treatment.

Vemurafenib acts as an aryl hydrocarbon receptor antagonist: Implications for inflammatory cutaneous adverse events.

In recent years, the BRAF inhibitor vemurafenib has been successfully established in the therapy of advanced melanoma. Despite its superior efficacy, the use of vemurafenib is limited by frequent inflammatory cutaneous adverse events that affect patients' quality of life and may lead to dose reduction or even cessation of anti-tumor therapy. To date, the molecular and cellular mechanisms of vemurafenib-induced rashes have remained largely elusive. In this study, we deployed immunohistochemistry, RT-qPCR, flow cytometry, lymphocyte activation tests, and different cell-free protein-interaction assays. We here demonstrate that vemurafenib inhibits the downstream signaling of the canonical pathway of aryl hydrocarbon receptor (AhR) in vitro, thereby inducing the expression of proinflammatory cytokines (eg, TNF) and chemokines (eg, CCL5). In line with these results, we observed an impaired expression of AhR-regulated genes (eg, CYP1A1) and an upregulation of the corresponding proinflammatory genes in vivo. Moreover, results of lymphocyte activation tests showed the absence of drug-specific T cells in respective patients. Taken together, we obtained no hint of an underlying sensitization against vemurafenib but found evidence suggesting that vemurafenib enhances proinflammatory responses by inhibition of canonical AhR signaling. Our findings contribute to our understanding of the central role of the AhR in skin inflammation and may point toward a potential role for topical AhR agonists in supportive cancer care.

1057 Vemurafenib acts as an aryl hydrocarbon receptor antagonist

The BRAF-inhibitor vemurafenib has been successfully established in the therapy of advanced melanoma. Despite its superior efficacy, the use of vemurafenib is limited by frequent inflammatory cutaneous adverse events that affect patients quality of life and may lead to dose reduction or even cessation of anti-tumor therapy. To date, the molecular and cellular mechanisms of vemurafenib-induced rashes have remained largely elusive. Against this background, we here demonstrate that vemurafenib inhibits the downstream signaling of the canonical pathway of aryl hydrocarbon receptor (AhR) in vitro, thereby inducing the expression of proinflammatory cytokines (e.g. IL1B, TNF) and chemokines (e.g. CCL5). In line with these results we observed an impaired expression of AhR regulated genes (e.g. CYP1A1) and an upregulation of the corresponding proinflammatory genes in vivo. Moreover, results of lymphocyte activation tests showed the absence of drug-specific T cells in respective patients. Taken together, we obtained no hint of an underlying sensitization against vemurafenib but found evidence suggesting that vemurafenib enhances proinflammatory responses by inhibition of AhR signaling. Our findings contribute to our understanding of the central role of the AhR in skin inflammation and may point towards a potential role for topical AhR agonists in supportive cancer care.

The Function of HLA-B*13:01 Involved in the Pathomechanism of Dapsone-Induced Severe Cutaneous Adverse Reactions

Dapsone-induced hypersensitivity reactions may cause severe cutaneous adverse reactions, such as drug reaction with eosinophilia and systemic symptoms (DRESS). It has been reported that HLA-B*13:01 is strongly associated with dapsone-induced hypersensitivity reactions among leprosy patients. However, the phenotype specificity and detailed immune mechanism of HLA-B*13:01 remain unclear. We investigated the genetic predisposition, HLA-B*13:01 function, and cytotoxic T cells involved in the pathogenesis of dapsone-induced severe cutaneous adverse reactions. We enrolled patients from Taiwan and Malaysia with DRESS and maculopapular eruption with chronic inflammatory dermatoses. Our results showed that the HLA-B*13:01 allele was present in 85.7% (6/7) of patients with dapsone DRESS (odds ratio= 49.64, 95% confidence interval= 5.89-418.13; corrected P= 2.92× 10-4) but in only 10.8% (73/677) of general population control individuals in Taiwan. The level of granulysin, the severe cutaneous adverse reaction-specific cytotoxic protein released from cytotoxic T cells, was increased in both the plasma of DRESS patients (36.14 ± 9.02 ng/ml, P < 0.05) and invitro lymphocyte activation test (71.4%, 5/7 patients) compared with healthy control individuals. Furthermore, dapsone-specific cytotoxic T cells were significantly activated when co-cultured with HLA-B*13:01-expressing antigen presenting cells in the presence of dapsone (3.9-fold increase, compared with cells with no HLA-B*13:01 expression; P < 0.01). This study indicates that HLA-B*13:01 is strongly associated with dapsone DRESS and describes a functional role for the HLA-restricted immune mechanism induced by dapsone.

Delayed‐type hypersensitivity reactions induced by proton pump inhibitors: A clinical and in vitro T‐cell reactivity study

Proton pump inhibitors (PPIs) have been known to induce type I hypersensitivity reactions. However, severe delayed-type hypersensitivity reactions (DHR) induced by PPI, such as Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), or drug rash with eosinophilia and systemic symptoms (DRESS), are rarely reported. We conducted a study of a large series of PPI-related DHR, followed up their tolerability to alternative anti-ulcer agents, and investigated the T-cell reactivity to PPI in PPI-related DHR patients. We retrospectively analyzed patients with PPI-related DHR from multiple medical centers in Taiwan during the study period January 2003 to April 2016. We analyzed the causative PPI, clinical manifestations, organ involvement, treatment, and complications. We also followed up the potential risk of cross-hypersensitivity or tolerability to other PPI after their hypersensitivity episodes. Drug lymphocyte activation test (LAT) was conducted by measuring granulysin and interferon-γ to confirm the causalities. There were 69 cases of PPI-related DHR, including SJS/TEN (n=27) and DRESS (n=10). The LAT by measuring granulysin showed a sensitivity of 59.3% and specificity of 96.4%. Esomeprazole was the most commonly involved in PPI-related DHR (51%). Thirteen patients allergic to one kind of PPI could tolerate other structurally different PPI without cross-hypersensitivity reactions, whereas three patients developed cross-hypersensitivity reactions to alternative structurally similar PPI. The cross-reactivity to structurally similar PPI was also observed in LAT assay. PPIs have the potential to induce life-threatening DHR. In patients when PPI is necessary for treatment, switching to structurally different alternatives should be considered.

Non-immediate Cutaneous Reactions to Beta-Lactams: Approach to Diagnosis

Non-immediate cutaneous reactions (i.e., occurring at least 1h after the initial drug administration), particularly maculopapular exanthemas and urticarial eruptions, are common during beta-lactam treatments. A T cell-mediated pathogenic mechanism has been demonstrated in some cutaneous reactions, such as maculopapular exanthema, fixed drug eruption, acute generalized exanthematous pustulosis, and drug-induced hypersensitivity syndrome. In the diagnostic work-up, patch testing is useful, together with delayed-reading intradermal testing. Patch tests are a simple and safe diagnostic tool, which in the case of severe reactions should be used as the first line of investigation. However, patch tests are less sensitive than intradermal tests, which are preferable in subjects with mild reactions. Lymphocyte transformation or activation tests and enzyme-linked immunosorbent spot assays can be used as complementary tests. In selected cases of mild or moderate reactions, displaying negative results in the aforesaid allergy tests, a graded challenge with the implicated beta-lactam can be performed.

Methazolamide-induced toxic epidermal necrolysis confirmed by lymphocyte activation test

Methazolamide-induced toxic epidermal necrolysis confirmed by lymphocyte activation test

Allergic sensitization to pegylated interferon-α results in drug eruptions.

The introduction of pegylated interferon (PEG-IFN)-α in the treatment of chronic hepatitis C has led to an increase in sustained virological response. Despite reduced immunogenicity of the pegylated form in comparison with native interferon (IFN)-α, a high frequency of adverse cutaneous reactions has been reported with pegylated IFN-α. Here, we aimed to investigate the immunological mechanisms underlying pegylated IFN-α-induced drug eruptions. Hepatitis C patients suffering from drug eruptions in association with administration of pegylated interferons were enrolled in the study (n=22). Subjects were tested for sensitivity to pegylated IFN-α2a , pegylated IFN-α2b , or ribavirin using intradermal, scratch, and/or patch tests, as well as lymphocyte activation tests (LATs). Skin biopsies obtained from pegylated IFN-α-associated exanthemas, as well as from localized inflammatory skin reactions at pegylated IFN-α injection sites, were analyzed for the expression of relevant chemokines by quantitative real-time PCR and immunohistochemistry. A subset of patients suffering from pegylated IFN-α-associated exanthemas displayed positive intradermal tests to PEG-IFNs but not to conventional IFN (11/22). In selected patients, this observation correlated with the presence of pegylated IFN-specific T cells (3/11). Chemokine profiles of inflammatory skin reactions at the injection sites reflected an IFN-α-signature, whereas lesional skin of exanthemas showed induction of TH2-associated chemokines. Our results indicate that specific sensitizations are one cause of exanthemas under therapy with PEG-IFNs. Clinical proof-of-concept analyses demonstrate that affected patients may benefit from a switch to conventional,nonpegylated drugs, enabling IFN-α therapy continuation without drug-associated skin eruptions.

Two cases of cutaneous drug eruption associated with temozolomide therapy for glioblastoma.

Glioblastoma is the most common form of primary brain cancer. Its treatment involves surgery, radiotherapy, and chemotherapy with temozolomide (tmz), which is an oral alkylating agent. To the best of our knowledge, few dermatologic side effects of tmz have been described. We report two cases of cutaneous drug eruption caused by tmz during and after radiochemotherapy treatment. In the first case, all tests were negative, but the clinical history and the time of onset supported an allergy to tmz. In the second case, an allergy to tmz was proved by a positive lymphocyte activation test. In this context, our study is one of a very few trying to determine dermatologic side effects by applicable tests used in routine practice.

A New In Vitro Flow Cytometry Method For Detection Of Delayed Drug Sensitization To Local Anesthetics. The Lymphocyte Activation Test (LAT)

A New In Vitro Flow Cytometry Method For Detection Of Delayed Drug Sensitization To Local Anesthetics. The Lymphocyte Activation Test (LAT)

Diagnostic des réactions d’hypersensibilité allergique et non allergique aux médicaments courants de l’enfant : arbre décisionnel

Suspected allergic reactions to drugs and biological substances (anti-infectious drugs and antipyretics, non opioid analgesics and nonsteroidal anti-inflammatory drugs especially) are reported in 5 to 12% of children. Most frequent reactions are morbilliform/maculopapular rashes, urticaria and angioedema. Other cutaneous and respiratory reactions, and severe allergic and non-allergic anaphylactic reactions are rare. The results of studies based on allergological tests and/or microbiological/serological tests strongly suggest that, except for a few types of reactions (anaphylactic and/or immediate reactions, potentially harmful toxidermias) and for very specific drugs (i.e. latex and myorelaxants), most reactions to commonly used drugs and biological substances in children do not result from drug hypersensitivity, but are rather a consequence of the infectious and/or inflammatory diseases for which the drugs have been prescribed. Non-immediate reactions may also result from complex interactions between drugs, immune system and “danger signals” provided or induced by infectious and/or inflammatory diseases. Diagnosis is based above all on a detailed analysis of clinical history, skin tests (if validated), and challenge tests (if indicated). At present, the diagnostic and predictive values of in vitro tests exploring immediate (specific IgE determination, histamine and leukotriene release tests, basophil activation test) and non-immediate type (lymphocyte activation tests, and cytokine assays in the supernatant of activated blood mononuclear cells) of drug hypersensitivity are not validated.

Diagnosis and management of drug hypersensitivity reactions

The present article addresses the advances in the diagnosis and management of drug hypersensitivity reactions that were discussed in the 4th Drug Hypersensitivity Meeting held in Rome in April 2010. Such reactions can be classified as immediate or nonimmediate according to the time interval between the last drug administration and onset. Immediate reactions occur within 1 hour, and nonimmediate reactions occur after more than 1 hour. Clinical and immunologic studies suggest that type-I (IgE-mediated) and type-IV (T cell-mediated) pathogenic mechanisms are involved in most immediate and nonimmediate reactions, respectively. In diagnosis prick, patch, and intradermal tests are the most readily available tools. Determination of specific IgE levels is still the most common in vitro method for diagnosing immediate reactions. New diagnostic tools, such as the basophil activation test, the lymphocyte activation test, and enzyme-linked immunospot assays for analysis of the frequency of antigen-specific, cytokine-producing cells, have been developed for evaluating either immediate or nonimmediate reactions. The sensitivity of allergologic tests is not 100%; therefore in selected cases provocation tests are necessary. In the diagnosis of nonallergic hypersensitivity reactions to nonsteroidal anti-inflammatory drugs, the provocation test with the suspected drug still represents the "gold standard." However, there was no consensus regarding the use of this test in subjects with histories of hypersensitivity reactions to 1 (single reactors) or more (multiple reactors) nonsteroidal anti-inflammatory drugs. With regard to management, desensitization allows patients to be treated with irreplaceable chemotherapy agents, such as taxanes, platinum salts, and mAbs, to which they have presented hypersensitivity reactions. Desensitization also permits the use of aspirin in aspirin-sensitive patients undergoing revascularization and in subjects with aspirin-exacerbated respiratory disease.

Lymphocyte activation test for diagnosis of seronegative Brucellosis in humans

Seronegative brucellosis is occasionally encountered in clinical practice especially in localized disease where diagnosis is reached mainly through positive blood culture. Cellular immune responses are pivotal for protection against intracellular bacteria such as Brucella. This study was performed to evaluate the expression of activation markers on peripheral blood mononuclear cells in response to in vitro stimulation by whole-cell suspension of Brucella melitensis for the diagnosis of brucellosis. Fifteen seronegative patients with positive blood cultures for Brucella and twenty-five unexposed healthy blood donors serving as controls were recruited for the study. Peripheral blood mononuclear (PBMC) cells were obtained by the method of Ficoll Hypaque density gradient and stimulated in vitro with Brucella antigen. Expression of activation markers was assessed by flow cytometry after staining of PBMC with mononuclear cells with relevant monoclonal antibodies. Incubation with mitogen induced expression of all the four markers was demonstrated in all blood samples. In contrast, samples from all patients of Brucellosis showed significant positive responses with the expression of activation markers (CD38, CD69, CD25, and CD71) on both CD4+ and CD8+ cells as compared with the control group (P < 0.001). It is inferred that there was a remarkable upregulation of activation markers on CD4+ and CD8+ in seronegative patients with Brucellosis. It is recommended that the method can be utilized as a novel diagnostic test for detection of brucellosis where serology is negative.

Para‐phenylenediamine‐specific lymphocyte activation test: a sensitive in vitro assay to detect para‐phenylenediamine sensitization in patients with severe allergic reactions

Patients sensitized to para-phenylenediamine (PPD) by semi-permanent tattoos increasingly develop threatening allergic reactions in response to black hair dye. The gold standard to diagnose allergic contact dermatitis is to perform epicutaneous patch tests, however, iatrogenic sensitizations and severe patch test reactions to PPD have been described, the latter especially in patients with severe allergic reactions. We examined nine patients with severe allergic reactions in response to permanent hair dyes. Patch tests using the standard concentration of 1% or 0.5% PPD resulted in severe and sometimes even bullous reactions in all patients responsive to PPD. Titration revealed that at 1% of the standard concentration (0.01% PPD), patch test sensitivity decreased and only 50% of patients responded. Consequently, we established an in vitro assay to diagnose PPD allergy. Freshly isolated peripheral blood mononuclear cells (PBMC) were cultured with titrated concentrations of PPD with or without IL-2 supplementation, and cell proliferation was determined by [3H]-thymidine incorporation. Lymphocyte activation test (LAT) detected PBMC cell proliferation specific to PPD, with at least 3.5-fold increase in [3H]-thymidine uptake in all PPD allergic patients. Most importantly, PPD-LAT without IL-2 supplementation remained negative in three out of eight PPD allergic patients. Thus, PPD-LAT with IL-2 supplementation demonstrated a sensitivity of 100%, remained unresponsive in controls not sensitized to PPD, and in one patient sensitive to other p-amino compounds. These data demonstrate that LAT with PPD can be used to detect PPD sensitization as a possible alternative to patch testing at least in patients with severe allergic reactions to PPD.