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Allergenic cross-reactivities in flea-reactive canine serum samples.

OBJECTIVES: To determine whether flea extract could be determined (via ELISA) to share allergenic epitopes with other insects, and to determine whether sera with different reactivities to insect extracts have different cross-reactivity patterns. SAMPLE POPULATION: 69 canine serum samples that were selected from samples submitted for routing ELISA allergy testing and had previously been found to have high reactivities to flea. PROCEDURE: Each serum sample was assessed by means of a direct ELISA for IgE binding to 11 common insects. Samples that were reactive primarily to flea extract alone were designated pool 1, samples that were reactive to small numbers of insects were designated pool 2, and samples that were reactive to all or almost all insects were designated pool 3. Samples that did not have any apparent patterns of cross-reactivity were not included in the rest of the study. Inhibition ELISA techniques were used with the 3 serum pools to determine whether multiple insect extracts inhibited reactivity on flea-coated ELISA plates. Those extracts were used to coat ELISA plates, and reciprocal inhibition studies were then performed. RESULTS: Black fly, black ant, and cockroach extracts were capable of > 50% inhibition of flea solid-phase IgE binding with all 3 serum pools. In the ELISA inhibition studies, flea extract was able to inhibit IgE binding to each extract with all pools, confirming reciprocal inhibition. CONCLUSIONS: Inhibition of IgE binding to solid-phase flea antigen by black ant, black fly, and cockroach extracts suggested sharing of allergenic epitopes among these species. Reciprocal inhibition studies further confirmed these findings. These results indicated in vitro cross-reactivity between flea, black ant, black fly, and cockroach extracts. These results need to be further investigated in vivo. CLINICAL RELEVANCE: It is possible that dogs may become sensitized to fleas via exposure to other insects, and flea allergenic dogs may have signs of pruritus, in the absence of fleas, if exposed to cross-reactive insects.

Animals↗

Oxime-induced reactivation of acetylcholinesterase inhibited by organophosphinates.

The comparative potency of oximes for reactivation of inhibited eel acetylcholinesterase (AChE) in vitro is dependent on the organophosphinate inhibitor. Some of the data, dealing with a reference organophosphonate, support the conclusion of other investigators that the oxime potency order is also dependent on the inhibiting phosphonate. This work was done to identify more clearly the nature of phosphinylated AChE with regard to oxime reactivation potency and the potential of phosphinates as pretreatment drugs to protect AChE against organophosphonate poisoning. We have determined the reactivation potency of four oximes--2-PAM, HI-6, TMB-4 and toxogonin--against four phosphinates--4-nitrophenyl methyl(phenyl)phosphinate (PMP), 4-nitrophenyl chloromethyl(phenyl)phosphinate (CPMP), 4-nitrophenyl trifluoromethyl(phenyl)phosphinate and 4-nitrophenyl bis(2-thienyl)phosphinate. For comparison, the phosphonate sarin (GB, isopropyl methylphosphonofluoridate) was included. Incubation of the inhibited enzyme (I-AChE) at 25 degrees C was with 0.30 microM oxime for PMP, 3.0 microM oxime for sarin and CPMP and 100 microM oxime for the two remaining phosphinates. AChE activity was assayed spectrophotometrically for 3.0 min at 272.5 nm at 25 degrees C in 0.10 M MOPS buffer (pH 7.60) using phenyl acetate as substrate. When sarin was the inhibitor (0% spontaneous recovery after a 2-h incubation), the order of oxime reactivation was 2-PAM (46%) greater than or equal to toxogonin (33%) = TMB-4 (31%) greater than HI-6 (9%) after 2-h incubations. For PMP (12% spontaneous recovery after a 2-h incubation) the oxime order was toxogonin (67%) greater than TMB-4 (53%) greater than 2-PAM (40%) after 2-h incubations.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗

Effects of aging on sex differences in psychomotor reminiscence and tracking proficiency.

The influence of aging on sex differences in psychomotor reminiscence and practice distribution effects was investigated in young (18 to 22 year) and elderly (57 to 86 year) participants within the context of reactive inhibition theory. As predicted by the theory in conjunction with hormonal considerations, aging eliminated the sex differences typically found in young adults, reduced the absolute amount of reminiscence, and diminished the gains in task proficiency arising from practice distribution, progressively so as practice increased. Significant age effects on task proficiency were detectable even within the age span of the elderly adults.

Adolescent↗

Kinetic studies and structure-activity relationships of bispyridinium oximes as reactivators of acetylcholinesterase inhibited by organophosphorus compounds.

The kinetics of the reactivation of acetylcholinesterase inhibited by isopropyl methylphosphonofluoridate was studied. The reactivators used include nine bispyridinium monooximes and three bispyridinium dioximes. The dissociation constant (Kd) and the rate constant (k2) of dephosphorylation of the complex formed from the organophosphorus acetylcholinesterase (OP-AChE) and the oxime were measured. The reactivation parameters obtained from the in vitro kinetic studies were used to elucidate the structure-activity relationships. The hydrophobic property of a nonoxime substituent at the 3-position on the pyridinium ring can exert a positive effect on their binding affinity to OP-AChE. However, the rate constants (k2) of the nucleophilic displacement of OP-AChE by oximes depend negatively on these physical and structural factors of the oximes. The correlations of the in vivo antidotal efficacy (ED50) of these bispyridinium oximes have been analyzed with their pharmacological properties, e.g., reactivation potency, antimuscarinic activities, and antinicotinic activities. However, no satisfactory correlations were observed. It may be concluded that the detoxication mechanism of poisoning by isopropyl methylphosphonofluoridate is different from those of pinacolyl methylphosphonofluoridate and paraoxon.

Acetylcholinesterase↗

Some effects of ACTH 4-10 on performance during a serial reaction task.

Eighteen subjects worked continuously for half an hour in a self-paced reaction task. Half of the subjects were injected with ACTH 4-10 (30 mg/s.c.) and the other half with a placebo. The subjects injected with ACTH 4-10 showed a larger improvement in reaction time during the experimental session than the placebo group. However, this effect disappeared in a short retest, which was given half an hour after the experimental session. These results suggest that ACTH 4-10 has no effect on skill acquisition, but counteracts the build-up of reactive inhibition during the experimental session; that is, the peptide suppresses the decrease in motivation, which usually occurs during continuous performance tasks. This notion is confirmed by an analysis of the frequency distributions of the reaction times. Treatment effects were only found for the long reaction times. Long reaction times tend to occur with increasing frequency as a function of time-on-task and are an indication for the level of motivation of the subject. It was concluded that ACTH 4-10 counteracts the usual decay in performance as a function of time-on-task due to increasing boredom and mental fatigue.

Adrenocorticotropic Hormone↗

Effect of organophosphorus hydrolysing enzymes on obidoxime-induced reactivation of organophosphate-inhibited human acetylcholinesterase.

The reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) by oximes results inevitably in the formation of highly reactive phosphyloximes (POX), which may re-inhibit the enzyme. An impairment of net reactivation by stable POX was found with 4-pyridinium aldoximes, e.g. obidoxime, and a variety of OP compounds. In this study the effect of organophosphorus hydrolase (OPH), organophosphorus acid anhydrolase (OPAA) and diisopropylfluorophosphatase (DFPase) on obidoxime-induced reactivation of human acetylcholinesterase (AChE) inhibited by different OPs was investigated. Reactivation of paraoxon-, sarin-, soman- and VX-inhibited AChE by obidoxime was impaired by POX-induced re-inhibition whereas no deviation of pseudo first-order kinetics was observed with tabun, cyclosarin and VR. OPH prevented (paraoxon) or markedly reduced the POX-induced re-inhibition (VX, sarin, soman), whereas OPAA and DFPase were without effect. Additional experiments with sarin-inhibited AChE indicate that the POX hydrolysis by OPH was concentration-dependent. The activity of OP-inhibited AChE was not affected by OPH in the absence of obidoxime. In conclusion, OPH may be a valuable contribution to the therapeutic regimen against OP poisoning by accelerating the degradation of both the parent compound, OP, and the reaction product, POX.

Animals↗

Stability studies of bis(pyridiniumaldoxime) reactivators of organophosphate-inhibited acetylcholinesterase.

Relative stability studies of three organophosphate-inhibited acetylcholinesterase reactivators, 1-(2-hydroximinomethyl-1-pyridinium)-3-(4-carbamoyl-1-pyridinium)- 2-oxapropane dichloride (HI-6), 1,1'-methylenebis(4-hydroximinomethylpyridinium) dichloride (MMB-4), and 1,1'-trimethylenebis(4-hydroximinomethylpyridinium) dibromide (TMB-4) were carried out by semiquantitative TLC and NMR methods. TMB-4 appears to be the most, and HI-6 the least stable of the three compounds. The extent of hydrolysis of HI-6, MMB-4, and TMB-4 in 0.05 M, pH 7 phosphate buffer was approximately 50, 25, and less than 1%, respectively, after 20 d at room temperature. The hydrolysis products of HI-6 were identified by NMR and MS (electron impact) as 2-pyridinealdoxime, picolinamide, and isonicotinamide, whereas that of MMB-4 was identified as 4-pyridinealdoxime. The stability of these reactivators decreases with increasing pH. TMB-4 was stable under both neutral and basic conditions at room temperature. Deuterium exchange of the methylene protons of MMB-4 in D2O and of the protons at the 2- and 6-positions of the pyridinium ring of TMB-4 in NaOD/D2O were observed.

Acetylcholinesterase↗

Allergy to goat and sheep cheese with good tolerance to cow cheese.

BACKGROUND: We report on a patient who experienced allergic reactions after eating goat cheese and after touching goat and sheep cheese, but not after consuming cow's milk dairy products. OBJECTIVE: To assess the allergenicity and IgE-binding capacity of the caseins from the three different species. METHODS: Skin prick tests were carried out using whole milk and caseins from three different species (goat, sheep and cow), and whey fractions of cow's milk. Total serum IgE and specific IgE to cow's milk proteins were measured by CAP system and specific IgE against caseins and whole milk were determined by ELISA technique. To evaluate allergenic cross-reactivity, inhibition of the IgE ELISA activity to goat's milk and goat casein was tested for the three caseins. SDS-PAGE and immunoblotting was used to determine IgE binding bands in caseins. RESULTS: Skin tests were positive to sheep and goat's milk, sheep and goat casein, as well as to sheep and goat cheese. Total serum IgE was 66 kU/L and IgE determinations by CAP were negative. IgE ELISA against the caseins from goat and sheep was strongly positive, whereas it was negative to cow casein. ELISA inhibition assays revealed a high degree of cross-reactivity between goat casein and sheep casein. Immunoblotting showed three IgE-binding bands in goat casein at 31, 27 and 22 kDa, which may correspond to alpha-, beta- and gamma-caseins. A band at about 31 kDa was observed in sheep casein and another band at 34 kDa was recognized in cow casein. CONCLUSION: This patient developed allergy to goat and sheep cheese with good tolerance to cow's milk. We identified goat casein as the main allergen causing sensitization in this patient as demonstrated by in vivo and in vitro tests. A high degree of cross-reactivity between goat and sheep casein was observed.

Allergens↗

Rheumatoid factors from patients with rheumatoid arthritis react with beta 2-microglobulin.

IgM rheumatoid factors (RF) from 18 sera of rheumatoid arthritis (RA) patients isolated from monomeric IgG affinity columns showed strongly positive ELISA reactions with human beta 2-microglobulin (beta 2m), as well as with recombinant beta 2m. When the same RA sera were adsorbed to beta 2m-Sepharose affinity columns, eluted material showed predominant IgM anti-Fc of IgG and anti-beta 2m reactivity. Inhibition reactions with "RF" obtained from IgG affinity columns showed slightly higher reactivity of RF for Fc over beta 2m; however, when RF from the same RA serum had been adsorbed to and eluted from beta 2m affinity columns, beta 2m showed greater inhibition than Fc for RF reacting with either beta 2m or Fc on ELISA plates. Thus two overlapping populations of RF were identified in RA sera showing reactivity with both beta 2m and Fc of IgG. When RF were isolated from IgG columns, affinity was slightly higher for Fc than beta 2m. Conversely, RF eluted from beta 2m Sepharose reacted slightly more with beta 2 m than Fc. Trypsin digests of a polyclonal RA IgM RF showed no beta 2m reactivity in Fc mu 5 fragments. Fab mu RF retained slight anti-Fc IgG but no residual anti-beta 2m activity. Monoclonal human IgM, IgG, or IgA RF either from mixed cryoglobulins or EBV-stimulated RA lymphoid cell lines showed negative or occasional weakly positive anti-beta 2m activity. Overlapping 7-mer peptide ELISA analysis of the entire 99-amino acid sequence of beta 2m showed a major RF-reactive linear hydrophilic sequence at positions 56-60 which included a 3-amino acid exact homology to positions 401, 403, and 404 of the C gamma 3 domain. A peptide encompassing this sequence produced 90% inhibition of RF binding to whole beta 2m. Substitution of neutral glycines for each amino acid throughout the reactive epitope at positions 56-66 indicated that lysine at position 58 aspartic acid at 59, and tryptophane at 60 represented major portions of the RF-reactive epitope. These findings indicate that human RF derived from patients with RA react with other epitopes besides those present on IgG Fc, including epitopes on human beta 2m. For many years serum RF3 found in patients with RA have been regarded as premier examples of autoantibodies to autologous IgG.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

Isolation of homologous restriction factor from human urine. Immunochemical properties and biologic activity.

A soluble form of homologous restriction factor (HRF-U) was isolated from normal human urine. With respect to m.w. (65,000) and immunoblotting characteristics, it resembled membrane HRF (HRF-M) that had been isolated from human E membranes. The protein exhibited limited cross-reactivity with the channel-forming proteins of C and cytotoxic lymphocytes. It inhibited reactive lysis of E by human C5b-9. Inhibition occurred at the attachment stage of C5b-7 to target cells, rather than at the C8 or C9 stage of membrane attack complex assembly which is inhibited by HRF-M. In this respect, HRF-U acts analogously to S protein of serum, but no immunochemical relationship between these two proteins was detected. HRF-U might be derived from the soluble HRF detected in cytoplasmic granules of killer lymphocytes.

Animals↗

In vitro reactivation of acetylcholinesterase inhibited by cyclosarin using bisquaternary pyridinium aldoximes K005, K033, K027 AND K048.

We have tested four new bisquaternary pyridinium acetylcholinesterase (AChE; EC 3.1.1.7) reactivators - K005 (1,3-bis(2-hydroxyiminomethylpyridinium) propane dibromide), K033 (1,4-bis(2-hydroxyiminomethylpyridinium) butane dibromide), K027 (1 -(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide) and K048 (1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide) as the potential reactivators of AChE inhibited by cyclosarin. Their reactivation potencies were studied using standard in vitro reactivation test. Rat brain homogenate was used as the source of the enzyme. Oxime K033 seems to be the most potent reactivator of cyclosarin-inhibited AChE. Its reactivation potency is significantly higher than the efficacy of all other tested AChE reactivators.

Acetylcholinesterase↗

Effects of C-reactive protein on platelet function. II. Inhibition by CRP of platelet reactivities stimulated by poly-L-lysine, ADP, epinephrine, and collagen.

It was recently demonstrated that C-reactive protein (CRP)4 inhibits the response of human platelets to heataggregated human gamma-globulin and thrombin and that this inhibition is characterized by a dose-dependent reduction in aggregation, activation of platelet factor 3 (PF3), and release of beta-glucuronidase. In the present experiments, CRP was found also to inhibit the ability of washed human platelets to aggregate in response to poly-L-lysine (PLL); in these experiments, the magnitude of the inhibitory effect was dependent upon the m.w. of PLL used as the stimulating agent, and was more effective with low (15,000 daltons) than with high (400,000 daltons) m.w. polymers. CRP similarly inhibited ADP- and epinephrine-stimulated platelet aggregation in platelet-rich plasma (PRP), and this was characterized by relatively minimal suppression of the primary wave of aggregation. CRP also inhibited the platelet aggregation induced by collagen in PRP, although it had no effect upon the adherence of platelets to collagen. Finally, CRP inhibited the activation of PF3 and the release of serotonin during stimulation of platelets with ADP, and this inhibition was temporally related to the onset of the secondary wave of aggregation. These experiments extend the platelet reactivities inhibited by CRP, show that CRP expresses its inhibitory capacity in platelet-rich plasma as well as upon isolated platelets, raise the possibility that CRP exercises its effects by inhibiting or interfering with the release and/or utilization of endogenous platelet ADP, and support the concept that CRP plays an important role in the control of platelet responsiveness to a variety of stimuli during acute inflammatory reactions.

Adenosine Triphosphate↗

TRANSPLANTATION TOLERANCE INDUCED IN ADULT MICE BY PROTEIN OVERLOADING OF DONORS.

Treatment of donor animals with unrelated antigens can regularly inhibit reactivity of spleen cells against the host, with subsequent induction of specific immune tolerance in the recipient animals if the barrier to histocompatibility is weak. Mice made tolerant in this way accept skin grafts from donor strain animals. In the case of strong differences (in H(2) locus) or heterologous (rat-mouse) combinations, the inhibition of the reactivity of the transferred lymphoïd cells against the host is partial, and the skin-graft survival time is significantly prolonged.

Animals↗

Interaction of lidocaine with reactive oxygen and nitrogen species.

BACKGROUND AND OBJECTIVE: To investigate the ability of lidocaine to inhibit reactive oxygen and/or nitrogen species generation by either human leukocytes or cell-free systems via luminol- and lucigenin-enhanced chemiluminescence. METHODS: Venous blood was obtained from healthy volunteers and leukocytes were isolated, from which chemiluminescence was generated. Also, chemiluminescence, induced by H(2)O(2), HOCl, peroxynitrite or ferrous iron, was generated in cell-free systems. RESULTS: Lidocaine produced a concentration-dependent inhibition in chemiluminescence generated by leukocytes (92 +/- 1%, 1 mM). In cell-free experiments, lidocaine (1 mM) markedly inhibited chemiluminescence of xanthine-xanthine oxidase (24 +/- 3%), while it slightly suppressed hypochlorous acid-induced chemiluminescence (9 +/- 2%). Peroxynitrite-induced luminol- and lucigenin-enhanced chemiluminescence were also inhibited by lidocaine at 1 mM (19 +/- 3% and 48 +/- 8%, respectively). Lidocaine did not affect chemiluminescence generated by FeSO(4). However, lidocaine produced a biphasic effect on H(2)O(2)-induced chemiluminescence (37 +/- 5% inhibition at 0.01 mM and 61 +/- 17% activation at 1 mM). CONCLUSIONS: Lidocaine can elicit direct scavenging activity at high concentrations that might be important at or near the site of injection in local anaesthetic use.

Acridines↗

Allergenic cross-reactivity between Callistemon citrinis and Melaleuca quinquenervia pollens.

Aqueous extracts of both Callistemon citrinis (bottlebrush) and Melaleuca quinquenervia (melaleuca) were analyzed for allergenic cross-reactivity. Inhibition analysis using the radioallergosorbent test (RAST) was performed on the ammonium bicarbonate extracts of bottlebrush (NH4B) and melaleuca (NH4M) pollens. RAST inhibition analysis demonstrated that the extracts contained allergenically cross-reactive components. Sephadex G-100 column chromatography of NH4B and NH4M extracts resulted in at least 4 distinct peaks for each extract analyzed. These fractions were designated NH4B1-NH4B4 and NH4M1-NH4M4. A modified dot-blot assay for detection of allergenic components was utilized to show that the first elution peaks of bottlebrush and melaleuca, NH4B1 and NH4M1, respectively, contained allergenic components. These allergenic components, NH4B1 and NH4M1, had estimated molecular weights of 50,000 and 35,000 daltons, respectively.

Animals↗

Inactivation-reactivation of aconitase in Escherichia coli. A sensitive measure of superoxide radical.

The rapid inactivation of aconitase by O2-, previously seen to occur in vitro, was explored in vivo. A fraction of the aconitase in growing, aerobic, Escherichia coli is inactive at any instant but can be activated by imposition of anaerobic conditions. This reactivation occurred in the absence of protein synthesis and was inhibited by the ferrous chelator alpha,alpha'-dipyridyl. This fraction of inactive, but activatable, aconitase was increased by augmenting O2- production with paraquat, decreased by elevation of superoxide dismutase, and increased by inhibiting reactivation with alpha,alpha'-dipyridyl. The balance between inactive and active aconitase thus represented a pseudoequilibrium between inactivation by O2- and reactivation by restoration of Fe(II), and it provided, for the first time, a measure of the steady-state concentration of O2- within E. coli. On this basis, [O2-] was estimated to be approximately 20-40 pM in aerobic log phase E. coli containing wild type levels of superoxide dismutase and approximately 300 pM in a mutant strain lacking superoxide dismutase.

2,2'-Dipyridyl↗

Effective bisquaternary reactivators of tabun-inhibited AChE.

Two cholinesterase reactivators (K074 and K075) were synthesized and their reactivation efficacy against tabun-inhibited acetylcholinesterase of the rat brain was tested in vitro. Comparing this efficacy showed that commonly used oximes (pralidoxim, obidoxime and HI-6) were practically without reactivation potency. On the other hand, oximes K074, K075 and trimedoxime were satisfactorily effective. Moreover, K-oximes reactivated tabun-inhibited AChE at lower concentration (10(-4) and 10(-3) m) in comparison with trimedoxime (10(-3) and 10(-2) m). Thus, K-oximes can be considered as the most effective reactivators of tabun-inhibited AChE at present.

Acetylcholinesterase↗