Genome-wide pharmacogenetic investigation of a hepatic adverse event without clinical signs of immunopathology suggests an underlying immune pathogenesis.

One of the major goals of pharmacogenetics is to elucidate mechanisms and identify patients at increased risk of adverse events (AEs). To date, however, there have been only a few successful examples of this type of approach. In this paper, we describe a retrospective case-control pharmacogenetic study of an AE of unknown mechanism, characterized by elevated levels of serum alanine aminotransferase (ALAT) during long-term treatment with the oral direct thrombin inhibitor ximelagatran. The study was based on 74 cases and 130 treated controls and included both a genome-wide tag single nucleotide polymorphism and large-scale candidate gene analysis. A strong genetic association between elevated ALAT and the MHC alleles DRB1(*)07 and DQA1(*)02 was discovered and replicated, suggesting a possible immune pathogenesis. Consistent with this hypothesis, immunological studies suggest that ximelagatran may have the ability to act as a contact sensitizer, and hence be able to stimulate an adaptive immune response.

Comparison of batch and perfusion culture in combination with pilot-scale expanded bed purification for the production of soluble recombinant beta-secretase.

beta-Secretase is one of the prime targets for therapeutic intervention of Alzheimer's disease. For the development of a secretase inhibitor a steady supply of large quantities of a homogeneous and active recombinant beta-secretase is a prerequisite. Therefore various culture modes were investigated using HEK-293 cells stably transfected with soluble recombinant beta-secretase. The coupling of the Fc part of human IgG1 to the ectodomain of beta-secretase (residues 1-460) allowed a fast purification of the protein with rProtA expanded bed chromatography. Batch cultures of 5 to 50 L working volume run for 7 days showed reproducible cell growth and product yields of 3 mg/L purified protein. A 20 L perfusion culture was operated for 21 days, reaching a cell density of 30 x 10(6) cells/mL at a dilution rate of 2/d. The total product yield of the perfusion culture was 1.4 g of purified protein. The effect of different perfusion rates on cell growth, protein yield, and quality was investigated and compared to the results obtained in batch cultures. Protein quality was consistent as analyzed on 1D SDS-PAGE, and the final product contained both the mature and the pro form of beta-secretase. Although the cell specific protein expression was slightly reduced in perfusion culture, a substantial increase in specific activity of over 75% was achieved. Some of the increase in activity can be explained by an increase in the percentage of the mature form of the recombinant protein.

Cytokine production, lymphocyte proliferation and T-cell receptor Vbeta expression in primary peripheral blood mononuclear cell cultures from nickel-allergic individuals.

BACKGROUND: Clinical history and patch test constitute the two cornerstones in the diagnosis of nickel (Ni) allergy. Due to technical and interpretative limits of the patch test, the in vitro lymphocyte transformation test (LTT) has been developed for confirming contact allergy; however, most studies show an overlap in lymphocyte proliferation between Ni-allergic and nonallergic subjects using the LTT. The aim of this study was to see if the secretion of cytokines, especially interleukin (IL)-10 and IL-17, or the use of T-cell receptor (TCR) Vbeta families in Ni-stimulated primary peripheral blood mononuclear cell (PBMC) cultures might be more useful for discriminating between allergic and nonallergic subjects. METHODS: Ni(2+)-stimulated primary PBMC cultures derived from female subjects diagnosed as Ni-allergic (n = 5) or nonallergic (n = 5) on the basis of a positive or negative patch test were assessed for cell proliferation by tritiated thymidine incorporation and for production of interferon-gamma, IL-4, IL-10 and IL-17 in the culture supernatant by ELISA. The immunophenotype and TCR-Vbeta family affiliation of the Ni(2+)-induced lymphoblasts were determined by flow cytometry. RESULTS: Lymphocytes from Ni-allergic individuals challenged with a high and a low concentration of Ni showed significantly higher cell proliferation than lymphocytes from nonallergic individuals, but all subjects showed a positive LTT result (stimulation index >2). We found a significantly higher release of IL-10 in Ni(2+)-treated cultures from Ni-allergic compared with nonallergic subjects that provided better separation between individuals in the two groups than did lymphocyte proliferation. The proliferating lymphoblasts were predominantly CD4+, and in 2 of the 5 Ni-allergic subjects, but in none of the 5 nonallergic subjects, the CD4+ lymphoblasts showed a dominance of TCR-Vbeta17. CONCLUSIONS: Determination of IL-10 production in primary PBMC cultures is a potentially promising in vitro method for discrimination of Ni allergy in females, as compared with cell proliferation.

Flow cytometric enumeration of micronucleated reticulocytes: high transferability among 14 laboratories.

This laboratory previously described a single-laser flow cytometric method, which effectively resolves micronucleated erythrocyte populations in rodent peripheral blood samples. Even so, the rarity and variable size of micronuclei make it difficult to configure instrument settings consistently and define analysis regions rationally to enumerate the cell populations of interest. Murine erythrocytes from animals infected with the malaria parasite Plasmodium berghei contain a high prevalence of erythrocytes with a uniform DNA content. This biological model for micronucleated erythrocytes offers a means by which the micronucleus analysis regions can be rationally defined, and a means for controlling interexperimental variation. The experiments described herein were performed to extend these studies by testing whether malaria-infected erythrocytes could also be used to enhance the transferability of the method, as well as control intra- and interlaboratory variation. For these studies, blood samples from mice infected with malaria, or treated with vehicle or the clastogen methyl methanesulfonate, were fixed and shipped to collaborating laboratories for analysis. After configuring instrumentation parameters and guiding the position of analysis regions with the malaria-infected blood samples, micronucleated reticulocyte frequencies were measured (20,000 reticulocytes per sample). To evaluate both intra- and interlaboratory variation, five replicates were analyzed per day, and these analyses were repeated on up to five separate days. The data of 14 laboratories presented herein indicate that transferability of this flow cytometric technique is high when instrumentation is guided by the biological standard Plasmodium berghei.

Mercury intolerance and lymphocyte transformation test with nickel sulfate, palladium chloride, mercuric chloride, and gold sodium thiosulfate.

The peripheral lymphocytes of 10 patients referred to as mercury intolerant and 9 patients referred to as tolerant with regard to presence or absence of psychosomatic symptoms when percutaneously exposed to low patch test doses of mercury were stimulated in vitro with four metal salts. In addition, cells from 7 subjects with no anamnestic mercury intolerance or allergy to metals as well as free from dental alloys were included as controls. Lymphocyte transformation test was done by in vitro challenge with five concentrations of gold sodium thiosulfate, nickel chloride, palladium chloride, and seven concentrations of mercuric chloride. Stimulation with palladium chloride and mercuric chloride showed a difference between the mercury-intolerant and -tolerant patients on one hand and the controls on the other, but there was no difference between the two patient groups. With regard to nickel sulfate, there was a significant dose-dependent stimulation in all the three groups but no difference between the groups could be seen. Gold sodium thiosulfate did not stimulate the lymphocytes at all. Based on these results, we therefore conclude that lymphocyte transformation test performed with the four metal salts cannot be used to further differentiate between mercury-intolerant and -tolerant patients.

Characterization of primary recall in vitro lymphocyte responses to bacampicillin in allergic subjects.

BACKGROUND: Antigen-specific cell lines or clones are often used as models of drug-specific allergy. However, cloning procedures are time consuming, and the repeated antigen stimulation cycles as well as the addition of various growth enhancers may affect the in vivo relevance of these systems. OBJECTIVE: Using bacampicillin-allergic subjects, we wanted to investigate the applicability of primary recall in vitro lymphocyte responses to characterize type I and type IV allergy. The sensitivity and specificity of LTT (Lymphocyte transformation test), when used as an in vitro diagnostic tool, were also assessed. METHODS: A total of 39 patients with symptoms of type I (rhinitis) or type IV (allergic contact dermatitis, ACD) allergy following occupational exposure to bacampicillin, were included. Ten individuals without penicillin allergy or occupational exposure to bacampicillin served as controls. All subjects were LTT tested. Four patients with rhinitis and two patients with ACD were available for studying the immunophenotype and the TCR-Vbeta repertoire of bacampicillin induced lymphoblasts as well as the cytokine profiles and expression of the activation markers CD23 and CD134 in primary PBMC cultures. RESULTS: LTT was positive in 87% and at least one of the skin tests was positive in 85% of the patients with allergic symptoms. 69% of the patients with type I allergies were patch test-positive. Results from LTT and skin test correlated in 87% of the cases. The combined sensitivity of LTT and skin tests was 92%. The specificity of LTT was 90% in healthy controls. Bacampicillin induced lymphoblasts were mainly CD4 + in both ACD and rhinitis patients. The TCR-Vbeta profiles of the predominant CD4 + lymphoblasts were heterogeneous with individual skewing towards Vbeta2, Vbeta3, Vbeta5.1 and/or Vbeta14. An increased expression of IFNgamma was detected in bacampicillin treated PBMC cultures from the ACD but not from rhinitis patients. IL-5 was detected in bacampicillin exposed PBMC cultures from all patients but not from healthy controls. This Th2 environment could also be verified by CD23 and CD134 expression. CONCLUSION: LTT and skin tests are equally sensitive in identifying bacampicillin allergic subjects. When the two tests are combined, the sensitivity increases. The patch test is useful not only for detection of type IV but also for the identification of type I allergies. When using primary PBMC cultures, IFNgamma is the most suitable cytokine to discriminate between type I and type IV allergy. IL-5 can possibly be used as a general marker for bacampicillin induced allergy. Thus, primary cell cultures may be considered as an alternative to T-cell lines or clones for the study of drug induced allergy.

Characterization of mercuric mercury (Hg2+)-induced lymphoblasts from patients with mercury allergy and from healthy subjects.

Hg2+ induces lymphocyte proliferation when added to cell cultures from both healthy and mercury-allergic subjects. Consequently, when measuring DNA synthesis a possible Hg2+-specific response, resulting from proliferating memory cells, cannot be discriminated from a non-allergic response. The mechanism behind this non-allergic response is unknown but a superantigenic effect of Hg2+ has been suggested. In this study, five mercury-allergic patients, with oral lichen planus (OLP) lesions adjacent to dental amalgam and a positive patch test to Hg0, and five healthy subjects without amalgam were examined. The immunophenotype and the T cell receptor Vbeta (TCR Vbeta) repertoire of Hg2+-induced lymphoblasts as well as the expression of the lymphocyte activation markers CD23 and CD134 were analysed for possible differences between healthy and allergic subjects. The mechanism of Hg2+-induced proliferation was examined by comparing the TCR Vbeta expression of Hg- and staphylococcal enterotoxin B (SEB)-activated lymphoblasts, the latter used as a positive superantigen control. It was not possible to discriminate between mercury-allergic and healthy subjects using the immunophenotype or the TCR Vbeta profile of the Hg2+-induced lymphoblasts or the expression of CD23 and CD134. However, Hg2+-induced CD4+ lymphoblasts showed a skewing towards Vbeta2. This relative increase in Vbeta2 was only detected in the CD4+ but not in the CD8+ lymphoblast population. In conclusion, Hg2+ induced a proliferation-dependent skewing towards CD4+ but not CD8+ lymphocytes expressing Vbeta2. In this respect Hg2+ differs from the classical bacterial superantigen SEB, which also stimulates unique TCR Vbeta families among CD8+ cells.

Serotonin production in lymphocytes and mercury intolerance.

Patients with suspected illness due to mercury in dental amalgam were classified as tolerant or intolerant depending on their psychosomatic responses following in vivo epicutaneous provocation with low doses (patch test doses) of metallic mercury and phenylmercuric acetate. Ten intolerant patients and nine tolerant patients plus seven healthy amalgam-free and metal non-allergic controls were recruited to the study. Peripheral blood lymphocytes were exposed in vitro to three concentration of mercuric chloride (0.92, 1.83 and 3.68 microM) with and without 10 microg phytohaemagglutinine (PHA)/ml and the release of serotonin into the supernatant was measured. Lymphocytes exposed only to HgCl(2) showed no significant dose-dependent increase of serotonin, but the response of the tolerant patients was significantly higher compared with the controls. No other differences were found. Co-culture with mercuric chloride and PHA showed a statistically significant dose-dependant release of serotonin, but no differences between the three clinical groups could be detected. Thus, our results could not validate the concept of mercury tolerance and intolerance.

In vitro lymphoproliferative assays with HgCl2 cannot identify patients with systemic symptoms attributed to dental amalgam.

Dental amalgam is suspected, by some exposed individuals, to cause various systemic psychological, sensory, and neurological symptoms. Since not all amalgam-bearers experience such reactions, an individual characteristic--for example, a susceptible immune system--might explain these conditions. In vitro lymphocyte proliferation is a valuable tool in the diagnosis of allergy. With HgCl2 as the antigen, however, the test is hampered, because Hg2+ can cause unspecific lymphocyte proliferation, optimal at 1.4 to 9.5 micrograms HgCl2/mL. Recently, the use of suboptimal HgCl2 concentrations (< or = 0.5 microgram/mL) has been suggested to circumvent these problems. The main aim of this study was to investigate whether patients with systemic symptoms alleged to result from the presence of dental amalgam differ from healthy controls, with reference to in vitro lymphoproliferative responses to HgCl2 < or = 0.5 microgram/mL. Three different test protocols--lymphocyte transformation test (LTT) in micro- and macro-cultures, and the memory lymphocyte immunostimulation assay (MELISA)--were used. Other immune parameters--such as a standard patch test for dental materials, the number of T- and B-lymphocytes, monocytes, granulocytes, and NK cells in peripheral blood, allergic symptoms, and predisposition--were also investigated. Twenty-three amalgam patients, 30 healthy blood donors with amalgam, ten healthy subjects without amalgam, and nine patients with oral lichen planus (OLP) adjacent to dental amalgam and a positive patch test to Hg0 were tested. None of the investigated immune parameters revealed any significant differences between amalgam patients and controls. The sensitivity of in vitro lymphocyte proliferation ranged from 33 to 67%, with the OLP patients as a positive control group, and the specificity from 0 to 70% for healthy controls with a negative patch test to Hg0. Thus, despite the use of HgCl2 < or = 0.5 microgram/mL, a high frequency of positive results was obtained among healthy subjects with or without dental amalgam. Consequently, in vitro lymphocyte proliferation with HgCl2 cannot be used as an objective marker for mercury allergy in dental amalgam-bearers.

In vitro lymphocyte proliferation in the diagnosis of allergy to phenoxymethylpenicillin.

BACKGROUND: The aim of this study was to investigate in vitro lymphocyte proliferation in the diagnosis of allergy to phenoxymethylpenicillin (PcV), comparing chemically reactive PcV, added to cell cultures in unconjugated form, to a PcV-PLL (poly-L-lysine) conjugate as antigens. Side-chain specificity of lymphoproliferative responses was investigated with reactive benzylpenicillin (PcG) and bacampicillin. METHODS: Seventeen patients with a history of hypersensitivity reactions in connection with PcV treatment were studied by means of the lymphocyte transformation test (LTT), the radioallergosorbent test (RAST), skin tests (prick and intracutaneous), and oral challenge with PcV. LTT was also performed in 20 control subjects exposed to PcV therapeutically, and in eight subjects with occupational exposure to this penicillin. RESULTS: Nine patients had a positive in vivo test to PcV (five by oral challenge, three by intracutaneous test, and one by both tests), and six were challenge-negative. When reactive PcV was used as antigen in LTT, positive LTT responses were observed in five of the nine patients with a positive in vivo test, and two of them were also side-chain specific. Positive LTT responses with reactive PcV also correlated with a positive RAST in five of seven subjects. None of the six patients with a negative challenge test, and only one of the 28 controls showed a positive LTT result with reactive PcV. Thus, the specificity of LTT with reactive PcV was 96%. In contrast, when PLL-conjugated PcV served as antigen, four challenge-negative subjects and 11 controls were LTT-positive. CONCLUSIONS: The results of this study indicate that LTT with chemically reactive PcV could be useful as an in vitro complement in the diagnosis of PcV allergy and as a tool to reveal the side-chain specificity of peripheral blood lymphocytes. A positive LTT to PLL-conjugated PcV may be an indicator of immunization, but not necessarily allergy, to the penicilloyl structure.

Allergic nephropathy associated with norfloxacin and ciprofloxacin therapy. Report of two cases and review of the literature.

Allergic nephropathy associated with quinolone antibiotics has been reported in an increasing number of cases. The mechanism might be a hypersensitivity reaction. Norfloxacin has been incriminated previously as a cause once only, with acute interstitial nephritis (AIN) as the histopathological finding. Ciprofloxacin-associated nephropathy has been reported in 28 cases, with AIN as the main histopathological finding. This report describes a second case of AIN associated with norfloxacin treatment and another ciprofloxacin-associated renal interstitial drug adverse reaction. Clinicians should be aware of quinolone-associated AIN, which is a rare but potentially dangerous renal complication.

In vitro lymphocyte proliferation as compared to patch test using gold, palladium and nickel.

BACKGROUND: A conventional lymphocyte transformation test (LTT) was compared to the commercially available MELISA (memory lymphocyte immunostimulation assay), a lymphoproliferative assay that has been suggested to be a valuable instrument for the diagnosis of metal allergy. Sensitivity and specificity of the two assays were calculated using a patch test as a reference method. METHODS: 34 patients were patch-tested for gold sodium thiosulfate, palladium chloride and nickel sulfate, and the lymphocyte proliferation to these metals was tested in vitro using mononuclear cells from peripheral blood. RESULTS: No significant differences regarding sensitivity and specificity were found between MELISA and conventional LTT. The sensitivity varied between 55 and 95% and the specificity between 17 and 79%. CONCLUSIONS: The low specificity of the two in vitro assays suggests that they are not useful for diagnosis of contact allergy to the metals gold, palladium and nickel, since a large number of false-positive results will be obtained.

Mercury-specific lymphocytes: an indication of mercury allergy in man.

In this study, 18 patients with oral lichen planus (OLP), adjacent to amalgam fillings, were tested in vitro with an optimized lymphocyte proliferation test, MELISA (memory lymphocyte immunostimulation assay) and with a patch test. Twenty subjects with amalgam fillings but without oral discomfort and 12 amalgam-free subjects served as controls. The results show that patients with OLP have significantly higher lymphocyte reactivity to inorganic mercury, a corrosion product of amalgam, compared to control groups. Removal of amalgam fillings resulted in the disappearance of oral mucosal changes, thus indicating a causal relationship. Positive responses to phenylmercury (phenyl-Hg), a bactericidal agent in root fillings and in pharmaceutical preparations, were also noted in the oral lichen group but not in the control groups. Thus, low-grade chronic exposure to mercury may induce a state of systemic sensitization as verified by Hg-specific lymphocyte reactivity in vitro.

MELISA-an in vitro tool for the study of metal allergy.

The sensitizing properties of metals widely used in medical and dental care have been studied with the help of an optimized lymphocyte proliferative assay, MELISA. MELISA (memory lymphocyte immuno-stimulation assay) was originally developed for the screening of allergenic epitopes of drugs and other chemicals of low molecular weight, but has recently been adapted for the study of metal-induced sensitization. The patients studied suffered from various oral mucosal problems which were suspected to be caused by the release of metal ions from dental restorations. They were also troubled by chronic fatigue persisting over many years. One patient was also occupationally exposed to metals while working in a dental practice. Healthy subjects without any discomfort due to metal devices served as controls. In addition to metals used in dentistry, lymphocyte responses to organic mercurials used widely as preservatives in vaccines, eye/nose drops and contact lense fluids were studied. The results indicated that mercurials, as well as other metals such as gold or palladium, induce strong lymphocyte proliferative responses in patients with oral or systemic symptoms, but not in similarly exposed unaffected subjects. The results of MELISA performed with a pair of identical twins with chronic fatigue syndrome (CFS) indicated that metal-specific responses may be dependent on the genetics of the patient. Thus, many metals that are today accepted for use in medicine and dentistry carry a definite sensitizing risk for certain genetically predisposed individuals. Therefore, the use of these metals should be limited in the future.