Immunohistological assessment of cutaneous drug hypersensitivity in patients with HIV infection.

The pathogenesis of drug hypersensitivity in patients with HIV infection is unknown. To study further the nature of hypersensitivity, the histopathological features of morbilliform drug hypersensitivity reactions were examined in a group of HIV-infected patients. Skin sections from 23 HIV-infected subjects with morbilliform drug hypersensitivity reactions were examined by light microscopy, direct immunofluorescence and immunohistochemistry, to determine the nature of the inflammatory infiltrate and the role of immunoglobulin, complement and cytokines. The principal light microscopic findings were spongiosis, hydropic generation of the basal layer, civatte bodies, an epidermal lymphocytic infiltrate (48%), and a perivascular dermal infiltrate of lymphocytes (87%) and macrophages (52%). Two patients had findings consistent with toxic epidermal necrolysis. Immunohistochemistry demonstrated that the lymphocytic infiltrate consisted of CD8+, HLA-DR+ T lymphocytes (some of which also stained for CD38), a marked depletion of epidermal Langerhans cells (90%), and strong cytoplasmic staining of keratinocytes for IL-6 (60%), IL-1 beta (50%), tumour necrosis factor-alpha (TNF-alpha) (45%) and to a lesser degree, interferon-gamma (IFN-gamma) (35%). Immunofluorescence did not demonstrate any significant deposition of immunoglobulin or complement. The histological findings were independent of the responsible drug, the duration of either therapy or the rash, and of peripheral blood CD4+ and CD8+ cell counts. These findings suggest that activated CD8+ lymphocytes and perhaps epidermal production of cytokines are involved in the pathogenesis of cutaneous drug hypersensitivity in HIV-infected patients. The common histological features, regardless of the causative drug, suggest a common pathogenesis.     

In vitro analysis of metabolic predisposition to drug hypersensitivity reactions

Idiosyncratic hypersensitivity reactions may account for up to 25% of all adverse drug reactions, and pose a constant problem to physicians because of their unpredictable nature, potentially fatal outcome and resemblance to other disease processes. Current understanding of how drug allergy arises is based largely on the hapten hypothesis: since most drugs are not chemically reactive per se, they must be activated metabolically to reactive species which may become immunogenic through interactions with cellular macromolecules. The role of drug metabolism is thus pivotal to the hapten hypothesis both in activation of the parent compound and detoxification of the reactive species. Although conjugation reactions may occasionally produce potential immunogens (for example, the generation of acylglucuronides from non-steroidal anti-inflammatory drugs such as diclofenac), bioactivation is catalysed most frequently by cytochrome P450 (P450) enzymes. The multifactorial nature of hypersensitivity reactions, particularly the role of often unidentified, reactive drug metabolites in antigen generation, has hampered the routine diagnosis of these disorders by classical immunological methods designed to detect circulating antibodies or sensitized T cells. Similarly, species differences in drug metabolism and immune system regulation have largely precluded the establishment of appropriate animal models with which to examine the immunopathological mechanisms of these toxicities. However, the combined use of in vitro toxicity assays incorporating human tissues and in vivo phenotyping (or, ultimately, in vitro genotyping) methods for drug detoxification pathways may provide the metabolic basis for hypersensitivity reactions to several drugs. This brief review highlights recent efforts to unravel the bases for hypersensitivity reactions to these therapeutic agents (which include anticonvulsants and sulphonamides) using drug metabolism and Immunochemical approaches. In particular, examples are provided which illustrate breakthroughs in the identification of the chemical nature of the reactive metabolites which become bound to cellular macromolecules, the enzyme systems responsible for their generation and (possibly) detoxification, and the target proteins implicated in the subsequent immune response.     

CD69 upregulation on T cells as an in vitro marker for delayed-type drug hypersensitivity

Background:  T cells play a key role in delayed-type drug hypersensitivity reactions. Their reactivity can be assessed by their proliferation in response to the drug in the lymphocyte transformation test (LTT). However, the LTT imposes limitations in terms of practicability, and an alternative method that is easier to implement than the LTT would be desirable.
Methods:  Four months to 12 years after acute drug hypersensitivity reactions, CD69 upregulation on T cells of 15 patients and five healthy controls was analyzed by flow cytometry.
Results:  All 15 LTT-positive patients showed a significant increase of CD69 expression on T cells after 48 h of drug-stimulation exclusively with the drugs incriminated in drug-hypersensitivities. A stimulation index of 2 as cut-off value allowed discrimination between nonreactive and reactive T cells in LTT and CD69 upregulation. T cells (0.5–3%) showed CD69 up-regulation. The reactive cell population consisted of a minority of truly drug reactive T cells secreting cytokines and a higher number of bystander T cells activated by IL-2 and possibly other cytokines.
Conclusions:  CD69 upregulation was observed after 2 days in all patients with a positive LTT after 6 days, thus appearing to be a promising tool to identify drug-reactive T cells in the peripheral blood of patients with drug-hypersensitivity reactions.     

Delayed-type hypersensitivity responses to HIV Gag p24 relate to clinical outcome after peptide-based therapeutic immunization for chronic HIV infection

Therapeutic immunization in chronic HIV infection aims to induce durable, HIV-specific immune responses capable of controlling disease progression, but immunological correlates of clinical efficacy are poorly defined. We have previously immunized 38 patients with a mixture of four short Gag p24-like conserved peptides (Vacc-4x) targeting skin dendritic cells. Antiretroviral treatment (ART) was initially stopped after completed immunizations and resumed post-protocol during regular clinical follow-up according to current guidelines. Four years after enrolment, Vacc-4x-specific cellular responses were evaluated in vivo by delayed-type hypersensitivity (DTH) skin test, and in vitro by a T-cell proliferation assay. Kaplan-Meier product-limit estimates were used to analyse time until ART was resumed. Peptide-specific cellular immune responses induced by Vacc-4x had persisted 4 years after the last immunization in terms of unchanged DTH independent of ART and detectable proliferative T-cell responses which correlated to the native peptides (R = 0.73, p = 0.01). Circulating bifunctional (IFN-γ and IL-10) Vacc-4x-specific T-cell clones were detected in 43% of patients. Subjects with the strongest post-immunization DTH responses appeared to start ART later compared with DTH low responders (p = 0.07). These data suggest that DTH responses should be further evaluated as a new and convenient tool for predicting clinical efficacy in trials testing therapeutic immunizations.     

Impaired in vitro survival of monocytes from patients with HIV infection.

In vitro survival of monocytes (MO) was studied in 59 patients with HIV infection of different clinical stages. MO from 61 donors and 12 healthy seronegative homosexual men were also examined. Compared with the number of MO seeded, the percentage of adherent monocyte-derived macrophages (MDM) present after 10 days was significantly lower in patients with HIV infection than in the controls. However, the number of viable, non-adherent MO/MDM was similar in patients and controls. Our data indicate markedly decreased in vitro survival of MO from patients with HIV infection. After 10 days, the MDM population in the patient cultures was significantly less differentiated than the control cells, assessed by immunocytochemical staining with monoclonal antibodies against differentiation antigens. Reduced in vitro survival of MO/MDM was associated with low numbers of CD4+ and CD8+ lymphocytes in blood, reduced lymphocyte mitogen responses, presence of HIV p24 antigen in serum and advanced clinical stage. Decreased in vitro survival of MO/MDM may be associated with HIV replication in the cells. Although the level of HIV replication in the cultures was low as assessed by measurement of HIV p24 antigen in culture supernatants and staining of MO/MDM for HIV antigens, cytopathogenic effects of HIV or HIV products cannot be ruled out.     

In vitro detection and characterization of drug hypersensitivity using flow cytometry

Background:  The lymphocyte transformation test (LTT) is the only in vitro test for detecting drug sensitization at the cellular level irrespective of the reaction's phenotype. However, the LTT includes working with radioactive substances and is considered impracticable for routine laboratory investigation.
Objective:  The aim of this study was to assess drug-specific cytokine production by means of flow cytometry as an alternative nonradioactive approach which may be more appropriate for routine testing and may provide in addition more information about the pathophysiology of the reaction than proliferation-based assays, like the LTT.
Method:  Peripheral blood mononuclear cells of 19 patients were incubated with culprit drugs ( n  = 28) or irrelevant antigens ( n  = 10). Ten healthy persons served as controls for all different drugs ( n  = 15). Intracellular interleukin (IL)-5, interferon (IFN)-γ and IL-10 production was investigated using flow cytometry. Accuracy of the flow cytometry test system was confirmed using different statistical tests, i.e. receiver operating characteristic curve and Mann–Whitney rank test. In addition, drug-specific secretion of IL-5, IL-2 and IFN-γ were analysed using enzyme-linked immunosorbent assay (ELISA).
Results:  Drug-specific cytokine production could be demonstrated in 75% of the patients using flow cytometry and in 79% using ELISA respectively. Combining ELISA and flow cytometry increased the sensitivity to 100%. Analysis of involved T-cell subsets [e.g. CD4⁺ or CD8⁺; T helper (TH) 1 or TH 2] allowed characterization of the in vitro lymphocyte reactivity pattern.
Conclusions:  Analysis of drug-specific cytokine production by means of flow cytometry proved a useful and reliable approach for the in vitro detection and characterization of drug hypersensitivities.     

Ethanol as a cause of hypersensitivity reactions to alcoholic beverages

BACKGROUND: Adverse reactions after ingestion of alcoholic beverages are common. Metabolic differences in individuals and also the histamine content in alcoholic beverages have been implicated. By contrast pure ethanol has rarely been reported as a cause of hypersensitivity reactions and its mechanism has not been clarified yet. OBJECTIVE: To determine whether ethanol itself accounts for alcohol hypersensitivity in patients with anaphylactic reactions after alcohol intake. In search of possible pathomechanisms all patients were analysed by skin prick testing and sulfidoleukotriene production of peripheral leucocytes using ethanol and its metabolites. METHODS: Double-blind, placebo-controlled food challenges with a cumulated amount of 30 mL ethanol were performed in 12 adult patients with a positive history of adverse reactions after consumption of different alcoholic beverages. Skin prick tests and measurement of sulfidoleukotriene production were performed using different concentrations of ethanol and acetaldehyde from 50 to 1000 mm. RESULTS: Oral challenges with pure ethanol were positive in six out of eleven patients. All challenge-positive patients, but also four out of five challenge-negative patients, showed an increased sulfidoleukotriene production in-vitro compared with healthy controls. Skin prick tests using alcoholic beverages, ethanol, acetaldehyde and acetic acid were negative in all patients (12/12). CONCLUSION: Our study shows that ethanol itself is a common causative factor in hypersensitivity reactions to alcoholic beverages. These reactions occur dose-dependent and a non-IgE-mediated pathomechanism is likely, because skin prick tests were negative in all cases. Increased sulfidoleukotriene production was determined in some patients, but is no reliable predictor. Therefore oral provocation tests remain indispensable in making the diagnosis of ethanol hypersensitivity.     

In vitro tests of T cell-mediated drug hypersensitivity

Allergic side-effects of drugs are a major problem of drug therapy. Diagnosis of drug-hypersensitivity reactions in patient is still very difficult as a broad spectrum of different drugs can elicit various immune-mediated diseases with distinct pathomechanisms. In this review, we summarize current in vitro techniques with an emphasis on diagnosis of T cell- mediated drug allergies and discuss recent research finding that may be applied to develop tests allowing for a better diagnosis of drug hypersensitivity in the future.     

In vitro immunomodulatory properties of tucaresol in HIV infection

The immunomodulatory properties of tucaresol (compound 589C80) were tested on in vitro antigen- and mitogen-stimulated proliferation and cytokine production by peripheral blood mononuclear cells (PBMC) of HIV-infected individuals and healthy controls (HC). Results showed that tucaresol: (1) increases influenza A virus-, gp 160 peptide-, and HLA alloantigen-stimulated proliferation as well as interleukin (IL)-2 and interferon gamma (IFN gamma) production by PBMC of HIV-infected individuals with higher CD4 counts (>500/microl) but had only a marginal immunomodulatory effect on PBMC of patients with lower CD4 counts (<500/microl); (2) did not modify IL-10 production; (3) augmented CD25 expression on mitogen-stimulated T cells of HC but not of HIV-infected individuals; and (4) marginally increased CTL activity. The immunomodulatory properties of tucaresol were confirmed by PCR analyses; additional data showed that tucaresol costimulated CD3-dependent triggering of T cells and that this stimulation was independent of CD28 costimulation. The immunomodulatory effects of tucaresol on T cell functions are characterized by a bell-shaped dose response curve; the action of the compound is optimal in the 100 to 300 microM range. Analyses of mitogen-stimulated apoptosis demonstrated that the lack of effect of tucaresol at higher doses is not the result of increased cell death, suggesting a role of functional impairment. These data confirm that tucaresol can stimulate T helper cell function and enhance the production of type 1 cytokines, thus eliciting cell-mediated immunity, and warrant its potential utility in the therapy of HIV infection.     

Skin testing in patients with hypersensitivity reactions to iodinated contrast media – a European multicenter study

Background:  Iodinated contrast media cause both immediate and nonimmediate hypersensitivity reactions. The aim of this prospective study was to determine the specificity and sensitivity of skin tests in patients who have experienced such reactions.
Methods:  Skin prick, intradermal and patch tests with a series of contrast media were conducted in 220 patients with either immediate or nonimmediate reaction. Positive skin tests were defined according to internationally accepted guidelines. Seventy-one never-exposed subjects and 11 subjects who had tolerated contrast medium exposure, served as negative controls.
Results:  Skin test specificity was 96–100%. For tests conducted within the time period from 2 to 6 months after the reaction, up to 50% of immediate reactors and up to 47% of nonimmediate reactors were skin test positive. For immediate reactors, the intradermal tests were the most sensitive, whereas delayed intradermal tests in combination with patch tests were needed for optimal sensitivity in nonimmediate reactors. Contrast medium cross-reactivity was more common in the nonimmediate than in the immediate group. Interestingly, 49% of immediate and 52% of nonimmediate symptoms occurred in previously unexposed patients. Many of these patients were skin test positive, indicating that they were already sensitized at the time of first contrast medium exposure.
Conclusions:  These data suggest that at least 50% of hypersensitivity reactions to contrast media are caused by an immunological mechanism. Skin testing appears to be a useful tool for diagnosis of contrast medium allergy and may play an important role in selection of a safe product in previous reactors.     

Chronic delayed-type hypersensitivity reaction as a means to treat alopecia areata

The acute phase of alopecia areata (AA) is characterized by an increase in CD44v3+ and CD44v10+ skin-infiltrating leucocytes (SkIL). Induction of a contact eczema, one of the therapeutic options in AA, can be mitigated strongly by a blockade of CD44v10. The observation that induction of a delayed type hypersensitivity (DTH) reaction abrogates an autoimmune reaction, where both responses apparently use similar effector mechanisms, is surprising and prompted us to search for the underlying mechanisms. AA-affected C3H/HeJ mice were treated with the contact sensitizer SADBE (squaric acid dibutylester) and leucocyte subpopulations and their activation state was evaluated in SkIL and draining lymph nodes. AA-affected mice exhibited an increased number of SkIL with a predominance of T lymphocytes. After treatment with the contact sensitizer SADBE recovery of SkIL was reduced and monocytes predominated. However, a significantly increased number of leucocytes was recovered from draining lymph nodes. Draining lymph node cells from untreated and treated AA mice exhibited all signs of recent activation with high-level expression of co-stimulatory and accessory molecules and an increased percentage of CD44v3+ and CD44v10+ leucocytes. In contrast, SkIL of SADBE-treated AA mice contained relatively few activated T cells and reduced numbers of CD44v3+ and CD44v10+ cells. Thus, the activation state and the distribution of leucocyte subsets in SADBE-treated AA mice are consistent with a blockade of leucocyte extravasation. Accordingly, the therapeutic effect of long-term SADBE treatment may rely on impaired leucocyte traffic.     

In vitro induction of delayed-type hypersensitivity.

Murine spleen cells, cultured in vitro for 6 days in the presence of high concentrations of burro erythrocytes (BRBC), are sensitized to exhibit delayed-type hypersensitivity (DTH) specific for this antigen. Such cells, on being injected with antigen into the footpads of normal mice, cause a 24-h swelling reaction. This activity of the cultured cells requires the presence of BRBC both during the in vitro incubation and in the footpad. The activity of the sensitized cells in causing swelling is sensitive to anti-Thy-1 antibody and complement, and the kinetics of the swelling reaction are characteristic of a DTH response. In vivo low-dose priming of the spleen cell donors considerably enhances the ability of the cultured cells to cause swelling. This system provides a means of studying the regulation of the induction of DTH in vitro.     

In vitro delayed hypersensitivity in normal and hyporeactive patients

The in vitro macrophage migration inhibition test can be used to evaluate human delayed hypersensitivity. Using purified protein derivative of tuberculin (PPD) as the antigen, twenty of twenty-seven in vitro tests in non-anergic persons with negative PPD skin tests were negative and fifteen of sixteen in vitro tests in persons with positive skin tests were positive. In patients with drug or disease-induced cutaneous hyporeactivity, twelve of twenty-eight tests were positive despite negative skin tests. In two anergic patients with mucocutaneous candidiasis positive in vitro tests were obtained with Candida albicans antigen as well. Measurable levels of IgG were seldom detected in the test media.

The results indicate that the macrophage migration inhibition test measures delayed hypersensitivity in man and is sometimes positive in cases of reduced skin reactivity.

     

The significance of hypersensitivity to nuts in patients with birch pollen allergy

This study was performed in patients with allergic rhinitis/conjunctivitis to birch pollen to determine whether patients with additional hypersensitivity to nuts and apples differed from patients without such hypersensitivity; the determination was in terms of results of skin prick test (SPT), specific IgE antibodies (RAST), and symptoms during the pollen season. Forty-seven patients with birch pollen allergy were investigated by RAST against birch and hazel pollen and by SPT. They were treated in a randomized, double-blind, placebo-controlled study with fluticasone propionate aqueous nasal spray or placebo. The area of the SPTs was larger and the specific IgE values higher in patients with hypersensitivity to nuts and apples. These patients also had more symptoms during the pollen season. We conclude that hypersensitivity to nuts is an indication of a more severe allergy in patients with birch pollen allergic rhinitis.     

Possible consequences of elimination diets in asymptomatic immediate hypersensitivity to fish

The natural history of IgE antibodies to food without related symptoms is unknown. We have followed the progress of 7 children with various atopic diseases and asymptomatic immediate hypersensitivity to fish, treated with elimination diet in spite of full alimentary tolerance. During the diet period, between 24 and 113 months, all 7 patients presented immediate symptoms upon accidental exposure to or challenge tests with fish (skin symptoms in all 7 cases, digestive in 5, respiratory in 4, and anaphylaxis in 2), which differed from those related to atopic diseases previously present. The levels of fish-specific IgE (prick test, RAST) remained unchanged or were increased. These findings suggest that during elimination diet, and perhaps due to minimal and hidden contact with the allergen, the patients' degree of sensitization may increase, turning an asymptomatic into a symptomatic immediate hypersensitivity.     

Delayed hypersensitivity to azobenzenearsonate-N-acetyl-L-tyrosine. In vivo and in vitro study

Immunogenicity of azobenzenearsonate-N-acetyl-L -tyrosine (ABA-tyrosine) has been confirmed. This low-molecular weight conjugate, in a 50 μg dose, in complete Freund's adjuvant, regularly induces an apparently pure state of delayed hypersensitivity in guinea pigs. Binding of ABA-tyrosine to proteins seems to be very small. In detecting delayed hypersensitivity in vivo, ABA-tyrosine appears much less efficient than a protein conjugate (ABA-guinea pig serum albumin). With in vitro systems (macrophage migration and lymphocyte transformation) ABA-tyrosine is, on the other hand, as active as the protein conjugate. Experiments conducted with labeled antigens injected intradermally have shown that ABA-tyrosine was cleared from the skin far more rapidly than the protein conjugate. This fact probably accounts for its weak activity in detecting delayed hypersensitivity in vivo.     

Fish hypersensitivity. I. In vitro and oral challenge results in fish-allergic patients.

The purpose of this study was to determine whether patients allergic to one fish species can safely eat other fish species. Eleven atopic, food-allergic children and young adults with histories consistent with IgE-mediated fish hypersensitivity were skin prick tested to 10 fish species. Skin prick tests (SPTs) were positive to all 10 fish in eight of the 11 patients, and the remaining three patients had at least two positive fish SPTs. Positive oral challenges occurred to only one fish in seven of the patients, to two fish species in one patient, and to three fish species in two patients. One patient did not react to any of the fish tested. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses were performed on raw and cooked protein extracts from nine of the 10 fish species used in SPTs. Several protein bands in the raw-fish extracts appeared to denature with cooking and form high molecular weight conglomerates. Immunoblot analyses with sera from documented fish-allergic patients demonstrated specific IgE binding to protein bands from fish to which patients were not clinically allergic, as determined by oral challenge. In ELISA-inhibition assays, the concentration of fish antigen required to achieve 50% inhibition was similar for fish to which the patients were clinically allergic as compared to fish to which they were clinically tolerant. SPT and in vitro evidence of IgE-specific cross-reactivity does not necessarily correlate with symptomatic fish allergy. In addition, these fish-hypersensitive patients were able to consume one or more other fish species without adverse allergic reactions.