Etiology and pathogenesis of adverse drug reactions

In clinical routine, adverse drug reactions (ADR) are common, and they should be included in the differential diagnosis in all patients undergoing drug treatment. Only part of those ADR are immune-mediated hypersensitivity reactions and thus true drug allergies. Far more common are non-immune-mediated ADR, e.g. due to the pharmacological properties of the drug or to the individual predisposition of the patient (enzymopathies, cytokine dysbalance, mast cell hyperreactivity). In true drug allergiesT cell- and immunoglobulin E (lgE)-mediated reactions dominate the clinical presentation. T cell-mediated ADR usually have a delayed appearance and include skin eruptions in most cases. Nevertheless, it should not be forgotten that they may involve systemic T cell activation and thus take a severe, sometimes lethal turn. Clinical danger signs are involvement of mucosal surfaces, blistering within the exanthematous skin areas and systemic symptoms, e.g. fever or malaise. Drug presentation via antigen-presenting cells to T cells can either involve the classical pathway of haptenization of endogenous proteins or be directly mediated via noncovalent binding to immune receptors (MHC molecules or T cell receptors), the so-called p-i concept. Flare-up reactions during the acute phase of T cell-mediated ADR should not be mistaken for true drug allergies, as they only occur in the setting of a highly activated T cell pool. IgE-mediated ADR are less frequent and involve mast cells and/or basophils as peripheral effector cells. Recent data suggest that certain patients with drug allergy have a preexistent sensitization although they have never been exposed to the culprit drug, probably due to cross-reactivity. Thus, allergic drug reactions on first encounter are possible. In general, the extent of cross-reactivity is higher in IgE-compared to T cell-mediated ADR. Based on a specific ethnic background and only for severe T cell-mediated ADR to certain drugs, a strong HLA association has been established recently.

Evaluation of an in vitro sulphidoleukotriene release test for diagnosis of insect bite hypersensitivity in horses

REASONS FOR PERFORMING STUDY: Insect bite hypersensitivity (IBH) is an IgE-mediated allergic dermatitis caused by bites of Culicoides and Simulium species, and improved means of diagnosis are required. OBJECTIVES: The cellular antigen simulation test (CAST) with C. nubeculosus and S. vittatum extracts was assessed in a population of IBH-affected and healthy horses. Variations in test results over a one year period and possible cross-reactivity between different insect extracts was studied. METHODS: A total of 314 mature horses were studied using the CAST. Influence of severity of clinical signs, gender and age were evaluated, and 32 horses were tested repeatedly over one year. The kappa reliability test was used to assess agreement of the test results with different insect extracts. RESULTS: Horses with IBH had significantly higher sLT release than controls with C. nubeculosus and S. vittatum. The highest diagnostic sensitivity and specificity levels were attained when using adult C. nubeculosus extracts with the CAST (78% and 97%, respectively), suggesting that most horses with IBH are sensitised against Culicoides allergens. A proportion of IBH-affected horses was found to be sensitised to allergens of Simulium spp. in addition to those of C. nubeculosus. The CAST with C. nubeculosus had positive and negative predictive values > or = 80% for a true prevalence of IBH of 12-52%. In the follow-up study, the proportion of IBH-affected horses with a positive test result ranged from 90% in November to 68% in March. Severity of clinical signs or age did not influence test results significantly. However, IBH-affected males achieved significantly more positive test results than IBH-affected females. CONCLUSIONS: The CAST with adult C. nubeculosus has high specificity and good sensitivity for diagnosis of IBH. Horses with IBH are mainly sensitised to Culicoides allergens, and some horses are additionally also sensitised to allergens in Simulium spp. POTENTIAL RELEVANCE: The CAST is likely to be a useful test for diagnosis of IBH, even allowing the identification of IBH-affected but asymptomatic horses. This test may also help in further characterisation of allergens involved in this condition.

Noncovalent interactions of drugs with immune receptors may mediate drug-induced hypersensitivity reactions

Drug-induced hypersensitivity reactions are instructive examples of immune reactions against low molecular weight compounds. Classically, such reactions have been explained by the hapten concept, according to which the small antigen covalently modifies an endogenous protein; recent studies show strong associations of several HLA molecules with hypersensitivity. In recent years, however, evidence has become stronger that not all drugs need to bind covalently to the major histocompatibility complex (MHC)-peptide complex in order to trigger an immune response. Rather, some drugs may bind reversibly to the MHC or possibly to the T-cell receptor (TCR), eliciting immune reactions akin to the pharmacological activation of other receptors. While the exact mechanism is still a matter of debate, noncovalent drug presentation clearly leads to the activation of drug-specific T cells. In some patients with hypersensitivity, such a response may occur within hours of even the first exposure to the drug. Thus, the reaction to the drug may not be the result of a classical, primary response but rather be mediated by existing, preactivated T cells that display cross-reactivity for the drug and have additional (peptide) specificity as well. In this way, certain drugs may circumvent the checkpoints for immune activation imposed by the classical antigen processing and presentation mechanisms, which may help to explain the idiosyncratic nature of many drug hypersensitivity reactions.

Adverse side-effects to biological agents

Biological agents-like cytokines, monoclonal antibodies and fusion proteins are widely used in anti-inflammatory and tumour therapy. They are highly efficient in certain diseases, but can cause a great variety of adverse side-effects. Based on the peculiar features of biological agents a new classification of these adverse side-effects of biological agents is proposed - related but clearly distinct from the classification of side-effects observed with chemicals and drugs. This classification differentiates five distinct types, namely clinical reactions because of high cytokine levels (type alpha), hypersensitivity because of an immune reaction against the biological agent (beta), immune or cytokine imbalance syndromes (gamma), symptoms because of cross-reactivity (delta) and symptoms not directly affecting the immune system (epsilon). This classification could help to better deal with the clinical features of these side-effects, to identify possible individual and general risk factors and to direct research in this novel area of medicine.

Cloning and expression of the gene encoding the major surface protein 5 (MSP5) of Anaplasma phagocytophilum and potential application for serodiagnosis

BACKGROUND: Anaplasma phagocytophilum (formerly known as the human granulocytic ehrlichia, Ehrlichia equi and Ehrlichia phagocytophila) is an obligate intracellular organism causing clinical disease in humans and various species of domestic animals. OBJECTIVES: The objectives of this investigation were to sequence and clone the major surface protein 5 (MSP5) of A phagocytophilum and to evaluate the suitability of this antigen in the serologic diagnosis of anaplasmosis in humans and dogs. METHODS: The msp5 gene of A phagocytophilum was sequenced, cloned, and expressed in Escherichia coli. The predicted amino acid sequence homology of the various MSP5/major antigenic protein 2 orthologs was compared among various Anaplasma and Ehrlichia species. Recombinant MSP5 of A phagocytophilum was used in an ELISA to detect antibodies in serum samples from humans and dogs infected with the organism. RESULTS: Serum samples from 104 individuals previously diagnosed with A phagocytophilum infection, as well as samples from clinically healthy humans, were tested. In addition, multiple samples from 4 dogs experimentally infected with 2 different geographic isolates of A phagocytophilum and 5 dogs naturally infected with a Swiss isolate were tested using ELISA. Using this group of immunofluorescent antibody test-positive and immunofluorescent antibody test-negative samples, we found the overall agreement between assays to be >90%. CONCLUSIONS: These results indicate that recombinant MSP5 has potential for use as a diagnostic test antigen to detect infection with A phagocytophilum in both dogs and humans. However, sequence similarities among orthologs of MSP5 in related species of anaplasma and ehrlichia suggest that cross-reactivity among these pathogens is likely if the entire peptide is used as a test antigen.

Low prevalence of SV40 in Swiss mesothelioma patients after elimination of false-positive PCR results

The association of simian virus 40 (SV40) with malignant pleural mesothelioma is currently under debate. In some malignancies of viral aetiology, viral DNA can be detected in the patients' serum or plasma. To characterize the prevalence of SV40 in Swiss mesothelioma patients, we optimized a real-time PCR for quantitative detection of SV40 DNA in plasma, and used a monoclonal antibody for immunohistochemical detection of SV40 in mesothelioma tissue microarrays. Real-time PCR was linear over five orders of magnitude, and sensitive to a single gene copy. Repeat PCR determinations showed excellent reproducibility. However, SV40 status varied for independent DNA isolates of single samples. We noted that SV40 detection rates by PCR were drastically reduced by the implementation of strict room compartmentalization and decontamination procedures. Therefore, we systematically addressed common sources of contamination and found no cross-reactivity with DNA of other polyomaviruses. Contamination during PCR was rare and plasmid contamination was infrequent. SV40 DNA was reproducibly detected in only 4 of 78 (5.1%) plasma samples. SV40 DNA levels were low and not consistently observed in paired plasma and tumour samples from the same patient. Immunohistochemical analysis revealed a weak but reproducible SV40 staining in 16 of 341 (4.7%) mesotheliomas. Our data support the occurrence of non-reproducible SV40 PCR amplifications and underscore the importance of proper sample handling and analysis. SV40 DNA and protein were found at low prevalence (5%) in plasma and tumour tissue, respectively. This suggests that SV40 does not appear to play a major role in the development of mesothelioma.

A comprehensive test system to identify suitable antibodies against p53 for immunohistochemical analysis of canine tissues

The tumour suppressor p53 is commonly detected in tissues of companion animals by means of antibodies raised against the human protein. The following three-step procedure was devised to test the suitability of such antibodies for immunohistochemistry on canine tissues. (1) Western blot and immunohistochemical analyses on bacterially expressed recombinant canine protein to assess human-to-canine cross-reactivity. (2) Immunohistochemistry of cultured, UVB-irradiated canine keratinocytes to evaluate suitability for detection of endogenous p53. (3) Immunohistochemistry on tissue arrays to further substantiate suitability of the antibodies on a panel of normal and neoplastic human and canine tissues. Five of six antibodies cross-reacted with recombinant canine p53. Three of these (PAb122, PAb240, CM-1) also immunolabelled stabilized wild type p53 in cell cultures and elicited a consistent, characteristic labelling pattern in a subset of tumours. However, two alternative batches of polyclonal antibody CM-1 failed to detect p53 in cell cultures, while showing a characteristic labelling pattern of a completely different subset of tumours and unspecific labelling of normal tissues. The test system described is well suited to the selection of antibodies for immunohistochemical p53 detection. The results emphasize the need to include appropriate controls, especially for polyclonal antibodies.

Use of the immunodominant 18-kiloDalton small heat shock protein as a serological marker for exposure to Mycobacterium ulcerans

While it is well established that proximity to wetlands is a risk factor for contracting Buruli ulcer, it is not clear what proportion of a population living in an area where the etiologic agent, Mycobacterium ulcerans, is endemic is actually exposed to this disease. Immunological cross-reactivity among mycobacterial species complicates the development of a specific serological test. Among immunodominant proteins recognized by a panel of anti-M. ulcerans monoclonal antibodies, the M. ulcerans homologue of the M. leprae 18-kDa small heat shock protein (shsp) was identified. Since this shsp has no homologues in M. bovis and M. tuberculosis, we evaluated its use as a target antigen for a serological test. Anti-18-kDa shsp antibodies were frequently found in the sera of Buruli ulcer patients and of healthy household contacts but rarely found in controls from regions where the infection is not endemic. The results indicate that only a small proportion of M. ulcerans-infected individuals contract the clinical disease.

Recombinant major urinary proteins of the mouse in specific IgE and IgG testing

Background: Recombinant allergens are preferred over natural allergen extracts in measuring antibodies. We tested the use of recombinant variants of the major mouse allergen Mus m 1 in detection of mouse-specific antibodies in sera of laboratory animal workers and children. Methods: Six recombinant major urinary proteins (MUPs) were produced and antibody-binding capacity was compared to natural Mus m 1 and to mouse urine extract. In a specific subset, cross-reactivity of MUP with Mus m 1 and between the different recombinant MUPs was determined. Results: For IgE antibodies, MUP8 showed high cross-reactivity with Mus m 1. MUP8-specific IgE was found in 55% of the mouse urine IgE-positive sera. Specific IgG and IgG4 antibodies against natural Mus m 1 correlated strongly with antibodies against recombinant MUP8 and were cross-reactive. IgG4 levels against MUP8 and mouse urine extract correlated, but detection of mouse urine-specific IgG4 in the absence of MUP-specific IgG4 was not uncommon. Cross-reactivity of IgG antibodies between MUP8 and Mus m 1 as well as between the different MUPs was high and inhibition varied between 54 and 99%. Conclusion: The mouse allergen Mus m 1 can be replaced in antibody testing by recombinant MUP8. Other MUPs, except MUP4, are interchangeable with MUP8. However, mouse urine extract showed better detection of both mouse-specific IgE and IgG4 levels. Other components in the mouse urine, like mouse albumin and other yet unidentified components, also induce IgE and IgG(4) antibodies.

Design, expression, and processing of epitomized hepatitis C virus-encoded CTL epitopes

Hepatitis C virus (HCV) vaccine efficacy may crucially depend on immunogen length and coverage of viral sequence diversity. However, covering a considerable proportion of the circulating viral sequence variants would likely require long immunogens, which for the conserved portions of the viral genome, would contain unnecessarily redundant sequence information. In this study, we present the design and in vitro performance analysis of a novel "epitome" approach that compresses frequent immune targets of the cellular immune response against HCV into a shorter immunogen sequence. Compression of immunological information is achieved by partial overlapping shared sequence motifs between individual epitopes. At the same time, sequence diversity coverage is provided by taking advantage of emerging cross-reactivity patterns among epitope variants so that epitope variants associated with the broadest variant cross-recognition are preferentially included. The processing and presentation analysis of specific epitopes included in such a compressed, in vitro-expressed HCV epitome indicated effective processing of a majority of tested epitopes, although re-presentation of some epitopes may require refined sequence design. Together, the present study establishes the epitome approach as a potential powerful tool for vaccine immunogen design, especially suitable for the induction of cellular immune responses against highly variable pathogens.

Sensitization to wasp venom does not induce autoantibodies leading to infertility

Antigenic cross-reactivity has been described between the venom allergen (antigen 5) and mammalian testis proteins. Based on an allergen database we have previously shown that allergens can be represented by allergen motifs. A motif group was found containing venom antigen 5 sequences from different vespids. Using an optimized amino acid profile based on antigen 5 sequences for searching cross-reactive proteins, three human semen proteins belonging to the family of cysteine-rich secretory proteins (hCRISP) were found in the Swiss Protein database. To analyze antigenic cross-reactivity between antigen 5 and hCRISPs, antigen 5 from yellow jacket venom (Ves v 5) and two hCRISPs (CRISP-2 and -3) were chosen and produced as recombinant proteins in E. coli. A correlation was found between antibodies reacting with rVes v 5 and rhCRISP-2, -3 in a small human sera population indicating the presence of cross-reactive antibodies in human serum. Using intravenous immunoglobulin (IVIg), a therapeutic multidonor IgG preparation, cross-reactive antibodies were isolated that recognize rVes v 5, hCRISP-2 and -3 suggesting the presence of common epitopes between Ves v 5 and hCRISPs. However this cross-reactivity seems not to be linked to allergy to wasp venom as we could show no correlation between increasing CAP-class IgE level to wasp venom and IgG to sperm extract and hCRISPs. These data suggest that higher sensitization to wasp venom does not induce more antibodies against autoantigens and might not represent a higher risk to develop autoantibodies leading to infertility.

Antiaggregatory and proangiogenic effects of a novel recombinant human dual specificity anti-integrin antibody

BACKGROUND: beta(3)-Integrins are involved in platelet aggregation via alpha(IIb)beta(3) [glycoprotein (GP)IIb-GPIIIa], and in angiogenesis via endothelial alpha(V)beta(3). Cross-reactive ligands with antiaggregatory and proangiogenic effects, both desirable in peripheral vasculopathies, have not yet been described. OBJECTIVES: In vitro and in vivo characterization of antiaggregatory and proangiogenic effects of two recombinant human Fab fragments, with emphasis on beta(3)-integrins. METHODS: Recombinant Fab fragments were obtained by phage display technology. Specificity, affinity and IC(50) were determined by immunodot assays, enzyme-linked immunosorbent assay (ELISA), and Scatchard plot analysis, and by means of human umbilical vein endothelial cells (HUVECs). Functional analyses included ELISA for interaction with fibrinogen binding to GPIIb-GPIIIa, flow cytometry for measurement of activation parameters and competitive inhibition experiments, human platelet aggregometry, and proliferation, tube formation and the chorioallantoic membrane (CAM) assay for measurement of angiogenic effects. RESULTS: We observed specific and high-affinity binding to an intact GPIIb-GPIIIa receptor complex of two human Fab autoantibody fragments, with no platelet activation. Dose-dependent fibrinogen binding to GPIIb-GPIIIa and platelet aggregation were completely inhibited. One Fab fragment was competitively inhibited by abciximab and its murine analog monoclonal antibody (mAb) 7E3, whereas the other Fab fragment bound to cultured HUVECs, suggesting cross-reactivity with alpha(V)beta(3), and also demonstrated proangiogenic effects in tube formation and CAM assays. CONCLUSIONS: These Fab fragments are the first entirely human anti-GPIIb-GPIIIa Fab fragments with full antiaggregatory properties; furthermore, they do not activate platelets. The unique dual-specificity anti-beta(3)-integrin Fab fragment may represent a new tool for the study and management of peripheral arterial vasculopathies.

Normal human serum contains high levels of anti-Gal alpha 1-4GlcNAc antibodies

BACKGROUND: Natural xenoreactive antibodies (Abs) directed against the Bdi-epitope (Gal alpha 1-3Gal beta) on the cells of non-primate mammals take part in hyperacute rejection of xenotransplanted organs. We found that some Abs, which were one-step affinity purified on Bdi-Sepharose, cross-reacted with the disaccharide Gal alpha 1-4GlcNAc beta. The epitope Gal alpha 1-4GlcNAc has not been identified on mammals or bacterial polysaccharides yet. METHODS: To isolate the antibodies of the corresponding specificity the disaccharide was immobilized on Sepharose and antibodies were affinity purified from pooled serum of blood group O individuals. RESULTS: These one-step purified Abs cross-reacted with Bdi, but after a prior absorption step on Bdi-Sepharose no cross-reactivity with Bdi was observed any longer. Surprisingly, the quantity of anti-Gal alpha 1-4GlcNAc isolated from the same serum pool, 4-7 microg/ml, was equal to that of anti-Bdi or more. Independently of ABO blood groups all the tested healthy donors had anti-Gal alpha 1-4GlcNAc Abs at a similar level. Monospecific anti-Gal alpha 1-4GlcNAc Abs were not cytotoxic towards porcine cells. CONCLUSIONS: 1. The actual concentration of monospecific, xenoreactive Gal alpha 1-3Gal beta Abs in blood may be considerably lower than the value referred to in the literature for 'anti-alpha Gal' or 'anti-Galili' antibodies. 2. Anti-Gal alpha 1-4GlcNAc Abs seem not to be important for xenotransplantation.

Eczematous skin reaction to atopy patch testing with cockroach in patients with atopic dermatitis

BACKGROUND: Aeroallergens from house dust mite (HDM) may be an important trigger in a subgroup of patients with atopic dermatitis (AD). HDM and cockroach (CR) contain cross-reactive allergens, such as tropomyosin. OBJECTIVE: To investigate the diagnostic value of patch testing with an aeroallergen and the role of CR allergen and HDM allergen in persons with AD. METHODS: We performed skin prick tests (SPT) with a panel of common aeroallergens and total serum immunoglobulin (Ig)E and specific IgE tests for CR and HDM on 23 patients with AD and 9 nonatopic control participants. Atopy patch tests (APT) were performed with CR and HDM extracts on clinically uninvolved skin on the back, and evaluated after 48 and 72 hours. RESULTS: A positive APT reaction to CR was found in 10/23 (43%) patients with AD. No positive reactions were observed in the nonatopic control participants. Positive APT reactions for CR showed no significant correlation with SPT or specific IgE levels for this allergen. Twelve of the 23 (52%) patients with AD were also sensitized to HDM. There was no significant correlation between positive results for SPT, APT, and specific IgE to CR and HDM. CONCLUSION: We demonstrate that CR allergens can induce positive patch test reactions in patients with AD. The absence of a significant correlation to SPT and specific IgE antibodies suggests that T-cell- and IgE-sensitization may be mediated by different allergens. There was no significant relationship between CR and HDM sensitivity, thus indicating no major cross-reactivity.

Serologic and molecular evidence for Testudinid herpesvirus 2 infection in wild Agassiz's desert tortoises, Gopherus agassizii

Following field observations of wild Agassiz's desert tortoises (Gopherus agassizii) with oral lesions similar to those seen in captive tortoises with herpesvirus infection, we measured the prevalence of antibodies to Testudinid herpesvirus (TeHV) 3 in wild populations of desert tortoises in California. The survey revealed 30.9% antibody prevalence. In 2009 and 2010, two wild adult male desert tortoises, with gross lesions consistent with trauma and puncture wounds, respectively, were necropsied. Tortoise 1 was from the central Mojave Desert and tortoise 2 was from the northeastern Mojave Desert. We extracted DNA from the tongue of tortoise 1 and from the tongue and nasal mucosa of tortoise 2. Sequencing of polymerase chain reaction products of the herpesviral DNA-dependent DNA polymerase gene and the UL39 gene respectively showed 100% nucleotide identity with TeHV2, which was previously detected in an ill captive desert tortoise in California. Although several cases of herpesvirus infection have been described in captive desert tortoises, our findings represent the first conclusive molecular evidence of TeHV2 infection in wild desert tortoises. The serologic findings support cross-reactivity between TeHV2 and TeHV3. Further studies to determine the ecology, prevalence, and clinical significance of this virus in tortoise populations are needed.