Drug-protein conjugates--XII. A study of the disposition, irreversible binding and immunogenicity of penicillin in the rat

The disposition, irreversible binding and immunogenicity of benzylpenicillin (BP) were studied in male Wistar rats. [3H]BP, administered i.v. to anaesthetized rats at two doses (27 mumol/kg, 2.7 mmol/kg), showed dose-dependent kinetics: plasma and tissue concentrations of total BP were disproportionately increased at the higher dose. BP was rapidly cleared from the plasma at both doses (less than 0.05% of administered dose/ml plasma after 3 hr). In spite of the disproportionately elevated levels of total BP after the higher dose, covalent binding to plasma proteins was quantitatively similar as a percentage of the dose at both doses. Three hours after i.v. injection of 27 mumol/kg and 2.7 mmol/kg of the drug, 5.6% +/- 1.7% and 3.3% +/- 1.1% respectively of circulating BP was covalently bound, representing less than 0.004% of the administered dose bound per ml of plasma in each case. Covalent binding of BP to rat plasma proteins in vitro was of a similar magnitude to that observed in vivo: 1.6% +/- 0.4% of BP was bound to 25% rat plasma after 3 hr incubation at 37 degrees. In a separate series of experiments the immunogenicity of BP was studied by chronic administration of the drug to rats. Following daily i.v. or i.m. administration of BP (27 mumol/kg, 270 mumol/kg, 2.7 mmol/kg) for 4 consecutive days at 4-week intervals (three series of injections) neither IgG nor IgM anti-benzylpenicilloyl (BPO) antibodies were detected by enzyme-linked immunosorbent assay (ELISA). Intravenous administration of the high dose of BP was discontinued after the first series of injections due to local necrosis. In contrast to free BP, BPO-keyhole limpet haemocyanin (BPO-KLH, 42 nmol BP bound/mg KLH) administered by single i.v. injection at 4-week intervals at two doses (20 and 200 micrograms conjugate/kg, corresponding to 0.84 and 8.4 nmol BPO/kg) readily induced IgG and IgM anti-BPO antibody responses (median IgG titres were 872 and 5470 one week after the third injection of the low and high dose of conjugate respectively; corresponding IgM titres were 4513 and 22,866). The specificity of the IgG and IgM antibodies for the BPO determinant was confirmed by ELISA inhibition with BPO-aminocaproate. These experiments show that BP binds irreversibly, but to a limited extent, to plasma proteins in vivo, and that such a degree of conjugation appears to be insufficient to elicit a detectable anti-BPO antibody response.     

青霉素过敏病人血清特异性IgE和IgG抗体

采用放射过敏原吸附试验(RAST)和酶联免疫吸附试验(ELISA)测定52例青霉素过敏病人血清特异性IgE、IgG抗体，进一步探讨青霉素过敏反应机制。结果 52例过敏病人特异性IgE、IgG抗体的阳性率分别为50％和44．2％，若RAST与ELISA联合检测，IgE和IgG抗体总阳性率增至63.5％。荨麻疹组BPO—IgG水平高于过敏性休克组(P＜0.01)，过敏性休克病人BPA—IgG水平明显高于BPO—IgG(P＜0．01)，荨麻疹组内BPO—IgE水平与BPA—IgE无显著差异(P＞0．05)。但均比过敏性休克组高。研究结果提示，荨麻疹与BPO—IgE和BPA—IgE关系密切，过敏性休克与BPO—IgE和BPA—IgG关系密切；同时检测IgE和IgG抗体，可提高诊断阳性率。     

Fresh vs aged benzylpenicilin on non-IgE responses in mice

目的：研究新鲜青霉素和陈旧青霉素是否能诱导不同的非ＩｇＥ抗体应答．方法：酶联免疫吸附试验测定抗体应答，半抗原抑制试验检测被抗体识别的抗原分子和抗原性交叉反应．结果：新鲜青霉素免疫后诱导的特异性非ＩｇＥ抗体类型主要是ＩｇＭ，其次是ＩｇＧ和ＩｇＡ．部分抗体识别青霉素分子本身，而大部分抗体结合降解产物或转化产物．青霉素和氨苄西林间交叉反应性抗体主要是ＩｇＧ，青霉素和哌拉西林间交叉反应性抗体则主要是ＩｇＭ．结论：新鲜制备的青霉素与陈旧青霉素能诱导不同的非ＩｇＥ抗体应答．     

Drug-protein conjugates--XVII. The effect of storage on the antigenicity and immunogenicity of benzylpenicillin in the rat

The disposition and immunogenicity of freshly prepared and stored solutions of benzylpenicillin (BP) and benzylpenicillenic acid (BPE), a degradation product of BP, were studied. No IgG anti-benzylpenicilloyl (BPO) antibodies were detected by enzyme-linked immunosorbent assay (ELISA) following daily i.p. or i.m. administration to male Wistar rats of BP (2.7 mmol/kg) freshly dissolved in 0.5% glucose, for 4 consecutive days at 4-week intervals. In contrast, IgG anti-BPO antibodies were detected following both chronic i.p. and i.m. administration of BP (2.7 mmol/kg) stored for 24 hr at room temperature in 0.5% glucose. An IgG anti-BPO response was obtained only after the high dose, following daily i.m. administration of BPE (27 mumol/kg, 2.7 mumol/kg, 0.24 mumol/kg). The specificity of the IgG antibody for the BPO-determinant was confirmed by ELISA inhibition with BPO-amino-caproate. Circulating BPO plasma-protein antigens were detected by a modified ELISA following i.p. and i.m. administration of both stored and fresh BP. Significantly lower BPO-antigen levels were detected in serum following BPE administration. Irreversible binding of BP to 75% rat plasma proteins was of the same magnitude when freshly dissolved in phosphate buffer or in 0.5% glucose (2.63 +/- 0.32% and 2.55 +/- 0.25% bound, respectively after 3 hr incubation at 37 degrees). Irreversible binding was significantly greater (P less than 0.05) when the BP was stored prior to incubation with the protein (3.81 +/- 0.27%). The major degradation product of stored BP was benzylpenicilloic acid; a small amount of BPE (0.2% of incubated BP) was detected in stored but not fresh BP. Thus, the increased immunogenicity of BP stored for 24 hr at room temperature may be due to the formation of reactive degradation products such as BPE in vitro, which can then form immunogenic drug-protein conjugates in vivo. These experiments also show that although BP and BPE form drug-protein conjugates in vivo, circulating levels of antigen do not relate to the immunogenicity of either of the compounds.     

The presence of both antivirus and antiself antibodies in sera from patients with adenovirus and influenza B

Using the ELISA method we examined serum samples from 62 male patients aged 19-23 infected with adenovirus (serotype 7), 22 children aged 7-14 infected with influenza B (B/Norway 1/84) and 113 normal subjects aged 5-30. The infections were diagnosed serologically by complement fixation, by inhibition of hemagglutination, by ELISA and by viral culture. Moreover using enzyme-linked short-time culture assay, the production of specific antivirus antibodies and autoantibodies in vitro by spleen cells (1 x 10(6) cells/well) from normal mice and from mice immunized with adenovirus and influenza B was studied. At the same time their sera antibody titers were determined. All the serum samples were tested against the following antigens: adenovirus, influenza B, ds-DNA, actin, myosin, myoglobin, thyroglobulin, H. transferrin, H. interferon a and BSA. FV. For the further characterization of positive sera, an evaluation of specificity by competitive ELISA-test and by preparations of F(ab')2 fragments from patients' sera was also carried out. It was found that the percentage of positivity for the specific virus and other antigens was higher in the patients' samples than in the samples from the normal subjects. The specific antivirus antibody was of IgG class and their titers ranged from 1/4, 800 up to 1/19,200. Autoantibodies belonged to IgM, IgA, IgG classes and their titers ranged from 1/400 to 1/1,600. In comparison, titers of normal subjects' sera ranged from 1/150 to 1/600 and 1/150 to 1/300, respectively and both were IgG classes. Both specific virus antibodies and autoantibodies appeared at the same time. The competitive ELISA-test showed a marked inhibition (95-98%) of antivirus antibodies with the specific antigen, whereas autoantibodies were less inhibited (40-50%) by homologous antigens. The antigen-antibody reaction occurred at the Fab portion of the immunoglobulin molecule, since these fragments inhibited antibody reactivity. The same results were observed with spleen cells from immunized mice and the above-mentioned antigens when cultured in vitro.     

Cross-reactivity of anti-phosphorylcholine antibodies to neuromuscular blockers in a murine model of immunization

Hypersensitivity reactions to curare-like neuromuscular blocking agents (NMBA) used in anesthesiology are more frequent in females and often occur at the first exposure to these drugs. To evaluate the antibody response to a hapten sharing the same allergenic epitope with NMBA in a murine model of immunization with Nitrophenylphosphorylcholine (NPPC) coupled with Keyhole Limpet Hemocyanin (KLH). BALB/c mice from both sexes were intraperitoneally immunized with NPPC-KLH with alum and boosted twice, on 7 and 14 days. The antibodies were tested for specificity to PC and for cross-reactivity to the haptens, NPPC and PC, as well as to the NMBAs. Mice immunized with NPPC-KLH produced anti-PC antibodies mainly of IgM, IgG1 and IgG3 isotypes, at similar levels in both sexes. Different affinities for the haptens were detectable between isotypes, with anti-PC IgG3 antibody reactivity mostly related to the choline portion of the NPPC hapten. When comparing among sexes, females developed greater IgG2 affinity to the hapten than males. Cross-reactivity to NMBAs was predominant among the anti-PC IgG3 antibodies, mainly in females achieving 75% of inhibition with 16 mM of suxamethonium, while other isotypes achieved up to 30% and was absent for IgE. The investigation of antibody isotypes regarding sensitization to choline-derived structures could contribute to understanding the differential ability of females to produce antibodies that are cross-reactive with NMBAs.     

Adjuvant and immuno-suppressive effect of six monophthalates in a subcutaneous injection model with BALB/c mice.

The prevalence of allergic airway diseases is rapidly increasing in Western Europe and North America. This increase in disease prevalence may be associated with environmental pollutants. The present study investigated the adjuvant and immuno-suppressive effect of a series of monophthalates which are considered to be important metabolites of commonly used phthalate plasticizers. The effects were studied in a screening model. Ovalbumin (OA), used as the model antigen, was injected subcutaneously in the neck region of BALB/cJ mice with or without one of the test substances, mono-n-butyl phthalate (MnBP), monobenzyl phthalate (MBnP), mono-n-octyl phthalate (MnOP), mono-2-ethylhexyl phthalate (MEHP), mono-iso-nonyl phthalate (MiNP) or mono-iso-decyl phthalate (MiDP). The levels of OA-specific IgE, IgG1 and IgG2a in sera were measured by ELISA. Immuno-suppressive effect, defined as a statistically significant reduction in IgE or IgG1 antibody production, was observed with MEHP (1000 microg/ml, IgE and IgG1), MnOP (1000 microg/ml, IgE and IgG1), MiNP (1000 microg/ml, IgE and 10 microg/ml, IgG1) and MiDP (100 microg/ml, IgE and IgG1). Adjuvant effect, defined as a statistically significant increase in IgE or IgG1 antibody level, occurred with MEHP (10 microg/ml, IgE), MnOP (100 microg/ml, and 10 microg/ml, IgG1) and MiNP (100 microg/ml, IgE). No statistically significant immune modulating effect was seen with MBnP and MnBP.     

Albumin binding in uraemia: quantitative assessment of inhibition by endogenous ligands and carbamylation of albumin

Summary The binding capacity of human serum albumin (HSA) for small acidic molecules is known to be reduced in chronic renal failure (CRF). The contribution of competitive inhibition by accumulated endogenous ligands and of structural changes in HSA has now been evaluated. In a fluorimetric in vitro assay using HSA and two dansylated amino acids the inhibitory properties of various endogenous ligands were determined in concentration-effect studies. The effect of carbamylation of HSA on binding was also examined. The mode of inhibition, including binding parameters n and K_a, was determined. Finally, HSA binding in sera from controls and dialysis patients was compared in a modified assay.Thirty three substances were tested and were placed in 3 groups: strong inhibitors (IC₅₀ < 3*10^–5 mol · 1^–1, e. g. indolyl acids, furanoic acids), medium inhibitors (IC₅₀ > 3*10^–5, eg. vanillic acid), and no inhibition (e.g. urea, creatinine, guanidino compounds). Complete ( > 80 %) carbamylation of HSA reduced binding by 67 in a non-competitive mode. There was a significant reduction in the binding capacity of HSA from the dialysis patients ( 24 %), irrespective of medication.It is concluded that the uraemic binding defect of HSA is caused by competitive inhibition by the many physiological ligands accumulated in CRF and structural modifications of HSA. The assay presented proved useful for the rapid analysis of possible HSA binding inhibitors and for testing large groups of patients, e. g. comparison of dialysis treatments, and pharmacological binding studies.     

Enzyme-linked immunosorbent assay, immunofluorescent assay, and recombinant immunoblotting in the serodiagnosis of early Lyme borreliosis

Serum samples from Bulgarian patients with physician-diagnosed erythema migrans (EM) (n=105) were examined using Borrelia burgdorferi ELISA (Boehring, Germany) after previous absorption with Treponema phagedenis. For IgM antibody detection sera were additionally pretreated with anti-IgG serum (RF absorbent). Serum samples of 93% of persons from healthy control group were IgM negative and all were IgG negative. Out of 105 patients with EM, 49% were IgM positive and 14 % were borderline. IgG ELISA showed positive results for 17% and borderline for 6% of the patients. Positive and borderline serum samples were examined further by immunofluorescent assay (IFA) and immunoblot test with recombinant B. burgdorferi proteins from strain PKo (B. afzelii) - p100, flagellin, OspA and OspC, and internal flagellin fragments from strains PKo and PBi (B. garinii) [B.Wilske, V.Fingerle, P. Herzer et al. 1993. Med. Microbiol. Immunol. 182:255]. IFA detected IgM antibodies against B. burgdorferi in 47 % of the positive and in none of the borderline by IgM ELISA serum samples as well as IgG antibodies in 83% of the positive and in 50% of the borderline by IgG ELISA samples. Presence of specific antibodies was confirmed by immunoblot in 71 % of the IgM ELISA postive and in 67 % of the IgG ELISA positive sera. In addition, anti-B. burgdorferi antibodies were detected in 60 % of the borderline by IgM ELISA serum samples. IgM serum reactivity was directed mainly against OspC antigen and flagellin and IgG antibodies were directed mainly against flagellin and p100. These findings clearly showed advantages of the ELISA test based on previous pretreatment of sera and capable to detect specific antibodies in more than half of patients with early Lyme borreliosis despite the well-known delayed immune response. IFA was less sensitive than ELISA in detection of anti-B. burgdorferi antibodies. An additional examination of ELISA borderline sera by immunoblot revealed more positive results. Serum reactivity to a single OspC antigen seems to be a sufficient criterion for positive IgM immunoblot.     

Drug-protein conjugates--XVIII. Detection of antibodies towards the antimalarial amodiaquine and its quinone imine metabolite in man and the rat

A specific enzyme-linked immunosorbent assay (ELISA) was developed for the detection and characterisation of antibodies directed against amodiaquine (AQ), an anti-malarial drug associated with agranulocytosis and liver damage in man. The assay incorporated an antigen which was produced by the reaction of amodiaquine quinone imine (AQQI), a protein reactive product produced from AQ by silver oxide oxidation, and metallothionein. The protein-conjugate (AQ-MT) had a ratio of AQ to protein of 5.2:1. Specific anti-drug antibody was defined as the differential binding to AQ-MT and unconjugated MT which was inhibitable by AQ-mercapturate (5 microM). Following administration of AQ (0.27 mmol/kg; for 4 days) to male Wistar rats there was a significant increase in the IgG anti-AQ activity on day 18 (P less than 0.05, 0.596 +/- 0.410, N = 7) compared to pre-injection levels (0.111 +/- 0.074, N = 7). This activity was shown to be specific for the AQ determinant by hapten inhibition with AQ (IC50 250 nM) and AQ-mercapturate (IC50 310 nM). Following administration of AQQI (27 mumol/kg; i.m.; 4 days) there was a significant increase in IgG anti-AQ antibody activities on day 18 (0.584 +/- 0.161, N = 7) compared to pre-injection levels (0.078 +/- 0.048, N = 7). This activity was inhibited by AQ (IC50 150 nM) and AQ-mercapturate (IC50 180 nM). In addition IgG anti-AQ antibodies were detected in four patients who exhibited agranulocytosis and one patient who exhibited hepatitis (range 0.017-0.842) whilst receiving AQ at a dose of 400 mg weekly for several weeks, but not in individuals who had not received the drug (-0.014 +/- 0.022, N = 7). There was no increase in IgG anti-AQ antibody activities in patients who had not exhibited an adverse reaction whilst receiving the drug for the treatment of malaria (-0.059 +/- 0.074 on day 0 and -0.053 +/- 0.068 on day 7, N = 13). Thus, we have shown that AQ is immunogenic in the rat and that the formation of a chemically reactive metabolite (AQQI) is involved in the generation of the antibody response. Furthermore, drug-specific antibodies were detected in sera from five patients with severe adverse reactions to the drug.     

A spectrum of antibody (IgG. IgG1, IgM) response in mice infected with trichinella spiralis treated with L-mimosine

The purpose of this study was to demonstrate the anti-inflammatory effects of L-mimosine on chronic inflammation, by investigating its effect on the immunological response of BALB/c mice infected with the nematode parasite Trichinella spiralis. Specific anti-parasite immunoglobulins (IgG, IgG1 and IgM) were detected by the ELISA method in the serum of both the treated and the untreated animals at different periods of time for 60 days post infection. Two groups consisting of 18 mice each were used. The mice were 6 weeks of age. Both groups were infected with 220 larvae (L1-T. spiralis) per os: one group was administered an intraperitoneal injection of L-mimosine (200 &mgr;g/100 ml/dose) for 27 days (the first injection started 7 days before infection) and the second group was administered an intraperitoneal injection of saline solution (100 &mgr;l/dose). Parasite specific IgG, IgG1 and IgM levels were determined in the sera of infected, untreated mice. The levels of IgG and IgG1 were increased following infection and remained elevated throughout the experimental period, while IgM was significantly decreased on the 50th day post-infection. These levels were found to be lower in the L-mimosine treated infected mice, compared to the untreated mice. The inhibition started from day 10 and continued until day 60. In healthy animals, the production of immunoglobulins was not measurable. Non-infected animals treated with L-mimosine also showed no detectable anti-parasite specific immunoglobulins.     

Improved ELISA method for screening human antigen-specific IgE and its application for monitoring specific IgE for novel proteins in genetically modified foods

For monitoring the occurrence of IgE antibody specific for novel proteins in genetically modified (GM) foods, ELISA is the most convenient method. The levels of IgE specific for recombinant proteins, phosphinothricin-N-acetyltransferase (PAT), CP4-EPSPS, and Cry9C were determined by ELISA using the sera from patients allergic to known allergens. Ovalbumin (OVA) and OVA-positive patient sera were used as positive control. In the ELISA, 20-fold-diluted sera tested were mostly negative for the specific IgE. However, the PAT-specific, but not CP4-EPSPS- or Cry9C-specific IgE in some patients was apparently higher than that of the healthy volunteers. To clarify the binding specificity of the antibody, we pre-incubated the sera with soluble PAT, but the inhibition was marginal, suggesting that the binding was non-specific. Therefore, we used 1M NaCl as a washing buffer to remove IgE non-specifically bound to the coated PAT. This washing step efficiently decreased non-specific binding. In contrast, OVA-specific IgE binding to OVA-coated plate was not affected by the washing. Finally, in this pilot study significant levels of IgE antibodies specific for the three proteins were not detected in the sera of Japanese food-allergy patients.     

新鲜与陈旧 霉素诱导小鼠体内非1gE抗体应答的对比

AIM: To study whether or not the freshly prepared benzylpenicillin could induce different non-IgE antibody response from aged benzylpenicillin. METHODS: Antibody response was determined by enzyme-linked immunosorbent assay (ELISA). Antigen molecules recognized by antibodies and antigenic cross reactions were tested by hapten inhibition assay. RESULTS: Isotypes of specific non-IgE antibodies induced by freshly prepared benzylpenicillin were mainly IgM, and then IgG and IgA. Some parts of specific antibodies recognized benzylpenicillin molecule and major parts combined with degraded or transforming products. Isotypes of antibodies responsible for cross reaction were mainly IgG between benzylpenicillin and ampicillin and IgM between benzylpenicillin and piperacillin. CONCLUSION: Freshly prepared and aged benzylpenicillin induced different non-IgE antibody response.     

Polyclonal IgM and IgA block in vitro complement deposition mediated by anti-ganglioside antibodies in autoimmune neuropathies

Intravenous immunoglobulin (IVIG), consisting of IgG, is the first-line treatment for Guillain–Barré syndrome and multifocal motor neuropathy. IgG, but neither IgM nor IgA, has been demonstrated in vitro to inhibit complement deposition mediated by anti-ganglioside autoantibodies in sera from patients with both conditions. The objective of this study is to investigate the in vitro effectiveness of IgM and IgA in inhibiting complement deposition to ganglioside/anti-ganglioside antibody complexes. Serum samples were obtained from patients with multifocal motor neuropathy associated with anti-GM1 IgM antibodies, Guillain–Barré syndrome associated with anti-GM1 IgG antibodies and Miller Fisher syndrome associated with anti-GQ1b IgG antibodies. Inhibition of complement deposition using different immunoglobulin preparations was measured by enzyme-linked immunosorbent assay. IgM/A-enriched IVIG and immunoglobulin isotypes (polyclonal IgM and IgA) showed higher potential in inhibiting complement deposition than standard IVIG. Although the safety concerns about the use of IgM and IgA for an immunotherapy still remain, IgM and IgA may serve as an alternative immunotherapy in those anti-ganglioside antibody-mediated neuropathies.     

Diisocyanate antigens that detect specific antibodies in exposed workers and guinea pigs

Evaluation of the immunologic contribution to the pathogenesis of isocyanate lung disease necessitates preparation of isocyanate-protein conjugates to detect anti-isocyanate antibodies. The preparation and characterization of the conjugates were described in the preceding paper in this issue. Sera were obtained from two guinea pigs immunized with 4,4'-diisocyanatodiphenylmethane (MDI) and from three workers with occupational exposure to MDI. By use of Western blot analysis, guinea pig IgG antibodies were best detected by the monomeric component of MDI-guinea pig serum albumin. ELISA additionally indicated that conjugates which contained a high density of hapten detected greater amounts of antibody than did conjugates containing low amounts of hapten. The same procedures were then used to assess the amount of MDI-specific IgG and IgE antibody in sera from symptomatic workers. Effective MDI-HSA antigens were those that were monomeric and had high haptenic content. Highly substituted conjugates of monoisocyanates (phenyl isocyanate and p-tolyl isocyanate) with serum albumins were also effective in detecting antibodies to MDI. These results indicate the importance of the composition of isocyanate conjugate antigens in detecting antibodies to diisocyanates and suggest that standards be developed for preparation and characterization of these diagnostic serologic reagents.     

Characteristics of antibody responses induced in mice by protein allergens

Whereas many foreign proteins are immunogenic, only a proportion is also allergenic, having the capacity to induce the quality of immune response necessary to support the production of IgE antibody. We have demonstrated previously that intraperitoneal administration to mice of proteins such as ovalbumin (OVA) or the industrial enzyme A. oryzae lipase, which possess significant allergenic potential, stimulates the production of both IgG and IgE antibody. Identical exposure to bovine serum albumin (BSA), a protein with limited potential to cause immediate respiratory or gastrointestinal hypersensitivity reactions, induced IgG responses only. In the current investigations, the quality of immune responses induced following exposure to these proteins via mucosal tissue (intranasal) has been compared with those provoked following administration via a non-mucosal (intraperitoneal) route of exposure. Intranasal or intraperitoneal administration of BSA, OVA or A. oryzae lipase elicited in each case vigorous IgG and IgG1 antibody responses. For all three proteins, at every concentration tested, and via both routes of exposure, IgG1 antibody titres paralleled closely IgG titres. However, the three materials displayed a differential potential to provoke IgE responses and this correlated with their known allergenic potential in humans. Thus, OVA and A. oryzae lipase stimulated strong IgE antibody responses, whereas BSA provoked low titre IgE only at the highest concentration tested (5% administered intraperitoneally). The quality of induced responses was not affected by the route of exposure. It would appear, therefore, that the stimulation of IgG and IgG1 antibody responses is a reflection of protein immunogenicity whereas protein allergenicity is associated with the induction of strong IgE responses.     

Interaction of various Piper methysticum cultivars with CNS receptors in vitro.

Methanolic leaf and root extracts of the Hawaiian kava (Piper methysticum Forst.) cultivars, Mahakea, Nene, Purple Moi and PNG, were tested on binding affinities to CNS receptors including GABAA (GABA and benzodiazepine binding site), dopamine D2, opioid (mu and delta), serotonin (5-HT6 and 5-HT7) and histamine (H1 and H2). HPLC analysis was carried out in order to determine the amount of the main kavalactones kavain, 7,8-dihydrokavain, methysticin, 7,8-dihydromethysticin, yangonin and 5,6-demethoxyyangonin. The most potent binding inhibition was observed for leaf extracts to GABAA receptors (GABA binding site) with IC50 values of approximately 3 micrograms/ml, whereas root extracts were less active with IC50 values ranging from 5 micrograms/ml (Nene) to 87 micrograms/ml (Mahakea). Since the leaf extracts generally contained lower amounts of the kavalactones than the root extracts, there might exist additional substances responsible for these activities. Leaf extracts also inhibited binding to dopamine D2, opioid (mu and delta) and histamine (H1 and H2) receptors more potently than the corresponding root extracts with IC50 values ranging from 1 to 100 micrograms/ml vs. > or = 100 micrograms/l, respectively. Significant differences in the potential of binding inhibition were also observed between cultivars. Binding to serotonin (5-HT6 and 5-HT7) and benzodiazepine receptors was only weakly inhibited by both root and leaf extracts of all four cultivars. In conclusion, our investigation indicates that the GABAA, dopamine D2, opioid (mu and delta) and histamine (H1 and H2) receptors might be involved in the pharmacological action of kava extracts. Since the cultivars contained similar amounts of kavalactones, while their pharmacological activities differed markedly, other constituents may play a role in the observed activities. Additionally, leaves generally exhibited more potent binding inhibition than roots, therefore leaf of P. methysticum might be an interesting subject for further pharmacological studies.     

The effect of disodium 4-chloro-2-iminodibenzoate (CCA) on IgE levels and anaphylactic shock

The effect of disodium 4-chloro-2,2-iminodibenzoate (CCA) on IgE antibody response was examined in C3H/A and (BALB/c x C57BL/6J) F1 hybrid mice immunized with low doses of ovalbumin (OA) adsorbed on aluminium hydroxide gel. CCA administered orally at the doses of 5 and 50 mg/kg/day reduced IgE antibody production in these mice as determined by PCA test. High doses of CCA (100 mg/kg/day) given from day 7 before immunization of C57BL mice and during 1 week after immunization of mice with OA and Bordetella Pertussis Vaccine reduced the mortality of these mice subjected to anaphylactic shock on day 7 of immunization. CCA treatment was ineffective in anaphylactic shock of C57BL mice immunized with very high dose of OA, known to elicit little or no IgE antibody production but high IgG antibody response. The treatment of OA-immunized Guinea pigs with one oral dose of CCA (100 mg/kg) did not reduce mortality in protracted anaphylactic shock. Our results demonstrate that CCA inhibits IgE production as well as IgE mediated hypersensitivity reactions in mice.     

Immunological evaluation in children with juvenile chronic arthritis treated with auranofin

An immunological evaluation was performed before therapy and every four months during the first year of treatment with auranofin in 6 children with juvenile chronic arthritis. The immunological tests included: IgG, IgA, IgM, IgE and "natural" antibody serum levels, CH50 of the classical and alternative complement pathways, PWM-induced IgM production in vitro, and polymorphonuclear neutrophil functions. A reduction of the in vitro IgM synthesis and in the CH50 of the classical pathway of complement, and a normalization of impaired chemotaxis, occurred in patients who presented a clinically significant improvement during auranofin treatment.