SIRT1 is increased in affected brain regions and hypothalamic metabolic pathways are altered in Huntington disease.

AIMS: Metabolic dysfunction is involved in modulating the disease process in Huntington disease (HD) but the underlying mechanisms are not known. The aim of this study was to investigate if the metabolic regulators sirtuins are affected in HD. METHODS: Quantitative real-time polymerase chain reactions were used to assess levels of SIRT1-3 and downstream targets in post mortem brain tissue from HD patients and control cases as well as after selective hypothalamic expression of mutant huntingtin (HTT) using recombinant adeno-associated viral vectors in mice. RESULTS: We show that mRNA levels of the metabolic regulator SIRT1 are increased in the striatum and the cerebral cortex but not in the less affected cerebellum in post mortem HD brains. Levels of SIRT2 are only increased in the striatum and SIRT3 is not affected in HD. Interestingly, mRNA levels of SIRT1 are selectively increased in the lateral hypothalamic area (LHA) and ventromedial hypothalamus (VMH) in HD. Further analyses of the LHA and VMH confirmed pathological changes in these regions including effects on SIRT1 downstream targets and reduced mRNA levels of orexin (hypocretin), prodynorphin and melanin-concentrating hormone (MCH) in the LHA and of brain-derived neurotrophic factor (BDNF) in the VMH. Analyses after selective hypothalamic expression of mutant HTT suggest that effects on BDNF, orexin, dynorphin and MCH are early and direct, whereas changes in SIRT1 require more widespread expression of mutant HTT. CONCLUSIONS: We show that SIRT1 expression is increased in HD-affected brain regions and that metabolic pathways are altered in the HD hypothalamus.

Drug-specific cyclodextrins with emphasis on sugammadex, the neuromuscular blocker rocuronium and perioperative anaphylaxis: implications for drug allergy.

Cyclodextrins, oligosaccharides linked in a circular arrangement around a central cavity, are used extensively in the pharmaceutical industry to improve drug delivery. Their usefulness depends on their capacity to form a drug inclusion, or host-guest, complex within the cavity. In an attempt to improve the delivery of the widely used neuromuscular blocking drug (NMBD) rocuronium, a rocuronium inclusion complex was formed with a chemically modified γ-cyclodextrin. The high binding affinity and specificity of the modified carrier (named sugammadex) for rocuronium (and other aminosteroid NMBDs) led to its use in anaesthesia as an innovative and useful agent for rapid reversal of rocuronium-induced neuromuscular block by sequestering the drug as an inclusion complex. This, in turn, led to the suggestion that sugammadex might be useful to remove the NMBD from the circulation of patients experiencing rocuronium-induced anaphylaxis, a suggestion subsequently supported in case reports where traditional treatment had failed. Successful resuscitations suggested that sugammadex might be a valuable new treatment for such intractable cases but, given the inappropriateness of clinical trials, confirmation or refutation will have to await the slow accumulation of results of individual case reports. Important questions related to antibody accessibility of drug allergenic structures on the rocuronium-sugammadex inclusion complex, and the competition between sugammadex and IgE antibodies (both free and cell bound) for rocuronium, also remain and can be investigated in vitro. The sugammadex findings indicate that the use of carrier molecules such as the cyclodextrins to improve drug delivery will sometimes give rise to changed immunologic and allergenic behaviour of some drugs and this will have to be taken into account in preclinical drug safety assessments of drug-carrier complexes. The possibility of encapsulating and removing other allergenic drugs, e.g., penicillins and cephalosporins, in cases of difficult-to-reverse anaphylaxis to these drugs is discussed.

Cloning and sequencing of a cDNA expressing a recombinant house dust mite protein that binds human IgE and corresponds to an important low molecular weight allergen.

A cDNA clone coding for a mite allergen of mol wt approximately 14,000 has been isolated and its DNA sequence determined. The native component from mite extracts encoded by this DNA was identified by immunoprobing blots of mite body extract with human IgE eluted from the electroblotted cloned fusion polypeptides derived from the expressed cDNA clone. The clone encodes a polypeptide with a deduced mol wt of 17,460. The deduced amino acid sequence was not homologous to any known protein sequences and it contains no cysteine or tryptophan. On blots, 21 of 38 sera from mite-allergic subjects recognized the cloned material, and this recognition strongly correlated with IgE-binding to the native component on protein blots of mite extract.

On the origin and specificity of antibodies to neuromuscular blocking (muscle relaxant) drugs: an immunochemical perspective.

Following the demonstration 25 years ago that substituted ammonium groups on neuromuscular blocking drugs (NMBDs) are the main allergenic structures recognized by IgE antibodies in the sera of some patients who experience anaphylaxis during anaesthesia, immunoassays for these drugs were quickly applied to supplement skin tests in the diagnostic assessment of suspected adverse reactions to anaesthetic agents. Many subjects who react to an NMBD do so on first exposure and this led to the speculation that the origin of allergic sensitization is an environmental agent(s) or another drug containing an ammonium ion. Direct antibody binding and hapten inhibition studies revealed that morphine, which contains a tertiary amino group, was strongly recognized by IgE in sera from anaphylactic patients and a morphine-solid phase immunoassay was found to be superior to NMBD-based assays for the detection of NMBD-reactive IgE antibodies. Extensive inhibition experiments indicate the likelihood of antibody combining site heterogeneity with recognition at the fine structural level of features additional, and adjacent to, ammonium ions. Further quantitative investigations are needed to identify these neighbouring groups on different NMBDs. Recent work has implicated the morphine analogue pholcodine as the sensitizing agent in Norway where, unlike Sweden, anaphylactic reactions to NMBDs are not uncommon and the medicament is available over-the-counter. This has led to the suggestion that allergenic sensitization to the ammonium group of pholcodine may account for the different incidences of anaphylaxis during anaesthesia in the two countries. This work is subjected to critical review and some alternative speculations on the nature and origin of the sensitizing agent(s) are presented.

Immediate hypersensitivity responses in flatfish.

Fungal extracts that precipitate with human C-reactive protein caused immediate erythema on subdermal injection into marine flatfish. Only species with calcium-dependent serum precipitins to these fungi showed skin reactions. Immediate hypersensitivity in a nonreactive species could be induced after injection with serum from reactive species. The transferable serum factor (or factors) was heat sensitive.

Fluorine Is a Major Constituent of the Marine Sponge Halichondria moorei.

Fluorine constitutes about 10 percent of the dry weight of the marine sponge Halichondria moorei. The fluorine occurs as potassium fluorosilicate, which is a potent anti-inflammatory agent. A closely related sponge living in the same habitat does not contain any fluorine. The habitat was found to be free of fluorine except for the small amount naturally present in seawater.

The investigation of bronchospasm during induction of anaesthesia.

BACKGROUND: The aim of this study was to ascertain whether anaesthetic induction-related anaphylactic bronchospasm could be distinguished from other types of bronchospasm by clinical features and response to treatment. Such features could then be used to identify a group of patients in whom skin testing is indicated. METHODS: We retrospectively studied data from 183 patients referred to an anaesthetic allergy clinic because of bronchospasm during induction. For the analysis, the patients were divided into two groups depending on whether there was evidence suggesting immunological anaphylaxis. RESULTS: When the patients in whom intradermal tests were positive were compared with those in whom intradermal tests were negative, the skin test-positive patients had significantly more severe reactions, and they were more commonly associated with other clinical signs. Mast cell tryptase (MCT) was an excellent discriminator between reactions likely to be allergic and those unlikely to be allergic. CONCLUSIONS: Anaphylactic bronchospasm related to induction of anaesthesia is more likely to be severe than bronchospasm due to non-immune causes. An allergic cause is more likely if there are associated features of anaphylaxis (skin changes, hypotension, angioedema) or elevated MCT. Patients with any of these features should undergo immuno-allergolical investigation.

Structural features of allergens large and small with emphasis on recombinant allergens.

The application of recombinant DNA techniques to the study of allergen structure has increased our knowledge of primary structures and B- and T-cell determinants. Thus, knowledge of the molecular bases of isoallergens and allergenic cross-reactivities is about to be rapidly expanded. Findings from the early applications of molecular cloning strategies to the study of some polypeptide allergens, together with a summary of our current knowledge of drug allergenic determinants, are presented here.

Role of adenosine A2 receptors in brain stimulation reward under baseline conditions and during cocaine withdrawal in rats.

The present experiments tested the hypothesis that adenosine A2 receptors are involved in central reward function. Adenosine receptor agonists or antagonists were administered to animals that had been trained to self-stimulate in a rate-free brain stimulation reward (BSR) task that provides current thresholds as a measure of reward. The adenosine A(2A) receptor-selective agonists 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido adenosine hydrochloride (CGS 21680) (0.1-1.0 mg/kg) and 2-[(2-aminoethylamino)carbonylethyl phenylethylamino]-5'-N-ethylcarboxamido adenosine (APEC) (0.003-0.03 mg/kg) elevated reward thresholds without increasing response latencies, a measure of performance. Specifically, CGS 21680 had no effect on response latency, whereas APEC shortened latencies. Bilateral infusion of CGS 21680 (3, 10, and 30 ng/side), directly into the nucleus accumbens, elevated thresholds but shortened latencies. The highly selective A(2A) antagonist 8-(3-chlorostyryl)caffeine (0.01-10.0 mg/kg) and the A2-preferring antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) (0.3-10.0 mg/kg) did not alter thresholds or latencies, but DMPX (1.0, 10.0 mg/kg) blocked the threshold-elevating effect of APEC (0.03 mg/kg). In another study, repeated administration of cocaine (eight cocaine injections of 15 mg/kg, i.p., administered over 9 hr) produced elevations in thresholds at 4, 8, and 12 hr after cocaine. DMPX (3 and 10 mg/kg), administered before both the 8 and 12 hr post-cocaine self-stimulation tests, reversed the threshold elevation produced by cocaine withdrawal. These results indicate that stimulating adenosine A(2A) receptors diminishes BSR without producing performance deficits, whereas blocking adenosine receptors reverses the reward impairment produced by cocaine withdrawal or by an A(2A) agonist. These findings indicate that adenosine, via A(2A) receptors, may inhibit central reward processes, particularly during the neuroadaptations associated with chronic drug-induced neuronal activation.

Molecular cloning and characterization of a major allergen (Myr p I) from the venom of the Australian jumper ant, Myrmecia pilosula.

Five IgE-binding components were identified in the venom of the Australian jumper ant, Myrmecia pilosula using SDS polyacrylamide gel electrophoresis and Western blotting. A cDNA clone which encodes the entire amino acid sequence of one of the major IgE-binding venom allergens has been nucleotide sequenced. The IgE-binding determinants of this allergen are located in its C-terminal domain. Database searches, however, did not reveal any homology with any other known nucleotide or protein sequence. The sequenced allergenic polypeptide has, according to the convention recommended by the International Union of Immunological Societies (IUIS), been named Myr p I.

Characterization of C-reactive protein from the eggs of the marine teleost. Cyclopterus lumpus L.

Further evidence is presented for the existence in teleost fish of proteins homologous with mammalian C-reactive protein. The amino acid composition is given for a C-reactive protein isolated from the eggs of a marine teleost, Cyclopterus lumpus, by extraction with lecithin in the presence of Ca2+, followed by electrofocusing. A molecular weight of 150,000 was calculated from gel filtration and electrophoresis at different polyacrylamide gel concentrations, while the s20,w was 7.4 S. The 1.5-S subunit had an apparent Mr of 20,000 by SDS-polyacrylamide gel electrophoresis and 21,000 by computer analysis based on amino acid composition. Comparison is made with the physicochemical properties of mammalian C-reactive protein.

Cloning and characterization of a major allergen of the house dust mite, Dermatophagoides pteronyssinus, homologous with glutathione S-transferase.

A major allergen of the house dust mite, Dermatophagoides pteronyssinus, has been identified and characterized from a lambda gt11 cDNA library of the mite. IgE antibodies from the sera of allergic patients that recognise the cloned polypeptide bind to an approximately 26 kDa polypeptide on a Western blot of reduced mite polypeptides. Nucleotides sequencing of the clone revealed a 219 amino acid open reading frame encoding a protein with a derived molecular mass of 25,589 Da and a pI of 6.3. Comparison of the deduced amino acid sequence with amino acid sequence databanks revealed a strong homology with glutathione S-transferases. The nucleotide sequence of the clone displayed a strong homology with the active glutathione binding site of glutathione transferases and contained all but one of the 19 positionally conserved amino acid residues found in glutathione transferases. The cloned polypeptide was expressed in Escherichia coli and affinity-purified on glutathione agarose.

Molecular cloning and characterization of the major allergen Myr p II from the venom of the jumper ant Myrmecia pilosula: Myr p I and Myr p II share a common protein leader sequence.

A major allergen Myr p II of the Australian jumper ant Myrmecia pilosula has been cloned, immunocharacterized and nucleotide sequenced. An open reading frame of 225 bases was identified and found to encode a deduced amino acid sequence of 75 residues which contained a typical hydrophobic peptide leader sequence. Expressed fusion proteins of Myr p II in both phage and plasmid vectors bind high levels of ant venom-specific IgE and the expressed clones are recognised by 35% of ant venom-allergic individuals. IgE antibodies that recognise the expressed clone have been shown to recognise IgE-binding bands in blots of native venom after separation by SDS-PAGE. The amino acid sequence of Myr p II shares close structural homology with the other major jumper ant allergen Myr p I, differing by only three amino acids in the first 47 residues of both sequences. However, N-terminal analysis of IgE-binding bands derived from Tricine-SDS-PAGE gel blots indicates that both Myr p I and Myr p II undergo extensive post-translational proteolytic processing to unique peptides of 45 and 27 residues, respectively.

Identification of an IgE-binding determinant of the major allergen Myr p I from the venom of the Australian jumper ant Myrmecia pilosula.

The structure of the single allergenic determinant of the major ant venom allergen, Myr p I from the Australian jumper ant Myrmecia pilosula has been determined by inhibition studies with synthetic peptides. A 14 amino-acid C-terminal peptide sequence has been shown to constitute this determinant. Half-maximal inhibition of binding of ant venom-specific IgE antibodies to the native venom was obtained with this peptide at a concentration of 5 x 10(-8) M. This allergenic determinant was invariant for all ant venom-allergic subjects tested whose IgE antibodies recognized this allergen.

Cytotoxicity of pilosulin 1, a peptide from the venom of the jumper ant Myrmecia pilosula.

The synthetic peptide pilosulin 1, corresponding to the largest defined allergenic polypeptide found in the venom of the jumper ant Myrmecia pilosula, inhibited the incorporation of [methyl-3H]thymidine into proliferating Epstein-Barr transformed (EBV) B-cells. The LD50 was four-fold lower in concentration than melittin, a cytotoxic peptide found in honey bee venom. Loss of cell viability was assessed by flow cytometry by measuring the proportion of cells that fluoresced in the presence of the fluorescent dye 7-aminoactinomycin D. Examination of proliferating EBV B-cells indicated that the cells lost viability within a few minutes exposure to pilosulin 1. Partial peptides of pilosulin 1 were less efficient in causing loss of cell viability and the results suggest that the 22 N-terminal residues are critical to the cytotoxic activity of pilosulin 1. Normal blood white cells were also labile to pilosulin 1. T- and B-lymphocytes, monocytes and natural killer cells, however, were more labile than granulocytes. Analysis of pilosulin 1 using circular dichroism indicated that, in common with melittin and other Hymenoptera venom toxins, it had the potential to adopt an alpha-helical secondary structure.

Anaphylaxis to muscle relaxant drugs: cross-reactivity and molecular basis of binding of IgE antibodies detected by radioimmunoassay.

IgE antibodies that bind the muscle relaxant alcuronium were found in sera from six patients who experienced anaphylactic-like reactions following administration of the drug during induction of anaesthesia. Drug-specific antibodies were detected by radioimmunoassay employing a covalently coupled alcuronium-Sepharose complex and 125I-labelled anti-human IgE. Quantitative inhibition studies undertaken with the sera revealed specificity differences between IgE antibodies from different patients. One serum reacted with alcuronium but not with five other muscle relaxants, decamethonium, gallamine, pancuronium, succinylcholine and tubocurarine. IgE antibodies in the other sera cross-reacted with the muscle relaxants, other quaternary ammonium compounds and some pharmacologically unrelated drugs including promethazine, morphine, neostigmine and pentolineum. The inhibition experiments revealed that the alcuronium-IgE reaction could be prevented or diminished by structures containing a substituted ammonium ion. As these ions occur widely in man's environment in drugs, cosmetics, disinfectants, foods and industrial materials, it seems possible that sensitization of patients may occur without previous exposure to muscle relaxants.

Identification of penicillin allergenic determinants that bind IgE antibodies in the sera of subjects with penicillin allergy.

Much of the literature on penicillin hypersensitivity is devoted to the identification of penicillin antigens rather than allergens. Human IgE-binding determinants on different penicillins have rarely been closely investigated with the view of defining fine structural allergenic features and differences. We have developed radioimmunoassays employing ampicillin, amoxicillin and ticarcillin solid phases for the detection of penicillin-reactive IgE antibodies. Quantitative hapten inhibition studies employed to identify IgE-binding regions on the penicillin molecules revealed a heterogeneous group of allergenic determinants consisting exclusively, or in part, of the alpha-aminobenzyl and benzyl side chain groups and the beta-lactam and thiazolidine rings of the penicillin nucleus.

Drugs as allergens: detection and combining site specificities of IgE antibodies to sulfamethoxazole.

An immunoassay was developed for the detection of sulfamethoxazole reactive IgE antibodies in the sera of patients who experienced life threatening anaphylactic reactions following the ingestion of co-trimoxazole (trimethoprim and sulfamethoxazole). Patients who had significant levels of sulfamethoxazole reactive IgE antibodies in their sera did not have IgE antibodies that reacted with trimethoprim-Sepharose. Inhibition experiments with a number of sulfonamides to determine the fine structural specificities of the sulfamethoxazole reactive IgE antibodies in three patients revealed that sulfamethoxazole and, depending on the serum, sulfamerazine and sulfamethizole, were the most potent inhibitors of IgE binding, whereas the parent sulfonamide, sulfanilamide, was a very poor inhibitor. From a detailed examination of structure-activity relationships, we concluded that the 5-methyl-3-isoxazolyl group on the sulfamethoxazole molecule was the allergenic determinant for all three patients with the 5-methyl group being particularly important for IgE antibody recognition. The assays for the detection of IgE antibodies to sulfamethoxazole and trimethoprim should prove useful for the diagnosis of immediate hypersensitivity to co-trimoxazole and perhaps for monitoring drug therapy in AIDS patients where a high incidence of adverse reactions to co-trimoxazole has been reported.