Clozapine--a dangerous drug in a clozapine-naïve subject.

Clozapine is a uniquely effective antipsychotic, but is very toxic in clozapine-naïve subjects. A 34-year-old male patient in a mental health facility, who was not prescribed clozapine, took 350 mg clozapine obtained from another patient at night. He was found dead the next morning. The presence of cardiomegaly related to obesity may have increased the risk of suffering an acute cardiac event after ingestion of clozapine. The medication prescribed to the patient was not thought to have contributed to the fatal outcome. Post mortem femoral blood clozapine and norclozapine concentrations were 0.48 and 0.20mg/L, respectively. By way of comparison, audit of 104,127 plasma samples (26,796 patients) assayed for therapeutic drug monitoring purposes 1993-2007, showed plasma clozapine 0.35 mg/L or more in 57.5% samples (8.4% 1mg/L or more). Those involved in the investigation of clozapine-associated deaths need to be aware that that death in an adult may occur after a single 'therapeutic' dose. A diagnosis of fatal clozapine poisoning cannot be made solely on the basis of a post mortem blood clozapine measurement.

Thirty years with Fc(sigma)R.

In his talk Henry Metzger [H. Metzger et al., Immunol. Lett. 68 (1999) 53-57] referred to a view recently expressed in an article by Koshland [D.E. Koshland Jr., Science 280 (1998) 852-853], that there are three stages in the study of biological systems: firstly, to describe what's happening; secondly, to define the molecules involved and what they are doing; thirdly, to quantitate these processes.

Levels of substance P and IgE decapeptide in nasal polyp fluid and matching sera: a preliminary study.

Sixteen unselected patients with nasal polyps had the levels of substance P and IgE decapeptide measured by ELISA in the oedema fluids and their matched sera. All 16 samples had low levels of substance P in their sera and had high level of substance P in eight samples of nasal polyp oedema. There was a considerable variation in the values of IgE decapeptide found in the sera but 14 polyp oedema fluids had high levels of IgE decapeptide. This study supports the idea that there is a linkage between the cellular and neurovascular responses. High levels of IgE decapeptide suggest that mast cell reactions occur in the majority of cases and that IgE may be implicated in the process of mast cell degranulation.

Microheterogeneity of alpha 1-antitrypsin in relation to the concentration of its complex with immunoglobulin A in the sera of patients with rheumatoid arthritis.

OBJECTIVE: Serum alpha 1-antitrypsin (alpha 1AT) is an acute-phase glycoprotein which contains three carbohydrate side chains, N-glycosidically linked to the asparagine molecules (Asn46, Asn83 and Asn247) of the single polypeptide unit. "Microheterogeneity", which is a varying proportion of bi- or triantennary heteroglycans attached to the glycosylation sites, has been observed in various inflammatory states including rheumatoid arthritis (RA), and may influence the properties of alpha 1AT. METHODS: In this study, we used affinity immunoelectrophoresis with the lectin concanavalin A (ConA) to investigate the possible role of alpha 1AT microheterogeneity in IgA-alpha 1AT complex formation. The concentrations of alpha 1AT and its glycosylation variants, the level of immunoglobulin A (IgA), and concentration of IgA-alpha 1AT complex were determined in the sera of 43 patients with RA. RESULTS: In seven patients, high concentrations of the IgA-alpha 1AT complex were found. This group did not differ from the remaining patients in sex, age, or disease activity. However, significantly higher concentrations of both the alpha 1AT variant 1a+1b (with a predominance of triantennary heteroglycan side chains) and IgA were found in patients with an elevated complex compared to those with low serum levels of IgA-alpha 1AT complex (p < 0.05 for both variables). In the entire group, there was a significant correlation between the IgA-alpha 1AT complex level and both serum IgA and the concentrations of alpha 1AT variants 1a+1b and 2 (r = 0.3473, p < 0.05; r = 0.4604, p < 0.005; r = 0.3176, p < 0.05, respectively). CONCLUSION: The results suggest that the microheterogeneity of alpha 1AT may play a part in the formation of the IgA-alpha 1AT complex in RA.

Cross-linking of a sequential epitope within the beta-chain of HLA-DR/DP molecules suppressing B lymphocyte growth and inducing homotypic cell aggregation.

A monoclonal antibody (SU2) directed against HLA class II antigens has been produced by immunizing BALB/c mice with a lymphoblastoid cell line, RPMI-8866 cells. The specificity of this mAb has been determined using a panel of HLA class II transfectants. SU2 stained all HLA-DR and HLA-DP transfectants tested, but reacted with only one HLA-DQ (HLA-DQw2). From comparison of the available data sequences of known HLA class II molecules, it appeared that the epitope recognized by the SU2 mAb contains a sequence of 6 amino acids (DSDVGE) at position 41-46 of the beta-chain of HLA-DR/DP. Functional studies indicated that SU2 mAb strongly induced cell aggregation and large clumping in resting tonsillar B cells. SU2 mAb inhibited the spontaneous growth and proliferation of B lymphoblastoid cell lines and drastically inhibited [3H]thymidine uptake by phorbol 12-myristate 13-acetate (PMA)-activated resting B cell. Results are discussed in relation to the dual recognition of HLA-DR/DP by SU2 mAb.

Epitope mapping of the site(s) of binding of Fc epsilon RII/CD23 within human IgE. Determination of the B lymphocyte-binding sites by use of synthetic peptides and anti-peptide antibodies representative of linear Fc sequences.

The work undertaken has investigated the structure-function relationship between IgE and its low affinity receptor on B lymphocytes. To identify sites on the IgE molecule which interact with the low affinity receptor (Fc epsilon RII/CD23), 10 different peptide sequences within the CH2, CH3 and CH4 domains of human IgE were selected according to charge, overall hydrophobicity and possible accessibility on native IgE sequences. Peptides representative of these were synthesized by the solid phase procedure; and their cytophilic activities were examined by determining their capacity to inhibit the binding of radiolabelled or erythrocyte-bound IgE to a Fc epsilon RII/CD23 positive B cell line (RPMI-8866). Moreover, these linear sequences were rendered immunogenic by conjugation to a protein carrier (KLH) and used to produced polyclonal antibodies in rabbits. The reactivity of the anti-peptide antibodies with both free peptides and native IgE bound to a solid phase, as well as their capacity to inhibit binding of IgE to a Fc epsilon RII/CD23 positive cell line, were investigated. Results from such use of peptides and anti-peptide antibodies indicate that two sequences, representative of residues 364-383 and 401-415, could be involved in the binding of IgE to both membrane-bound and soluble form Fc epsilon RII/CD23; indicating that the B lymphocyte-binding site on human IgE may be restricted to the CH3 domain.

Production and characterization of two murine monoclonal antibodies directed against epitopes exclusive to soluble fragments of Fc epsilon RII/CD23.

Two murine monoclonal antibodies (mAb) designated as SU1 and SU3 directed against soluble Fc epsilon RII/CD23 have been generated by fusing X.63.AG.8653 (a mouse myeloma cell line) with spleen cells from mice immunized with an Epstein Barr virus (EBV)-transformed B cell line (RPMI-8866). The antibodies have been shown to be capable of detecting affinity purified soluble Fc epsilon RII/CD23 in an enzyme-linked immunosorbent assay. Indirect immunofluorescence has shown that the SU1 and SU3 mAb do not stain RPMI-8866, a Fc epsilon RII/CD23+ B cell line. By studying the migration profiles of affinity purified SU1- and SU3-reactive molecules on sodium dodecylsulfate-polyacrylamide gel electrophoresis it has been shown that SU1 mAb immunoprecipitates 33- and 12-kDa components, while the SU3 mAb recognized 25- and 45-kDa proteins from culture supernatants of RPMI-8866 cells. Moreover, affinity purified SU1- and SU3-reactive proteins have been shown to be recognized by human IgE but not by the human IgG molecule. These results provide evidence that SU1 and SU3 mAb may recognize some putative post-cleavage epitopes on the N-terminal end of the low affinity receptor which appear, perhaps, following the process of fragmentation. In addition, the effect of these antibodies on continuous growth of a panel of lymphoblastoid cell lines indicates that SU1 mAb was found incapable of influencing the spontaneous proliferation of EBV-immortalized B cell lines; whereas SU3 mAb completely blocked the spontaneous growth and proliferation of all B cell lines tested. The results are discussed in relation to the appearance of a functional post-cleavage epitope on soluble Fc epsilon RII/CD23.

Synthetic peptides comprising defined sequences of CH-2 and CH-3 domains of human IgG1 induce prostaglandin E2 production from human peripheral blood mononuclear cells.

Synthetic peptides Y48 and Y75 comprising sequences at exposed sites within the CH-2 and CH-3 domains of human IgG1 at a concentration of 10(-5) M, increase PGE2 production by human peripheral blood mononuclear cell (PBMC) cultures. An increase of leukocyte migration inhibitory factor (LMIF) production in PBMC cultures--as a result of synthetic peptide treatment--was also observed. This LMIF activity, to some extent, is attributed to the PGE2 production by the cells; the inhibition of leukocyte migration being abolished by the presence of indomethacine or antibody to PGE2.

Effect of human IgG1 peptides on the antigen-specific antibody response of mice in vivo.

The effect of synthetic peptides--corresponding to the amino acid sequences 289-301 (Y48) and 293-301 (Y91) within the CH-2 domain in the human IgG1 was studied on the oxazolone-specific primary and secondary antibody response isotype distribution and on the sheep erythrocyte (SRBC)-specific primary IgM response. High responder (Balb/c) and low responder (C57Bl/6) mice to oxazolone hapten were treated intraperitoneally with various doses of peptides simultaneously with the first and second contact sensitization. The relative levels of oxazolone-specific IgM, IgG3, IgG1, IgG2a and IgG2b antibodies were determined by a solid phase radioimmunoassay. Y48 and Y91 peptides in a dose range of 10(-5) - 10(-8) M/animal enhanced the oxazolone-specific antibody response. This effect was more striking under suboptimal conditions: using smaller antigen dose for sensitization, cyclophosphamide pretreatment or using genetically low responder mice. SRBC-specific primary IgM response was enhanced by Y91 peptide, Y48 was ineffective.

Anti-IgG autoantibodies in HIV-infected hemophilia patients.

Sera of 76 HIV-negative hemophilia patients, 103 HIV-positive (HIV+) hemophilia patients free of AIDS or AIDS related complex (ARC), and 32 HIV+ hemophilia patients with AIDS/ARC were tested for four different anti-IgG activities. IgG-anti-F(ab')2 gamma, IgM-anti-F(ab')2 gamma, and IgG-anti-Fc gamma serum activities were significantly associated with the clinical stage of HIV infection, whereas IgM-anti-Fc gamma was not. IgG-anti-F(ab')2 gamma activity was found to be caused by cross-reaction of anti-HIV antibody with an epitope within the constant CH1 domain of human IgG. HIV+ hemophilia patients with severe thrombocytopenia (less than 50,000/microliters platelet counts) had significantly higher IgM-anti-IgG activity than patients with greater than 50,000/microliters platelets. Because anti-IgG antibodies possess immunoregulatory properties, our results may serve as a possible explanation for the frequent B cell disorders encountered in HIV-infected patients.

Affinity-purified soluble Fc epsilon RII/CD23 derived from a culture supernatant of an EBV-immortalized B-cell line induced a monophasic fever in rabbits.

A soluble form of Fc epsilon RII/CD23 is spontaneously released from most lymphoblastoid cell lines established by the Epstein-Barr virus (EBV). Such a product was purified on an IgE-Sepharose column and its pyrogenic effect was investigated in rabbits. This preparation induced a monophasic fever in rabbits, with a peak response appearing 75 min after injection. Since IgE was found to be capable of abrogating such an effect, it is suggested that IgE might be involved in the control of the effectiveness of this soluble protein.

Affinity-purified soluble Fc epsilon RII/CD23 derived from RPMI-8866 cells induces histamine release from human nasal polyp mast cells through a non-IgE-mediated mechanism.

Soluble Fc epsilon RII/CD23 (IgE-binding factor) is released spontaneously from activated B cells and most EBV-immortalised B cell lines. We have purified soluble Fc epsilon RII/CD23 from culture supernatants of RPMI-8866 cells on an IgE Sepharose column, and studied its ability to release histamine from human nasal polyp mast cells. Soluble Fc epsilon RII/CD23 induces release of a significant amount of histamine from nasal polyp mast cells in a dose-dependent manner. IgE, and a monoclonal antibody specific for the soluble form of this receptor, were shown to neutralise this effect. It was found that soluble Fc epsilon RII/CD23 was still capable of triggering histamine release from nasal polyp mast cells from which IgE had been eluted by incubation in a low pH buffer, suggesting that a non-IgE mediated mechanism was responsible for this effect.

The association and predictive value of the complex immunoglobulin A-alpha 1-antitrypsin in the development of erosions in early rheumatoid arthritis.

Immunoglobulin A-alpha 1 antitrypsin complex (IgA-AT), its constituent components and nine other clinical or laboratory variables were measured in thirty-three patients with early, non-erosive rheumatoid arthritis (RA) in order to assess their value in predicting the subsequent development of erosions. After 12 months, eighteen patients had developed erosions. Comparison of variables measured at outset between the group of patients subsequently developing erosions and those not, showed only the complex IgA-AT level to be significantly different, the mean being higher in the erosive group. In the subgroup of patients with high IgA-AT levels (greater than 3.0 arbitary units) all developed erosions. The possible therapeutic implications of these findings are discussed.

Modulatory effect of synthetic human IgG Fc peptides on the in vitro immune response of murine spleen cells.

Synthetic peptides representative of defined surface-exposed sequences within the CH-2 and CH-3 domains of human IgG1 induce IgM production by murine spleen cells, even in cultures depleted of T-lymphocytes. This stimulation was not altered by simultaneous administration of dextran sulphate in suboptimal concentration, its effect being additive to that of the peptides. Cell proliferation was augmented only at 10(-4) M doses of peptides. IL-1 production by adherent cells was also increased as a result of peptide treatment; whilst administration of exogenous IL-1, 4 h later, seemed to abrogate the effect of peptide treatment on the augmentation of IgM production. Peptide treatment failed to induce IL-2 and/or IL-4 production. The effect of IgG peptides seems to be exerted directly on B-cells at an early step of activation and to be mediated at least in part by IL-1.