Polyl-histidine

Poly-l-histidine (PHSTD) of molecular weight 26,000 induced the generation of large amounts of Superoxide (O ₂ ^– ) and hydrogen peroxide (H₂O₂) in human neutrophils (PMNs). Despite its low solubility at neutral pH, PHSTD was bound very rapidly to the PMN surfaces. Maximal generation of O ₂ ^– took place with 4–5 ×10^–6 M of PHSTD, starting after a lag of about 25 sec and proceeding for 15–17 min at a rate of 150 nmol/10⁷ PMNs/min, suggesting that this polycation is one of the most potent stimulators of O ₂ ^– generation known, PHSTD was found to be non-toxic for PMNs even at millimolar concentrations. Generation of O ₂ ^– by PHSTD depended on extracellular calcium; it was inhibited by calcium channel blockers and by trifluoperazine, and it triggered a sharp rise in intracellular calcium as determined by the Quin 2 fluorescence technique. The generation of both O ₂ ^– and H₂O₂ by PHSTD was partially inhibited by cytochalasin B or (CYB, CYE). On the other hand, CYB markedly enhanced the generation of both O ₂ ^– and H₂O₂ following stimulation of PMNs either by PHSTD, polyarginine, histone, or by antibody-opsonized group A streptococci. Electron microscopic analysis and NBT reduction tests revealed that both PHSTD and PHSTD-opsonized streptococci were avidly phagocytosed by PMNs. Since CYB totally inhibited internalization of both PHSTD and the PHSTD-opsonized streptococci, it was suggested that these agents stimulated oxygen radical generation mainly on the leukocyte surfaces. Complexes (CX) formed between PHSTD and polyanethole sulfonate (a strong polyanion) or between histone and the polyanion mimicked immune CX in their ability to trigger the generation of large amounts of O ₂ ^– which were inhibited by CYB. Generation of O ₂ ^– and chemiluminescence either by PHSTD or by PHSTD-opsonized streptococci were markedly inhibited by poly-l-glutamate, suggesting that PHSTD acted as a cationic agent which interacted via electrostatic forces with some negatively charged sites in the leukocyte membrane. Generation of H₂O₂ by PHSTD was also markedly inhibited by deoxyglucose, KCN, DASA, as well as by the lipoxygenase inhibitors nordihydroguaiaretic acid, phenidone, and propylgallate. On the other hand, cyclooxygenase inhibitors such as aspirin, indomethacin, and piroxicam were inactive, suggesting that arachidonic acid metabolism via lipoxygenase pathway might have been involved in the activation by PHSTD of the NADPH oxidase in PMNs. PHSTD may mimic the effects of antibodies both as an opsonin and as a potent stimulator of the respiratory burst in PMNs and may thus serve as a model for further study of leukocyte-bacteria interactions in infectious and inflammatory sites and of the pathogenicity of immune complexes.Supported by a research grant from Dr. S. M. Robbins of Cleveland, Ohio.     

PCR detection of amplified 132 bp repeats in Marek's disease virus type 1 (MDV-1) DNA can serve as an indicator for critical genomic rearrangement leading to the attenuation of virus virulence

A radioactive PCR test was developed that amplified the very virulent Marek's disease virus-1 (vvMDV-1) DNA sequence containing the 132 bp repeats. In apathogenic MDV-1 (CVI 988, Rispens), amplified DNA bands containing multiple copies of 132 bp repeats were identified. In the present study this PCR technique was used to monitor the passage level of vvMDV-1 in chicken embryo fibroblasts (CEF) in which the number of tandem 132 bp repeats was increased. It was found that at passage level 32 of vvMDV-1-B isolate, the 132 bp tandem repeat was already markedly amplified and its pattern resembled that of the MDV-1 (CVI 988, Rispens) vaccine virus DNA. In the vvMDV-1Z strain, amplification of the 132 bp repeat was not detectable at a similar passage level. The PCR test demonstrated that the apathogenic MDV-1 Md11/75c virus developed by extensive in vitro passaging has amplified 132 bp DNA repeats similar to those of the commercial vaccine virus (CVI 988, Rispense). It was also found that the pattern of viral RNA from infected cells detectable by Northern blot hybridization was markedly changed from a 2.4 kb RNA species in cells infected with vvMDV-1 viruses, to four RNA species (ranging from 2.2 to 4.4 kb) in cells infected with passage 32 of MDV-1-B strain, to a very large number of undefined RNA species synthesized in cells infected with attenuated MDV-1 viruses (CVI 988, Rispens and Md 11/75c).     

Identification and characterization of linear B-cell epitopes of beta-globulin, a major allergen of sesame seeds

BACKGROUND: The increased consumption of foods containing sesame seeds is paralleled by an increase in reported sesame-induced allergic reactions. OBJECTIVE: This study aimed at identifying and characterizing the linear B-cell epitopes of the 14-kd beta-globulin, the major allergen of sesame seed. METHODS: A peptide containing 71 amino acids (peptide B) was previously identified by us as the IgE-binding region on beta-globulin. To determine the amino acid sequence of the IgE-binding sites on peptide B, we synthesized overlapping peptides 20 and 10 amino acid residues long that span the entire length of peptide B, which were offset from each other by 10 and 2 amino acid residues, respectively. Sera from 20 subjects given diagnoses of allergy to sesame beta-globulin served to identify the epitopes by using the dot-blot test. RESULTS: At least 9 different IgE-recognition sites were identified on peptide B. Three of them, numbers 2, 3, and 13 (corresponding to amino acids 46-55, 48-57, and 76-86, respectively, in the beta-globulin sequence), appeared to be immunodominant IgE-binding epitopes. Also, these peptides were best recognized in terms of intensity of response. There was no obvious sequence motif shared by the 9 different IgE-binding epitopes of beta-globulin. However, approximately 60% of the amino acids represented in the epitopes are hydrophobic residues. CONCLUSION: Identification of the IgE-binding epitopes might provide a better understanding of the functional role the allergens play in the disease and might have implications for immunodiagnosis and probably immunotherapy.     

A novel point mutation (I137T) in the conserved 5-phosphoribosyl-1-pyrophosphate binding motif of hypoxanthine-guanine phosphoribosyltransferase (HPRTJerusalem) in a variant of Lesch-Nyhan syndrome

We identified a novel point mutation (I137T) in the hypoxanthine-guanine phosphoribosyltransferase (HPRT; EC 2.4.2.8) encoding gene, in a patient with partial deficiency of the enzyme (variant of Lesch-Nyhan syndrome). The mutation, ATT to ACT, resulting in substitution of isoleucine to threonine, occurred at codon 137 (exon 6), which is within the region encoding the binding site for 5-phosphoribosyl-1-pyrophosphate (PRPP). We suggest the mechanism by which the mutation-induced structural alteration of HPRT reduced the affinity of the enzyme for PRPP.     

Defective potassium currents in ataxia telangiectasia fibroblasts

Similarities exist between the progressive cerebellar ataxia in ataxia telangiectasia (AT) patients and a number of neurodegenerative diseases in both mouse and man involving specific mutations in ion channels and/or ion channel activity. These relationships led us to investigate the possibility of defective ion channel activity in AT cells. We examined changes in the membrane potential of AT fibroblasts in response to extracellular cation addition and found that the ability of AT fibroblasts to depolarize in response to increasing concentrations of extracellular K⁺ is significantly reduced when compared with control fibroblasts. Electrophysiological measurements performed with a number of AT cell lines, as well as two matched sets of primary AT fibroblast cultures, reveal that outward rectifier K⁺ currents are largely absent in AT fibroblasts in comparison with control cells. These K⁺ current defects can be corrected in AT fibroblasts transfected with the full-length ATM cDNA. These data implicate, for the first time, a role for ATM in the regulation of K⁺ channel activity and membrane potential.     

Growth hormone co-treatment for ovulation induction may enhance conception in the co-treatment and succeeding cycles, in clonidine negative but not clonidine positive patients

To investigate the effect of co-treatment with growth hormone(GH) for ovulation induction with human menopausal gonadotrophins(HMG) on conception, we compared the pregnancy rate and responseto co-treatment with GH versus HMG/human chorionic gonadotrophin(HCG) alone in a prospective, randomized, cross-over protocolof volation induction for either in-vivo or in-vitro fertilization(IVF). The main outcome measures were the amount of gonadotrophinused and conception. Co-treatment with GH was associated witha reduction of 30% in gonadotrophin requirement. In 24 clonidinenegative patients 14 pregnancies were achieved (58.3%) eitherin the GH/HMG/HCG cycle or in the succeeding one. GH co-treatmentdid not generate any pregnancy in eight clonidine positive patients.We conclude that growth hormone may increase the pregnancy ratewhen combined with HMG/HCG for ovulation induction, not onlyin the co-treatment cycle but also in the succeeding one. Thebeneficial, synergistic effect of GH co-treatment was detectedin clonidine negative but not in clonidine positive infertilepatients.     

The complete sequence of the coding region of the ATM gene reveals similarity to cell cycle regulators in different species

Ataxia-telangiectasia (A-T) is an autosomal recessive disorderinvolving cerebellar degeneration, immunodeficiency, radiationsensitivity, and cancer predisposition. A-T heterozygotes aremoderately cancer prone. The A-T gene, designated ATM, was recentlyidentified in our laboratory by positional cloning, and a partialcDNA clone was found to encode a polypeptide with a PI-3 kinasedomain. We report here the molecular cloning of a cDNA contigspanning the complete open reading frame of the ATM gene. Thepredicted protein of 3056 amino acids shows significant sequencesimilarities to several large proteins in yeast, Drosophilaand mammals, all of which share the PI-3 kinase domain. Manyof these proteins are involved in the detection of DNA damageand the control of cell cycle progression. Mutations in theirgenes confer a variety of phenotypes with features similar tothose observed in human A-T cells. The complete sequence ofthe ATM gene product provides useful clues to the function ofthis protein, and furthers understanding of the pleiotropicnature of the A-T mutations.     

A novel system to diagnose cutaneous adverse drug reactions employing the cellscan--comparison with histamine releasing test and Inf-gamma Releasing Test

Background: There are several mechanisms to describe allergic drug reactions yet the methods to diagnose them are limited. Objective: To compare several conventional clinical and laboratory methods to diagnose skin reactions to drugs to a new method of diagnosing drug reactions by the CellScan system. Methods: The study entailed 21 patients who were diagnosed as suffering from drug eruptions, and 105 healthy controls with no history of drug allergy. The drugs were classified into two groups according to suspicion of causing drug allergy: high and low. Most of the patients were on more than one drug, leading to 41 patient-drug interactions (assays). Histamine releasing test (HRT), interferon (INF)-γ releasing test and CellScan examination were performed on lymphocytes of the patients and controls. Results: The HRT was interpreted as positive in 9 out of 18 (50%) patients and in 13 out of 35 (37%) assays. Based on the INF-γ releasing test, positive results were observed in 16 out of 21 (76%) patients and in 24 out of 41 (59%) assays. In the CellScan test (CST), positive results were observed in 17 out of 21 (81%) patients and in 29 out of 41 (71%) assays. The rate of identifying the drug for eruption in the high suspicion level drugs was 9 out of 22 (41%) assays in the HRT, 20 out of 24 (83%) assays in the INF-γ releasing test, and 21 out of 24 (87%) studies with the CellScan method. The rate of determining of the drug that caused the eruption in the low suspicion level drugs was 4 out of 13 (31%) in the HRT, 4 out of 17 (24%) assays in the INF-γ releasing test, and 8 out of 17 (47%) analyses in the CST. When examined in the CellScan, 99 out of 105 (94%) controls were interpreted as negative. Conclusion: This preliminary study indicates that the CellScan seems to be an easy and promising method for the detection of drugs responsible for adverse skin reactions. In contrast to the HRT and to the Interferon-γ secretion test, the CellScan method is characterized by its ability to track and monitor the reaction of individual cells. By measuring the kinetic parameters of selected cells before and after adding the suspected drug, we were able to identify the culprit drug. The CellScan method had the highest sensitivity, and the interferon-γ secretion test had the highest specificity for detection of the culprit drug. In contrast, the analysis of 105 normal control sera disclosed a high specificity of 94% for the CellScan method.     

Reproducibility of skin testing and serum venom specific IgE in Hymenoptera venom allergy.

BACKGROUND: The decision regarding an immunotherapy regimen for venom-allergic patients is based on the results of skin testing and serum venom specific IgE measurements. However, their reliability has been questioned, and their reproducibility has not been examined. OBJECTIVE: To evaluate the reproducibility and reliability of the results of skin testing and serum venom specific IgE measurement in venom-allergic patients. METHODS: Patients with a systemic reaction after an insect sting were evaluated twice, 2 to 6 weeks apart, by intradermal skin tests and by determination of serum venom specific IgE to Hymenoptera venoms. RESULTS: Thirty-five patients were evaluated 1 to 168 months (mean, 23 months) after the sting reaction. Reproducibility of skin test results for all venoms at the 2 sessions was found in 23 patients (66%). Reproducibility of venom specific IgE results for all venoms was found in 16 (59%) of 27 patients from whom 2 blood samples were available for evaluation. Concordance between skin test and venom specific IgE results for all venoms was found in 30 (51%) of 59 samples available for evaluation. CONCLUSIONS: The reproducibility of venom skin test and serum venom specific IgE results is relatively poor. It is common practice for therapeutic decisions regarding venom immunotherapy to be based on a single diagnostic evaluation. Consequently, many patients are either overtreated or undertreated. Better diagnostic methods are required in venom allergy.