Double-blind placebo-controlled trial of the effect of omalizumab on basophils in chronic urticaria patients.

BACKGROUND: Omalizumab has been shown to be effective in treating chronic spontaneous urticaria (CSU). The reduction in FcεRI receptor density on the surface of basophils and mast cells is thought to play a major role in its effectiveness. We conducted a double-blind, randomized, placebo-controlled trial to investigate the mode of action of omalizumab in patients with antihistamine-resistant CSU. METHODS: Thirty patients were randomized in a 2:1 ratio to receive either 300 mg omalizumab or placebo. Four monthly applications of omalizumab/placebo were followed up with a visit 2 months after the last injection. The primary endpoint was the FcεRI receptor density change on basophils. RESULTS: Omalizumab led to a significant reduction in FcεRI receptor density on basophils as soon as 1 week after the first injection: baseline omalizumab vs placebo group, 80.31 ± 47.18 × 10³ vs 78.29 ± 45.09 × 10³ receptors/basophil ± SD; 1 week, 72.89 ± 47.79 × 10³ vs 27.83 ± 20.87 × 10³, P = .001. This effect continued during the treatment phase and persisted for 2 months after the last injection: 93.81 ± 56.50 × 10³ vs 21.09 ± 15.23 × 10³, P = .002. Values for basophil "releasability" and the basophil activation test (CU-BAT) of patient serum using donor basophils were unchanged despite treatment: CU-BAT, CD63 10.75% (7.35) in the placebo group vs 8.35% (15.20) in the omalizumab group, P = .778. CONCLUSION: We demonstrated a rapid reduction of FcεRI receptor density on basophils following treatment with omalizumab. Because CU-BAT using well-characterized, omalizumab-naïve donor basophils did not change during the treatment phase, autoreactive serum factors seem to remain unaltered. This points towards a cellular effect of omalizumab on basophils. To predict the omalizumab response time and to monitor disease, FcεRI density and CU-BAT might be promising cellular-based assays.

Minimal Invasive Dorsal Plate Osteosynthesis of the Tibia--Are Nerves and Vessels at Risk? An Anatomical Study.

PURPOSE OF THE STUDY: Purpose of this anatomic study was to develop a new and safe technique of minimal invasive dorsal plate osteosynthesis for tibia shaft fractures. MATERIAL AND METHODS: Sixteen paired adult lower limbs of eight different cadaveric specimens were examined. Anatomical prebending for each plate was done. Plates were inserted percutaneously, following plate fixation the neurovascular bundle was dissected out. The distance between the neurovascular bundle (posterior tibial nerve, posterior tibial artery) and the plate was measured at two different positions. The distance to the origin of the flexor digitorum longus muscle and the arch of the soleus muscle was measured. RESULTS: The mean distance between the neurovascular bundle and the plate amounted 1.4 cm (±0,2 cm; 1.0-1.7 cm) at hole number six and 1.1 cm (±0.4 cm; 0.6-2.0 cm) at hole number ten. The nerve was never directly in contact with the plate. The flexor digitorum longus muscle had its origin along the plate and was between the plate and the neurovascular bundle in all cases. CONCLUSIONS: Dorsal percutaneous plate insertion is a safe and easy method for osteosyntesis of tiba shaft fractures. Especially in case of poor skin and soft tissue conditions this technique offers a good alternative.

International Consensus on drug allergy.

When drug reactions resembling allergy occur, they are called drug hypersensitivity reactions (DHRs) before showing the evidence of either drug-specific antibodies or T cells. DHRs may be allergic or nonallergic in nature, with drug allergies being immunologically mediated DHRs. These reactions are typically unpredictable. They can be life-threatening, may require or prolong hospitalization, and may necessitate changes in subsequent therapy. Both underdiagnosis (due to under-reporting) and overdiagnosis (due to an overuse of the term 'allergy') are common. A definitive diagnosis of such reactions is required in order to institute adequate treatment options and proper preventive measures. Misclassification based solely on the DHR history without further testing may affect treatment options, result in adverse consequences, and lead to the use of more-expensive or less-effective drugs, in contrast to patients who had undergone a complete drug allergy workup. Several guidelines and/or consensus documents on general or specific drug class-induced DHRs are available to support the medical decision process. The use of standardized systematic approaches for the diagnosis and management of DHRs carries the potential to improve outcomes and should thus be disseminated and implemented. Consequently, the International Collaboration in Asthma, Allergy and Immunology (iCAALL), formed by the European Academy of Allergy and Clinical Immunology (EAACI), the American Academy of Allergy, Asthma and Immunology (AAAAI), the American College of Allergy, Asthma and Immunology (ACAAI), and the World Allergy Organization (WAO), has decided to issue an International CONsensus (ICON) on drug allergy. The purpose of this document is to highlight the key messages that are common to many of the existing guidelines, while critically reviewing and commenting on any differences and deficiencies of evidence, thus providing a comprehensive reference document for the diagnosis and management of DHRs.

Allopurinol hypersensitivity is primarily mediated by dose-dependent oxypurinol-specific T cell response.

BACKGROUND: Allopurinol is a main cause of severe cutaneous adverse reactions (SCAR). How allopurinol induces hypersensitivity remains unknown. Pre-disposing factors are the presence of the HLA-B*58:01 allele, renal failure and possibly the dose taken. OBJECTIVE: Using an in vitro model, we sought to decipher the relationship among allopurinol metabolism, HLA-B*58:01 phenotype and drug concentrations in stimulating drug-specific T cells. METHODS: Lymphocyte transformation test (LTT) results of patients who had developed allopurinol hypersensitivity were analysed. We generated allopurinol or oxypurinol-specific T cell lines (ALP/OXP-TCLs) from allopurinol naïve HLA-B*58:01(+) and HLA-B*58:01(-) individuals using various drug concentrations. Their reactivity patterns were analysed by flow cytometry and (51) Cr release assay. RESULTS: Allopurinol allergic patients are primarily sensitized to oxypurinol in a dose-dependent manner. TCL induction data show that both the presence of HLA-B*58:01 allele and high concentration of drug are important for the generation of drug-specific T cells. The predominance of oxypurinol-specific lymphocyte response in allopurinol allergic patients can be explained by the rapid conversion of allopurinol to oxypurinol in vivo rather than to its intrinsic immunogenicity. OXP-TCLs do not recognize allopurinol and vice versa. Finally, functional avidity of ALP/OXP-TCL is dependent on both the induction dose and HLA-B*58:01 status. CONCLUSIONS AND CLINICAL RELEVANCE: This study establishes the important synergistic role of drug concentration and HLA-B*58:01 allele in the allopurinol or oxypurinol-specific T cell responses. Despite the prevailing dogma that Type B adverse drug reactions are dose independent, allopurinol hypersensitivity is primarily driven by oxypurinol-specific T cell response in a dose-dependent manner, particular in the presence of HLA-B*58:01 allele.

In vitro drug causality assessment in Stevens-Johnson syndrome - alternatives for lymphocyte transformation test.

BACKGROUND: Patients with Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN) are often exposed simultaneously to a few potentially culprit drugs. However, both the standard lymphocyte transformation tests (LTT) with proliferation as the assay end-point as well as skin tests, if done, are often negative. OBJECTIVE: As provocation tests are considered too dangerous, there is an urgent need to identify the relevant drug in SJS/TEN and to improve sensitivity of tests able to identify the causative drug. METHODS: Fifteen patients with SJS/TEN with the ALDEN score ≥ 6 and 18 drug-exposed controls were included. Peripheral blood mononuclear cells (PBMC) were isolated and cultured under defined conditions with drugs. LTT was compared to the following end-points: cytokine levels in cell culture supernatant, number of granzyme B secreting cells by ELISpot and intracellular staining for granulysin and IFNγ in CD3(+) CD4(+), CD3(+) CD8(+) and NKp46(+) cells. To further enhance sensitivity, the effect of IL-7/IL-15 pre-incubation of PBMC was evaluated. RESULTS: Lymphocyte transformation tests was positive in only 4/15 patients (sensitivity 27%, CI: 8-55%). Similarly, with granzyme B-ELISpot culprit drugs were positive in 5/15 patients (sensitivity 33%, CI: 12-62%). The expression of granulysin was significantly induced in NKp46(+) and CD3(+) CD4(+) cells (sensitivity 40%, CI: 16-68% and 53%, CI: 27-79% respectively). Cytokine production could be demonstrated in 38%, CI: 14-68% and 43%, CI: 18-71% of patients for IL-2 and IL-5, respectively, and in 55%, CI: 23-83% for IFNγ. Pre-incubation with IL-7/IL-15 enhanced drug-specific response only in a few patients. Specificities of tested assays were in the range of 95 (CI: 80-99%)-100% (CI: 90-100%). CONCLUSIONS AND CLINICAL RELEVANCE: Granulysin expression in CD3(+) CD4(+) , Granzyme B-ELISpot and IFNγ production considered together provided a sensitivity of 80% (CI: 52-96%) and specificity of 95% (80-99%). Thus, this study demonstrated that combining different assays may be a feasible approach to identify the causative drug of SJS/TEN reactions; however, confirmation on another group of patients is necessary.

The three-dimensional morphometry of the odontoid peg and its impact on ventral screw osteosynthesis.

Ventral screw osteosynthesis is a common surgical method for treating fractures of the odontoid peg, but there is still no consensus about the number and diameter of the screws to be used. The purpose of this study was to develop a more accurate measurement technique for the morphometry of the odontoid peg (dens axis) and to provide a recommendation for ventral screw osteosynthesis. Images of the cervical spine of 44 Caucasian patients, taken with a 64-line CT scanner, were evaluated using the measuring software MIMICS. All measurements were performed by two independent observers. Intraclass correlation coefficients were used to measure inter-rater variability. The mean length of the odontoid peg was 39.76 mm (SD 2.68). The mean screw entry angle α was 59.45° (SD 3.45). The mean angle between the screw and the ventral border of C2 was 13.18° (SD 2.70), the maximum possible mean converging angle of two screws was 20.35° (SD 3.24). The measurements were obtained at the level of 66% of the total odontoid peg length and showed mean values of 8.36 mm (SD 0.84) for the inner diameter in the sagittal plane and 7.35 mm (SD 0.97) in the coronal plane. The mean outer diameter of the odontoid peg was 12.88 mm (SD 0.91) in the sagittal plane and 11.77 mm (SD 1.09) in the coronal plane. The results measured at the level of 90% of the total odontoid peg length were a mean of 6.12 mm (SD 1.14) for the sagittal inner diameter and 5.50 mm (SD 1.05) for the coronal inner diameter. The mean outer diameter of the odontoid peg was 11.10 mm (SD 1.0) in the sagittal plane and 10.00 mm (SD 1.07) in the coronal plane. In order to calculate the necessary screw length using 3.5 mm cannulated screws, 1.5 mm should be added to the measured odontoid peg length when anatomical reduction seems possible. The cross-section of the odontoid peg is not circular but slightly elliptical, with a 10% greater diameter in the sagittal plane. In the majority of cases (70.5%) the odontoid peg offers enough room for two 3.5 mm cannulated cortical screws.

Human leukocyte antigens (HLA) associated drug hypersensitivity: consequences of drug binding to HLA.

Recent publications have shown that certain human leukocyte antigen (HLA) alleles are strongly associated with hypersensitivity to particular drugs. As HLA molecules are a critical element in T-cell stimulation, it is no surprise that particular HLA alleles have a direct functional role in the pathogenesis of drug hypersensitivity. In this context, a direct interaction of the relevant drug with HLA molecules as described by the p-i concept appears to be more relevant than presentation of hapten-modified peptides. In some HLA-associated drug hypersensitivity reactions, the presence of a risk allele is a necessary but incomplete factor for disease development. In carbamazepine and HLA-B*15:02, certain T-cell receptor (TCR) repertoires are required for immune activation. This additional requirement may be one of the 'missing links' in explaining why most individuals carrying this allele can tolerate the drug. In contrast, abacavir generates polyclonal T-cell response by interacting specifically with HLA-B*57:01 molecules. T cell stimulation may be due to presentation of abacavir or of altered peptides. While the presence of HLA-B*58:01 allele substantially increases the risk of allopurinol hypersensitivity, it is not an absolute requirement, suggesting that other factors also play an important role. In summary, drug hypersensitivity is the end result of a drug interaction with certain HLA molecules and TCRs, the sum of which determines whether the ensuing immune response is going to be harmful or not.

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.

Multiple drug hypersensitivity: normal Treg cell function but enhanced in vivo activation of drug-specific T cells.

BACKGROUND: Up to 10% of patients with severe immune-mediated drug hypersensitivity reactions have tendencies to develop multiple drug hypersensitivities (MDH). The reason why certain individuals develop MDH and the underlying pathomechanism are unclear. We investigated different T cell subpopulations in MDH patients and compared them with patients allergic to a single drug and with healthy controls (HC). METHODS: We analyzed the in vitro reactivity of peripheral blood mononuclear cells from MDH patients (n=7), patients with hypersensitivity to a single drug (monoallergic, n=6), and healthy controls (HD) (n=6) to various drugs (mainly antibiotics and antiepileptics). By depleting and selectively re-adding CD4(+) CD25(bright) T cells (T regulatory cells, Treg), their effect on drug-specific T cell reactivity was analyzed. The phenotype of reacting T cells was determined ex vivo by staining for markers of activation (CD38) and cell exhaustion (PD-1). RESULTS: No functional deficiency of Treg cells was observed in all drug-allergic patients. Drug-reactive T cells from MDH patients were found in the CD4(+) CD25(dim) T cell fraction and showed enhanced CD38 and PD-1 expression, while those from monoallergic patients reside in the resting CD4(+) CD25(neg) T cell fraction. CONCLUSION: In patients with MDH, the drug-reactive T cells are contained in an in vivo pre-activated T cell fraction. Therefore, they may show a lower threshold for activation by drugs. The reason for this in vivo T cell pre-activation needs further investigations.

Continuous apple consumption induces oral tolerance in birch-pollen-associated apple allergy.

BACKGROUND: Patients with birch pollen allergy (major allergen: Bet v 1) have often an associated oral allergy syndrome (OAS) to apple, which contains the cross-reactive allergen Mal d 1. As successful birch pollen immunotherapy does not consistently improve apple related OAS symptoms, we evaluated whether regular apple consumption has an effect on OAS and immune parameters of Mal d 1 or Bet v 1 allergy. METHODS: A total of 40 patients with a clear history of birch pollen rhinoconjunctivitis and associated OAS to apple were included in an open, randomized, controlled clinical trial: 27 patients consumed daily defined amount of apple (1-128 g), doubling the amount every two to three weeks, while 13 patients remained untreated. Primary endpoint was the proportion of patients that achieved tolerance to at least 128 g of apple at the end of the study after 8 months. Exploratory endpoints were questionnaire about cross-reactive food and pollen allergy symptoms, conjunctival provocation test with birch pollen and Bet v 1, and in vitro tests (tIgE, sIgE, and IgG4 to Mal d 1 and Bet v 1; basophil activation test with both allergens). RESULTS: Seventeen of 27 patients in active group and none of 13 patients in control group (P = 0.0001) could tolerate a whole apple after the intervention. However, differences in endpoints reflecting systemic immune reactivity did not reach statistical significance. CONCLUSION: In patients with OAS to apple, tolerance can be safely induced with slowly, gradually increasing consumption of apple. However, the observation of a relapse after discounting of apple consumption and absence of immunologic changes suggest that induced tolerance is only transient.

NKp46+ cells express granulysin in multiple cutaneous adverse drug reactions.

BACKGROUND: The spectrum of cutaneous adverse drug reactions (cADRs) ranges from benign presentations to severe life-threatening forms such as toxic epidermal necrolysis (TEN). In TEN, granulysin has been shown to be the key cytotoxic molecule. Still, little is known about the expression of granulysin in other cADRs. As an important source of granulysin, natural killer (NK) cells are of major interest in cADRs. Recently, NKp46 has been identified as the most selective NK-cell marker. However, the role of NKp46(+) cells in cADRs and their contribution to granulysin expression remain to be elucidated. METHODS: Immunohistochemical and immunofluorescence staining of tissue sections from multiple cADRs were quantitatively and qualitatively evaluated. Further, in vivo and in vitro drug-stimulation tests were performed. RESULTS: Granulysin is expressed at different levels in multiple cADRs both by NKp46(+) cells and by CD8(+) T cells. Even in mild forms of cADRs, granulysin can be induced in vivo and in vitro in a drug-specific manner. NKp46(+) cells were found to infiltrate the dermal/epidermal junction particularly in TEN. CONCLUSION: The impressive clinical differences of cADRs may not be uniquely explained by the expression of granulysin. Additional factors such as drug-specific activation and recruitment of NKp46(+) cells to the epidermis may play a role in determining the severity of cADRs. Therefore, unraveling the effects of drugs on NK-cell activation and trafficking may help to better understand the cytotoxic mechanisms behind cADRs.

In vitro diagnosis of T cell-mediated drug allergy.

Diagnosis of drug hypersensitivity relies on history, skin tests, in vitro tests and provocation tests. In vitro tests are of great interest, due to possible reduction of drug provocation tests. In this review we focus on best investigated in vitro techniques for the diagnosis of T cell-mediated drug hypersensitivity reactions. As drug hypersensitivity relies on different pathomechanisms and as a single diagnostic test usually does not cover all possible reactions, it is advisable to combine different tests to increase the overall sensitivity. Recently, proliferation-based assays have been supplemented by a panel of novel in vitro tests including analysis of cytotoxic potential of effector cells (granzyme B, granulysin, CD107a), evaluation of cytokine secretion (IL-2, IL-5, IL-13, and IFN-γ) and up-regulation of cell surface activation markers (CD69). We discuss the latest findings and readout systems to identify causative drugs by detecting functional and phenotypic markers of drug-reacting cells, and their ability to enable a more conclusive diagnosis of drug allergy.

Robust expression of CCR3 as a single basophil selection marker in flow cytometry.

BACKGROUND: Basophil activation tests (BAT) rely on different combinations of basophil selection and activation markers. Whereas activation markers, especially CD63, are widely validated, the most suitable and robust marker for basophil selection is still a matter of debate. AIMS: Comparison of cell surface expression of two commonly used basophil selection markers (IgE, CD123/HLA-DR) with CCR3 in an unselected group of atopic and nonatopic donors in resting and activated basophils. METHODS: EDTA blood of 94 healthy adults, about half of them atopic by history, was analyzed using two different staining strategies: anti-CD123-PE/anti-HLA-DR-PerCP/anti-lin1-FITC and anti-IgE-FITC/anti-CD3-PerCP/anti-CCR3-PE. Additionally 40 pollen-allergic patients were recruited for the assessment of CCR3 expression after basophil activation. RESULTS: In resting basophils, cell surface expression of the three basophil selection markers was most constant for CCR3. IgE gating strategy showed the highest variation and up to 80% of nonbasophils in the selected gate in certain donors. During basophil activation, a shift of the mean fluorescence intensity for CCR3 toward the lower third of the CCR3-positive population could be demonstrated, but neither were CCR3-positive cells significantly lost for further analysis nor was differentiation between CCR3-positive and CCR3-negative cell populations hampered by this shift. CONCLUSIONS: CCR3 is a stable and highly expressed basophil selection marker, independent of the atopic background or basophil activation state and allows an accurate identification of basophils without need of a second marker. The basophil markers CD123/HLA-DR and IgE showed significantly higher interindividual variability in cell surface expression and are therefore less suited as selection markers.

General considerations on rapid desensitization for drug hypersensitivity - a consensus statement.

Drug hypersensitivity reactions can occur with most drugs, are unpredictable, may affect any organ or system, and range widely in clinical severity from mild pruritus to anaphylaxis. In most cases, the suspected drug is avoided in the future. However, for certain patients, the particular drug may be essential for optimal therapy. Under these circumstances, desensitization may be performed. Drug desensitization is defined as the induction of a temporary state of tolerance of a compound responsible for a hypersensitivity reaction. It is performed by administering increasing doses of the medication concerned over a short period of time (from several hours to a few days) until the total cumulative therapeutic dose is achieved and tolerated. It is a high-risk procedure used only in patients in whom alternatives are less effective or not available after a positive risk/benefit analysis. Desensitization protocols have been developed and are used in patients with allergic reactions to antibiotics (mainly penicillin), insulins, sulfonamides, chemotherapeutic and biologic agents, and many other drugs. Desensitization is mainly performed in IgE-mediated reactions, but also in reactions where drug-specific IgE have not been demonstrated. Desensitization induces a temporary tolerant state, which can only be maintained by continuous administration of the medication. Thus, for treatments like chemotherapy, which have an average interval of 4 weeks between cycles, the procedure must be repeated for every new course. In this paper, some background information on rapid desensitization procedures is provided. We define the drugs and drug reactions indicated for such procedures, describe the possible mechanism of action, and discuss the indications and contraindications. The data should serve as background information for a database (accessible via the EAACI-homepage) with standardized protocols for rapid desensitization for antibiotics, chemotherapeutic agents, monoclonal antibodies/fusion proteins, and other drugs.

Does the anteromedial or anterolateral approach alter the rate of joint puncture in injection of the ankle?: A cadaver study.

Injection or aspiration of the ankle may be performed through either an anteromedial or an anterolateral approach for diagnostic or therapeutic reasons. We evaluated the success of an intra-articular puncture in relation to its site in 76 ankles from 38 cadavers. Two orthopaedic surgical trainees each injected methylene blue dye into 18 of 38 ankles through an anterolateral approach and into 20 of 38 through an anteromedial. An arthrotomy was then performed to confirm the placement of the dye within the joint. Of the anteromedial injections 31 of 40 (77.5%, 95% confidence interval (CI) 64.6 to 90.4) were successful as were 31 of 36 (86.1%, 95% CI 74.8 to 97.4) anterolateral injections. In total 62 of 76 (81.6%, 95% CI 72.9 to 90.3) of the injections were intra-articular with a trend towards greater accuracy with the anterolateral approach, but this difference was not statistically significant (p = 0.25). In the case of trainee A, 16 of 20 anteromedial injections and 14 of 18 anterolateral punctures were intra-articular. Trainee B made successful intra-articular punctures in 15 of 20 anteromedial and 17 of 18 anterolateral approaches. There was no significant difference between them (p = 0.5 and p = 0.16 for the anteromedial and anterolateral approaches, respectively). These results were similar to those of other reported studies. Unintended peri-articular injection can cause complications and an unsuccessful aspiration can delay diagnosis. Placement of the needle may be aided by the use of ultrasonographic scanning or fluoroscopy which may be required in certain instances.

In vitro detection of cytotoxic T and NK cells in peripheral blood of patients with various drug-induced skin diseases.

BACKGROUND: Cytotoxic cells are involved in most forms of drug-induced skin diseases. Till now, no in vitro test addressed this aspect of drug-allergic responses. Our report evaluates whether drug-induced cytotoxic cells can be detected in peripheral blood of nonacute patients with different forms of drug hypersensitivity, and also whether in vitro detection of these cells could be helpful in drug-allergy diagnosis. METHODS: GranzymeB enzyme-linked immunosorbent spot-forming (ELISPOT) and cell surface expression of the degranulation marker CD107a were evaluated on peripheral blood mononuclear cells from 12 drug-allergic patients in remission state and 16 drug-exposed healthy controls. RESULTS: In 10/12 allergic patients culprit but not irrelevant drug elicited granzymeB release after 48-72 h stimulation. It was clearly positive in patients with high proliferative response to the drug, measured in lymphocyte transformation tests. In patients, who showed moderate or low proliferation and low drug-response in granzymeB ELISPOT, overnight preincubation with interleukin (IL)-7/IL-15 enhanced drug-specific granzymeB release and allowed to clearly identify the offending agent. CD107a staining was positive on CD4+/CD3+, CD8+/CD3+ T cells as well as CD56+/CD3- natural killer cells. None of the drug-exposed healthy donors reacted to the tested drugs and allergic patients reacted only to the offending, but not to tolerated drugs. CONCLUSION: GranzymeB ELISPOT is a highly specific in vitro method to detect drug-reacting cytotoxic cells in peripheral blood of drug-allergic patients even several years after disease manifestation. Together with IL-7/IL-15 preincubation, it may be helpful in indentifying the offending drug even in some patients with weak proliferative drug-response.

Processing-dependent and -independent pathways for recognition of iodinated contrast media by specific human T cells.

BACKGROUND: One to three percent of patients exposed to intravenously injected iodinated contrast media (CM) develop delayed hypersensitivity reactions. Positive patch test reactions, immunohistological findings, and CM-specific proliferation of T cells in vitro suggest a pathogenetic role for T cells. We have previously demonstrated that CM-specific T cell clones (TCCs) show a broad range of cross-reactivity to different CM. However, the mechanism of specific CM recognition by T cell receptors (TCRs) has not been analysed so far. OBJECTIVE: To determine how T cells specifically recognize CM. METHODS: CM-specific TCCs were generated from human blood of three CM-allergic patients and a specific TCR was transfected into a mouse T cell hybridoma. Functional analysis such as proliferation assays, IL-2 secretion assays, and calcium influx experiments were performed using irradiated, glutaraldehyde-fixed, CM-pre-incubated, human leucocyte antigen (HLA)-DR-matched or -mismatched antigen-presenting cells (APCs), and HLA-blocking antibodies. RESULTS: We identified two mechanisms of T cell stimulation: some TCCs and the transfectant reacted to CM independent of uptake by APCs because proliferation/IL-2 secretion occurred in the presence of glutaraldehyde-fixed APCs, and intracellular calcium increased within seconds after drug addition. Other TCCs required functional APCs, compatible with uptake and presentation of CM on MHC-class II molecules, as implied by three findings: (1) glutaraldehyde fixation of APCs abrogated presentation; (2) CM could not be washed away from CM-pre-incubated APCs; and (3) the optimal pulsing time was 10-20 h. Because allogeneic, MHC-matched, CM-pulsed APCs could induce proliferative responses as well, the ability of CM uptake and presentation is not unique to APCs from patients with CM-induced delayed hypersensitivity. CONCLUSION: Our data suggest that CM may be stimulatory for T cells either by direct binding to the MHC-TCR complex or by binding after uptake and processing by APCs. This questions the assumed inert nature of CM.