Nonclinical Tools to Assess Risk of Drug Hypersensitivity Reactions.

The term drug hypersensitivity refers to a category of adverse drug reactions mediated by various immunological and nonimmunological mechanisms. Small-molecule drugs and biotherapeutics have been associated with drug hypersensitivity reactions (DHRs), and the mechanisms driving the responses vary. Depending on the mechanism, some DHRs may be detected in nonclinical toxicology studies, and there may be tools and models in place that can be used as part of a risk assessment strategy. In contrast, for other mechanisms, particularly those that are not readily detected during nonclinical development, predictive tools and strategies for risk assessment are not well defined. This chapter provides an overview of the nonclinical tools currently available to assess the risk for developing DHRs.

Cadmium, copper, iron, and zinc concentrations in kidneys of grey wolves, Canis lupus, from Alaska, Idaho, Montana (USA) and the Northwest Territories (Canada).

Cadmium, copper, iron, and zinc levels were measured in the kidneys of 115 grey wolves (Canis lupus) from Idaho, Montana and Alaska (United States), and from the Northwest Territories (Canada). No significant differences in the levels of iron or copper were observed between locations, but wolf kidneys from more northern locations had significantly higher cadmium levels (Alaska > Northwest Territories > Montana ≈ Idaho), and wolves from Alaska showed significantly higher zinc than other locations. Additionally, female wolves in Alaska had higher iron levels than males, and adult wolves in Montana had higher copper levels than subadults.

Interaction of a protein phosphatase with an Arabidopsis serine-threonine receptor kinase.

A protein phosphatase was cloned that interacts with a serine-threonine receptor-like kinase, RLK5, from Arabidopsis thaliana. The phosphatase, designated KAPP (kinase-associated protein phosphatase), is composed of three domains: an amino-terminal signal anchor, a kinase interaction (KI) domain, and a type 2C protein phosphatase catalytic region. Association of RLK5 with the KI domain is dependent on phosphorylation of RLK5 and can be abolished by dephosphorylation. KAPP may function as a signaling component in a pathway involving RLK5.

Structure and expression of a calcium-binding protein gene contained within a calmodulin-regulated protein kinase gene.

We have determined the first genomic structure and characterized the mRNA and protein products of a novel vertebrate gene that encodes a calcium-binding protein with amino acid sequence identity to a protein kinase domain. The elucidation of the complete DNA sequence of this transcription unit and adjacent genomic DNA, Southern blot and polymerase chain reaction analyses of cellular genomic DNA, and examination of mRNA and protein species revealed that the calcium-binding kinase-related protein (KRP)-encoding gene is contained within the gene for a calmodulin-regulated protein kinase, myosin light-chain kinase (MLCK). The KRP gene transcription unit is composed of three exons and a 5'-flanking sequence containing a canonical TATA box motif. The TATA box, the transcription initiation site, and the first 109 nucleotides of the 5' noncoding region of the KRP mRNA correspond to an MLCK gene intron sequence. Both KRP and MLCK are produced in the same adult chicken tissue in relatively high abundance from a single contiguous stretch of genomic DNA and utilize the same reading frame and common exons to produce distinct mRNAs (2.7 and 5.5 kb, respectively) that encode proteins with dissimilar biochemical functions. There appears to be no precedent in vertebrate molecular biology for such a relationship. This may represent a mechanism whereby functional diversity can be achieved within the same vertebrate tissue by use of common exons to produce shuffled domains with identical amino acid sequences in different molecular contexts.

Membrane estrogen receptor engagement activates endothelial nitric oxide synthase via the PI3-kinase-Akt pathway in human endothelial cells.

17beta-Estradiol (E(2)) is a rapid activator of endothelial nitric oxide synthase (eNOS). The product of this activation event, NO, is a fundamental determinant of cardiovascular homeostasis. We previously demonstrated that E(2)-stimulated endothelial NO release can occur without an increase in cytosolic Ca(2+). Here we demonstrate for the first time, to our knowledge, that E(2) rapidly induces phosphorylation and activation of eNOS through the phosphatidylinositol 3 (PI3)-kinase-Akt pathway. E(2) treatment (10 ng/mL) of the human endothelial cell line, EA.hy926, resulted in increased NO production, which was abrogated by the PI3-kinase inhibitor, LY294002, and the estrogen receptor antagonist ICI 182, 780. E(2) stimulated rapid Akt phosphorylation on serine 473. As has been shown for vascular endothelial growth factor, eNOS is an E(2)-activated Akt substrate, demonstrated by rapid eNOS phosphorylation on serine 1177, a critical residue for eNOS activation and enhanced sensitivity to resting cellular Ca(2+) levels. Adenoviral-mediated EA.hy926 transduction confirmed functional involvement of Akt, because a kinase-deficient, dominant-negative Akt abolished E(2)-stimulated NO release. The membrane-impermeant E(2)BSA conjugate, shown to bind endothelial cell membrane sites, also induced rapid Akt and consequent eNOS phosphorylation. Thus, engagement of membrane estrogen receptors results in rapid endothelial NO release through a PI3-kinase-Akt-dependent pathway. This explains, in part, the reduced requirement for cytosolic Ca(2+) fluxes and describes an important pathway relevant to cardiovascular pathophysiology.

Gain of function mutations for yeast calmodulin and calcium dependent regulation of protein kinase activity.

Yeast calmodulin binds only three calcium ions in the presence of millimolar concentrations of magnesium due to a defective calcium-binding sequence in its carboxyl terminal domain. Yeast calmodulin's diminished calcium-binding activity can be restored to that of other calmodulins by the use of site-directed mutagenesis to substitute its fourth calcium-binding domain with that of a vertebrate calmodulin sequence. However, the repair of yeast calmodulin's calcium-binding activity is not sufficient to repair quantitatively yeast calmodulin's defective protein kinase activator activity. Yeast calmodulin's activator activity with smooth muscle and skeletal muscle myosin light chain kinases and brain calmodulin-dependent protein kinase II can be progressively repaired by additional substitutions of vertebrate calmodulin sequences, provided that the four calcium-binding sites remain intact. An unexpected result obtained during the course of these studies was the observation that myosin light chain kinases from smooth and skeletal muscle tissues can respond differently to mutations in calmodulin. These and previous results indicate that the binding of four calcium ions by calmodulin is necessary but not sufficient to bring about quantitative activation of protein kinases, and are consistent with the conformational selection/restriction model of the dynamic equilibrium among calcium, calmodulin and each calmodulin regulated enzyme.

Multiple gene products are produced from a novel protein kinase transcription region.

The nonmuscle/smooth muscle myosin light chain kinase (MLCK) and the kinase related protein (KRP) that lacks protein kinase activity are myosin II binding proteins encoded in the vertebrate genome by a true gene within a gene relationship. The genomic organization and expression result in the same amino acid sequence in different molecular contexts from two different sizes of mRNA. We report here the identification and characterization of a third size class of gene products. The protein appears to be a higher molecular weight form of MLCK with additional amino terminal tail sequence which might provide differential subcellular targeting characteristics.

Inhibition of interferon-gamma-mediated microvascular endothelial cell major histocompatibility complex class II gene activation by HMG-CoA reductase inhibitors.

BACKGROUND: Graft vascular disease, a major cause of late graft failure in cardiac transplant patients, is associated with the presence of class II major histocompatibility complex molecules on the endothelium. 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, e.g., simvastatin, have been shown to reduce the incidence of graft vascular disease. We studied the effect of simvastatin on interferon (IFN)-gamma-induced human leukocyte antigen (HLA)-DR expression in human microvascular endothelial cells (MVECs). METHODS AND RESULTS: Simvastatin pretreatment inhibited MVEC HILA-DR induction by IFN-gamma, as detected by flow cytometry. Simvastatin's inhibitory effect was reversed by the cholesterol synthesis pathway intermediates mevalonate and geranylgeranyl pyrophosphate but not squalene, indicating the involvement of protein prenylation in this process. Reverse transcription-polymerase chain reaction analysis demonstrated that induction of class II transactivator (CIITA), and consequently, HLA-DRalpha mRNA, is abrogated by simvastatin. Although signal transducer and activator of transcription (STAT)-1 is a critical CIITA gene transactivator, immunofluorescence studies, Western blotting, and electrophoretic mobility shift assays demonstrated that IFN-gamma-induced STAT-1 phosphorylation, nuclear translocation, and DNA binding are not affected by simvastatin. However, simvastatin inhibited IFN-gamma-induced transactivation of a CIITA promoter IV reporter construct, indicating the involvement of this promoter in the inhibitory effect of simvastatin. CONCLUSIONS: Simvastatin pretreatment inhibits CIITA and consequent HLA-DR induction by IFN-gamma in MVECs through interference with protein prenylation. This inhibitory effect occurs at the level of transcription and is directed, at least in part, at the CIITA promoter IV. These results explain some of the beneficial effects of HMG-CoA reductase inhibitors in cardiac transplantation.

Nonfatal disseminated mucocutaneous herpes simplex type 2 infection in a healthy woman.

Disseminated mucocutaneous herpes simplex virus (HSV) infection in nonpregnant and healthy women is a rare manifestation of primary genital inoculation. The literature contains relatively few cases of this type of infection in nonimmunocompromised adults. Furthermore, the majority of adult disseminated mucocutaneous infections have fulminant courses and terminate in death. In this report, a case of nonfatal disseminated mucocutaneous HSV type 2 infection is described in a nonimmunocompromised woman with a childhood history of immune thrombocytopenia purpura and splenectomy.

Differential induction of two potato genes, Stprx2 and StNAC, in response to infection by Phytophthora infestans and to wounding.

To find out more about the interaction between potato and Phytophthora infestans at the molecular level, we screened for genes induced early after infection using mRNA differential display. Among the twenty cDNA clones recovered in the screen, two were found to represent plant genes whose transcript levels increased during infection of intact plants. These two genes differed strikingly in their response to wounding. Stprx2, a putative peroxidase, responded slowly and transiently to wounding, and its expression pattern was similar to that of gst1, a well-described pathogen-induced gene of potato. The second gene, StNAC, was induced rapidly and strongly after wounding but not systemically. Transcript levels reached a maximum after around 1 h and returned to basal levels after ca. 24 h. StNAC has strong similarity to the ATAF subfamily of NAC domain proteins, a large family of putative transcriptional activators. Arabidopsis ATAF1 and ATAF2 were also shown to be induced by wounding. This implies that the ATAF genes are not merely structurally similar but also share a conserved role in stress responses.

Isolation of an Arabidopsis thaliana casein kinase II beta subunit by complementation in Saccharomyces cerevisiae.

Casein kinase II is thought to play an essential role in the control of cell division and differentiation in all eukaryotes. Through complementation of a defective casein kinase II catalytic subunit gene from Saccharomyces cerevisiae, we isolated an Arabidopsis thaliana casein kinase II regulatory subunit homologue, CKB1. A second regulatory subunit was identified by low-stringency hybridization with CKB1. Casein kinase II from S. cerevisiae is composed of two catalytic (alpha) and two regulatory (beta) subunits. Simultaneous disruption of the genes for the alpha and alpha' subunits, CKA1 and CKA2, respectively, is lethal. Strain YDH8 has disruptions of CKA1 and CKA2; its viability depends on a temperature-sensitive allele of CKA2, cka2-8, carried on a centromeric plasmid. We screened an A. thaliana cDNA library, whose inserts are under the control of the galactose-inducible GAL10 promoter, for cDNAs which enabled YDH8 cells to grow at the restrictive temperature. One cDNA, CKB1, was isolated by this screen which had homology to cDNAs of casein kinase II beta subunits. A second cDNA, CKB2, was isolated by hybridization and was also able to suppress the YDH8 mutant phenotype. The proteins encoded by CKB1 and CKB2 are 80% identical. The carboxy-terminal two thirds of both proteins is ca. 54% identical to the regulatory beta subunits of casein kinase II from other species. The amino termini are unrelated to any other known proteins. CKB1 and CKB2 lack the conserved autophosphorylation site characteristic of animal beta subunits, but have potential casein kinase II phosphorylation sites in the same region. Suppression of the cka1 delta cka2-8 mutant phenotype occurs by interaction of CKB1 with the defective, cka2-8-encoded, catalytic subunit. Cells with disruptions in CKA1 and CKA2 are not rescued by expression of CKB1.

Expression of human poly(ADP-ribose) polymerase in Saccharomyces cerevisiae.

The coding sequence for human poly(ADP-ribose) polymerase was expressed inducibly in Saccharomyces cerevisiae from a low-copy-number plasmid vector. Cell free extracts of induced cells had poly(ADP-ribose) polymerase activity when assayed under standard conditions; activity could not be detected in noninduced cell extracts. Induced cells formed poly(ADP-ribose) in vivo, and levels of these polymers increased when cells were treated with the alkylating agent N-methyl-N'-nitro-N- nitrosoguanidine (MNNG). The cytotoxicity of this agent was increased in induced cells, and in vivo labelling with [3H]adenine further decreased their viability. Increased levels of poly(ADP-ribose) found in cells treated with the alkylating agent were not accompanied by lowering of the NAD concentration.

The location of calmodulin in the pea plasma membrane.

Plasma membrane has been prepared from pea seedlings in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA). Calmodulin has been detected in these plasma membrane preparations using calcium overlay techniques, immunoblots, quantitation with antibodies raised against spinach calmodulin, phosphodiesterase activation, mobility shift, and heat stability. EGTA-stable calmodulin represents 0.5-1% of the total plasma membrane protein, and it is the only detectable calcium-binding protein in plasma membrane isolated under these conditions. The anti-spinach calmodulin reacts only with the N-terminal region of spinach calmodulin representing residues 1-106. The positioning of EGTA-stable calmodulin in the plasma membrane has been probed with trypsin and anti-spinach calmodulin. The data suggest that the calmodulin N-terminal region representing residues 1-106 projects from the membrane and could be available for binding other proteins. Calcium-dependent calmodulin binding to the plasma membrane has also been detected. Calcium-dependent calmodulin-binding proteins have been characterized using calmodulin overlay methods. The exposure of calmodulin-binding domains of most of these proteins from the plasma membrane is further suggested by their reaction with azidoiodinated calmodulin.

Identification of potato genes induced during colonization by Phytophthora infestans.

Summary Suppression Subtractive Hybridization (SSH) was applied in a search for genes induced during the compatible interaction between Phytophthora infestans and potato. Using potato leaves that had been treated with benzo(1,2,3)thiadiazole-7-carbothioic acid S-methylester (BTH) as the control tissue, a low redundancy library with a relatively low frequency of the classic plant Pathogenesis-Related (PR) genes was generated. 288 of the clones were screened for induced sequences using Inverse Northern analysis (hybridizing the arrayed clones with radiolabelled cDNA populations). Of the 75 clones that were detectable by this method, 43 appeared to be induced. Eleven of these clones were then analysed by total RNA blot analysis, and elevation of transcript levels during P. infestans infection was confirmed for 10 of them. Some of the cDNAs analysed by RNA blot analysis have homology to genes already known to be induced during infection, e.g. to beta-1,3-glucanase. Another group of cDNAs have homology to enzymes involved in detoxification: gamma-glutamylcysteine synthetase, cytochrome P450, glutathione S-transferase and an MRP-type ABC transporter. Other infection induced cDNAs encode putative proteins that have not previously been reported to be induced by infection: e.g. the ER-located chaperone BiP, and a homologue of Aspergillus nidulans SudD, which was isolated as a suppressor of a mutation in chromosome disjunction. The differential library therefore presents the opportunity to analyse the metabolic changes occurring during infection, and the disease process itself in more detail.

Cryptosporidiosis in a hospital-associated day care center.

Cryptosporidium was identified in the stools of infants and toddlers during an outbreak of diarrheal illness at a hospital-associated day care center. Stools from 42 of 54 children were examined for parasites, Salmonella, Shigella, Campylobacter and rotavirus; 27 children (64%) had Cryptosporidium. One child also had Salmonella and two other children were also found to have Giardia. Information regarding the severity and duration of symptoms was obtained from parents by means of a questionnaire. The most frequently reported symptoms included diarrhea, fever, anorexia and malaise. Of 9 children whose stools were examined during the time they were reported by parents to be symptomatic, 8 were positive for Cryptosporidium oocysts. Symptoms lasted from 1 day to 4 weeks and children excreted oocysts for up to 48 days after the onset of symptoms. Staff at the facility and family members were also affected. Cryptosporidiosis should be considered in the differential diagnosis of young children with diarrheal illness, especially those attending a day care facility.

Class II transactivator-independent endothelial cell MHC class II gene activation induced by lymphocyte adhesion.

NK cells induce MHC class II molecules on the surface of allogeneic endothelial cells in an adhesion-dependent, IFN-gamma-independent manner. Here, we demonstrate that NK cells induce HLA-DR on the surface of a mutant cell line that is defective in IFN-gamma-induced MHC class II expression. RNA analysis in these cells and in a cell line that is defective in class II transactivator (CIITA) demonstrates that NK cell-induced HLA-DR alpha mRNA expression is also CIITA-independent. The Janus kinase-1-deficient cell line U4A expresses HLA-DR alpha mRNA in response to NK cell activation, and HLA-DR alpha promoter constructs transfected into these cells are induced by NK cells but not IFN-gamma. These data indicate that the IFN-gamma-independent component of the target cell HLA-DR expression induced by lymphocyte adhesion uses a signaling pathway that is distinct from the IFN-gamma-dependent mechanism and also suggest that CIITA is not required.

Endoglycosidic cleavage of branched polymers by poly(ADP-ribose) glycohydrolase.

Post-translational modification of nuclear proteins with poly(ADP-ribose) modules chromatin structure and may be required for DNA processing events such as replication, repair and transcription. The polymer-catabolizing enzyme, poly(ADP-ribose) glycohydrolase, is crucial for the regulation of polymer metabolism and the reversibility of the protein modification. Previous reports have shown that glycohydrolase digests poly(ADP-ribose) via an exoglycosidic mechanism progressing from the protein-distal end of the polymer. Using two independent approaches, we investigated the possibility that poly(ADP-ribose) glycohydrolase also engages in endoglycosidic cleavage of polymers. First, partial glycohydrolase digestion of protein-bound poly(ADP-ribose) led to the production of protein-free oligomers of ADP-ribose. Second, partial glycohydrolase digestion of a fixed number of protein-free poly(ADP-ribose) polymers resulted in a transient increase in the absolute number of polymers while polymer size continuously decreased. Furthermore, endoglycosidic activity produced linear polymers from branched polymers although branch points themselves were not a preferential target of cleavage. From these data, we propose a mechanism whereby poly(ADP-ribose) glycohydrolase degrades polymers in three distinct phases; (a) endoglycosidic cleavage, (b) endoglycosidic cleavage plus exoglycosidic, processive degradation, (c) exoglycosidic, distributive degradation.

Simvastatin modulates cytokine-mediated endothelial cell adhesion molecule induction: involvement of an inhibitory G protein.

Endothelial cell adhesion molecules (CAMs) E-selectin, ICAM-1, and VCAM-1 play variably important roles in immune-mediated processes. They are induced by the proinflammatory cytokines IL-1 and TNF-alpha, and NF-kappaB is required for the regulated expression of all three genes. Regulators of this pathway could potentially be potent immune modulators. We studied the effect of a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, simvastatin, on cytokine-induced expression of CAMs in HUVEC. Unexpectedly, pretreatment with simvastatin potentiated the induction of all three endothelial CAMs by IL-1 and TNF, but not LPS or PMA, as detected by flow cytometry. Northern blot analysis demonstrated an increase in steady state IL-1-induced E-selectin mRNA levels in cells pretreated with simvastatin. This was associated with an increase in nuclear translocation of NF-kappaB, as detected by EMSA. The effect of simvastatin was reversed by mevalonate and geranylgeranyl pyrophosphate but not squalene, indicating that an inhibitory prenylated protein is involved in endothelial responses to proinflammatory cytokines. Pertussis toxin mimicked the effect of simvastatin, and the G protein activator NaF inhibited the cytokine-induced expression of endothelial CAMs, indicating that a Gialpha protein is involved. These results demonstrate that cytokine-mediated activation of the endothelium, and specifically CAM induction, can be modulated by a heterotrimeric G protein-coupled pathway. This may represent a "basal tone" of endothelial inactivation, which can either be disinhibited or amplified, depending on the stimulus.

Posttranscriptional regulation of urokinase plasminogen activator receptor messenger RNA levels by leukocyte integrin engagement.

As an adhesion receptor, the beta2 integrin lymphocyte function-associated antigen-1 (LFA-1) contributes a strong adhesive force to promote T lymphocyte recirculation and interaction with antigen-presenting cells. As a signaling molecule, LFA-1-mediates transmembrane signaling, which leads to the generation of second messengers and costimulation resulting in T cell activation. We recently have demonstrated that, in costimulatory fashion, LFA-1 activation promotes the induction of T cell membrane urokinase plasminogen activator receptor (uPAR) and that this induced uPAR is functional. To investigate the mechanism(s) of this induction, we used the RNA polymerase II inhibitor 5, 6-dichloro-1-beta-D-ribobenzimidazole and determined that uPAR mRNA degradation is delayed by LFA-1 activation. Cloning of the wild-type, deleted and mutated 3'-untranslated region of the uPAR cDNA into a serum-inducible rabbit beta-globin cDNA reporter construct revealed that the AU-rich elements and, in particular the nonameric UUAUUUAUU sequence, are crucial cis-acting elements in uPAR mRNA degradation. Experiments in which Jurkat T cells were transfected with reporter constructs demonstrated that LFA-1 engagement was able to stabilize the unstable reporter mRNA containing the uPAR 3'-untranslated region. Our study reveals a consequence of adhesion receptor-mediated signaling in T cells, which is potentially important in the regulation of T cell activation, including production of cytokines and expression of proto-oncogenes, many of which are controlled through 3' AU-rich elements.