Treating pediatric neuromuscular disorders: The future is now

Pediatric neuromuscular diseases encompass all disorders with onset in childhood and where the primary area of pathology is in the peripheral nervous system. These conditions are largely genetic in etiology, and only those with a genetic underpinning will be presented in this review. This includes disorders of the anterior horn cell (e.g., spinal muscular atrophy), peripheral nerve (e.g., Charcot–Marie–Tooth disease), the neuromuscular junction (e.g., congenital myasthenic syndrome), and the muscle (myopathies and muscular dystrophies). Historically, pediatric neuromuscular disorders have uniformly been considered to be without treatment possibilities and to have dire prognoses. This perception has gradually changed, starting in part with the discovery and widespread application of corticosteroids for Duchenne muscular dystrophy. At present, several exciting therapeutic avenues are under investigation for a range of conditions, offering the potential for significant improvements in patient morbidities and mortality and, in some cases, curative intervention. In this review, we will present the current state of treatment for the most common pediatric neuromuscular conditions, and detail the treatment strategies with the greatest potential for helping with these devastating diseases.     

阻断型抗人IgE单克隆抗体的制备及其表位分析

目的 利用基因重组抗原免疫小鼠,制备抗人IgE单克隆抗体,研究其特异性和功能.方法 用表达的重组人IgE-Fc免疫BLAB/c小鼠,取其脾细胞与Sp2/0细胞融合,以间接ELISA法筛选杂交瘤上清.用ELISA阻断分析鉴定其生物学功能;通过丙氨酸突变扫描分析确定其抗原表位.结果 利用杂交瘤技术获得了1株抗人IgE的单克隆抗体178,它具有IgE抗体特异性反应,其识别的抗原表位位于IgE与其受体结合的关键部位.经鉴定其具有体外阻断IgE与其受体结合的活性.结论 利用基因重组抗原制备了1株抗人IgE的单克隆抗体178,此单克隆抗体具有体外阻断IgE与其受体结合的生物活性.     

Mouse monoclonal IgE antibodies specific for ragweed pollen antigens

In order to obtain monoclonal IgE antibody specific for the major allergens in ragweed pollen extracts, hybridomas were constructed from spleens of mice immunized with ragweed antigen E (AgE). Two hybridomas were selected for thorough study, both secreting antibodies of the IgE class. Large quantities of IgE antibodies were isolated by affinity purification using Sepharose 4B conjugated with ragweed pollen proteins (Fraction A). Both MAbs were found to bind to a high molecular weight and heterogeneous population of proteins, but not to monomeric AgE as demonstrated by protein blot analysis. It is suspected that both MAbs react with low affinity with AgE determinants and binding could be demonstrated only with aggregated forms of AgE. Although the specific antigens with which they react are unknown, these monoclonal IgE antibodies should be useful reagents, complementing the previously obtained monoclonal anti-dinitrophenyl IgE antibody, for studying various aspects of the mouse and human IgE antibody systems.     

Targetting VEGF in anti-angiogenic and anti-tumour therapy: Where are we now?

Since the recognition of the importance of the vascular bed for growth and metastasis of solid tumours, many researchers have investigated the approach of attacking the tumour vascular bed instead of the tumour cells themselves in anti-cancer therapy. Such approaches have become possible with the increasing knowledge of the angiogenic process and the factors that regulate it. Especially the potent angiogenic factor VEGF has been the subject of extensive study in this regard. A number of studies showed that inactivation of this factor or its receptors led to a profound negative effect on the development of experimental tumours. However, despite the encouraging results obtained in animal studies, it remains to be established whether human tumours, which might be in a state of relative quiescence, are as sensitive to anti-VEGF treatment as the fast-growing tumours that are generally used in animal studies. If so, anti-VEGF treatment might certainly represent a powerful tool in anti-cancer therapy, either or not in combination with other blockers of angiogenesis.     

Dot-immunobinding assay with monoclonal anti-IgE antibodies for the detection and quantitation of human IgE

This paper describes a dot immunobinding assay for determining total human IgE with a tandem of monoclonal anti-IgE antibodies. Minute quantities of monoclonal anti-IgE antibodies were adsorbed on nitrocellulose discs. IgE bound to this solid phase monoclonal anti-IgE antibody was detected by a second monoclonal antibody conjugated with horseradish peroxidase. Using 4-chloro-1-naphthol as a chromogen results in a stable colour reaction that can be semiquantitatively analysed by the naked eye. The colour intensities of the reaction were also analysed by densitometry, yielding a very reproducible quantitation of human serum IgE. Using a serum dilution of 1:50, IgE could be detected in the range of 12.5-2500 U/ml. Using non-diluted serum samples IgE levels between 0.05-50 U/ml were reproducibly measured. Total serum IgE as determined by this dot assay correlated very well with IgE determinations performed by the commercial PRIST assay.     

Quantitative determination of total and specific human IgE with the use of monoclonal antibodies

We have used two monoclonal antibodies (BS17 and Le27) that recognize two different epitopes of the constant region of human IgE in order to determine the total and specific IgE contained in human serum. Both types of assay are based on classic PRIST and RAST procedures and involve one or two monoclonal antibodies labeled with 125I iodine. The performance of these two assays were compared systematically with those obtained with a commercially available polyclonal tracer. We first investigated the 125I-labeling conditions for monoclonal antibodies. In our hands the best results were obtained by use of a tracer of specific radioactivity close to 10 microCi/micrograms. We have been able to demonstrate that as a consequence of its limited affinity, the radioactive tracer is always incompletely bound to the solid-phase IgE. Nonetheless, the sensitivity of the assay is comparable to that obtained with the polyclonal antibody, since less than 0.5 IU/ml can be measured by the PRIST procedure. In contrast, the dilution curves obtained in the RAST with the monoclonal antibodies are very different from those observed with the polyclonal tracer. In fact, these curves are strictly parallel to the dilution curve and to the standard curve derived from the PRIST method, thus indicating that the monoclonal antibodies recognize all IgE equally well regardless of the way in which they are bound to the solid phase. On the basis of this observation, we propose a quantitative assay of specific IgE with the PRIST standard curve as reference. Our results demonstrate that this approach is indeed possible if high-capacity, solid-phase as well as long incubation times are used.     

Production of monoclonal mouse IgE antibodies with DNP specificity by hybrid cell lines

IgE anti-DNP antibody-secreting hybridomas were obtained by polyethylene glycol fusion of murine myeloma cells with spleen cells from mice, primed with DNP-KLH and challenged with DNP-Nippostrongylus brasiliensis extract. The in vivo and in vitro continuously growing hybridomas are producing high amounts of monoclonal IgE anti-DNP antibodies, which are heat-labile, hapten-specific and have rat mast cell sensitizing capacity.     

Characterization of monoclonal antibodies produced by immunization with partially purified IgE receptor complexes

A series of monoclonal antibodies (mAb) were produced following the immunization of mice with partially purified IgE receptors from the rat basophilic leukemia cell line (RBL-2H3). Twelve hybridoma cell lines were selected that secreted monoclonal antibodies capable of binding RBL-2H3 plasma membranes. These antibodies were all of the IgG1, or IgG2a subclass. All 12 antibodies bound to either intact or glutaraldehyde-fixed RBL-2H3 cells. Only one monoclonal (mAb 2AC3) inhibited 125I-labeled IgE binding (IC50 = 65 micrograms/ml compared to 1.0 microgram/ml for unlabeled IgE). This same mAb weakly precipitated the alpha component of the receptor from 125I-surface-labeled cells and directly triggered histamine secretion when incubated with RBL-2H3 cells. Therefore, this hybridoma most likely represents a low affinity anti-receptor antibody. Among the other 11 monoclonals, two caused direct histamine secretion from RBL-2H3 cells. These same two, as well as four others, released greater than 10% of total cellular histamine when rabbit anti-mouse antibody was added to cross-link mAb bound to the cell surface. One monoclonal (mA 1AD3) did not trigger histamine secretion but did inhibit IgE-mediated histamine release when incubated with pre-sensitized RBL-2H3 cells. Except for mAb 2AC3, none of the other monoclonal antibodies that caused or inhibited histamine secretion immunoprecipitated receptor or other protein components from either 125I-surface-labeled or intrinsically-labeled cells. However, two monoclonal antibodies (mAb 1CC4 and 1CD1) immunoprecipitated 45,000 and 55,000 proteins from 125I-surface-labeled cells. One hybridoma (mAb 2AA2) that failed to immunoprecipitate surface-labeled proteins did precipitate a 20,000 band from intrinsically-labeled cells. This band increased slightly in apparent mol. wt after reduction and, therefore, was not the previously described gamma component of the receptor. Because several mAb were capable of modulating histamine secretion, it appeared that some of the present monoclonal antibodies bound to undefined membrane components that are crucial to the secretory process of rat basophilic leukemia cells.     

The use of monoclonal and polyspecific antibodies in the IgE sandwich ELISA

The use of antibody to link antigen to microtitre plates in the enzyme-linked immunosorbent assay (ELISA) has been extended to include mouse monoclonal antibodies and polyspecific rabbit antibodies. Small amounts of both reagents could be used. The capacity of the microtitre plate for the antibody was found to be critical and irradiated plates generally gave better results. Both rabbit anti-IgE conjugated to horseradish peroxidase, and monoclonal mouse anti-IgE with alkaline phosphatase-conjugated rabbit anti-mouse IgG could be used as detection reagents. Comparison with the radioallergosorbent (RAST) test showed a good agreement with the sandwich ELISA. The sandwich ELISA using polyspecific rabbit antibody was substantially more sensitive than conventional ELISA and also marginally more sensitive than RAST.     

Inhibition of IgE binding to mast cells and basophils by monoclonal antibodies to murine IgE

In an attempt to identify the site on IgE which binds with high affinity to the Fc? receptor (Fc?R) on mast cells, we established monoclonal anti-IgE antibodies (mAb) by fusion of myeloma cells with rat splenocytes immunized with purified murine IgE mAb. Six individual mAb were found to react with various IgE mAb of different specificities and not with immunoglobulins of other classes. Three different clusters of epitopes on the Fc? portion could be detected by antibody competition studies. These antigenic determinants were expressed on the Fc? portion and required the two heavy chains in their native conformation. Two groups of mAb and their Fab′ fragments completely inhibited the binding of ¹²⁵I-labeled IgE to rat basophilic leukemia cells (RBL), and one mAb inhibited the specific IgE binding only partially (55–65%). Likewise, the Fab′ fragments of the purified mAb inhibited the antigen-mediated, IgE-dependent, serotonin release of RBL cells. These in vitro findings were confirmed by in vivo experiments, which demonstrated that the anti-IgE mAb could specifically block passive cutaneous anaphylaxis reaction when injected i.d., before challenging with the antigen. The differences in blocking reactivity of the various anti-IgE mAb are discussed in view of heterogeneity in the IgE-Fc?R interaction.     

The future is now: Technology's impact on the practice of genetic counseling

Smartphones, artificial intelligence, automation, digital communication, and other types of technology are playing an increasingly important role in our daily lives. It is no surprise that technology is also shaping the practice of medicine, and more specifically the practice of genetic counseling. While digital tools have been part of the practice of medical genetics for decades, such as internet- or CD-ROM-based tools like Online Mendelian Inheritance in Man and Pictures of Standard Syndromes and Undiagnosed Malformations in the 1980s, the potential for emerging tools to change how we practice and the way patients consume information is startling. Technology has the potential to aid in at-risk patient identification, assist in generating a differential diagnosis, improve efficiency in medical history collection and risk assessment, provide educational support for patients, and streamline follow-up. Here we review the historic and current uses of technology in genetic counseling, identify challenges to integration, and propose future applications of technology that can shape the practice of genetic counseling.     

In vitro IgE inhibition in B cells by anti-CD23 monoclonal antibodies is functionally dependent on the immunoglobulin Fc domain

CD23, the low affinity receptor for IgE (FcvarepsilonRII), is involved in regulation of IgE synthesis by B-lymphocytes. Five monoclonal antibodies to human CD23 were generated from cynomolgus macaques immunized with purified soluble CD23 (sCD23). Four of the five primate antibodies blocked the binding of IgE complexes to CD23 positive cells and also inhibited the production of IgE in vitro by IL-4 induced human peripheral blood mononuclear cells (PBMC). The variable domains of several primate antibodies were utilized to construct chimeric macaque/human (PRIMATIZED((R))) monoclonal antibodies. PRIMATIZED((R)) p5E8G1, containing human gamma 1 constant region, inhibited IgE production in vitro as efficiently as the parent primate antibody, but the human gamma 4 constant version, PRIMATIZED((R)) p5E8G4, was not as effective in IgE inhibition. An F(ab')(2) of p5E8G1 did not inhibit IgE production but did interfere with IgE inhibition by the intact anti-CD23 antibody in a dose dependent fashion. The murine monoclonal antibody MHM6 recognizes human CD23 at a different epitope than primate antibody 5E8, and inhibits IgE production by IL-4 induced PBMC. As with the F(ab')(2) of p5E8G1, the F(ab')(2) of MHM6 also failed to inhibit IgE production. These data imply that the mechanism by which anti-CD23 antibodies inhibit IgE production requires cross-linking of CD23 to an IgG receptor. These data also imply that neither bivalent cross-linking of CD23 alone or inhibition of CD23 binding to its natural ligands is sufficient to inhibit IgE production.     

Production of syngeneic autoreactive monoclonal antibodies specific for isotypic determinants of IgE

Methods are described for the production of syngeneic mouse anti-IgE monoclonal antibodies (mAb). Hybridomas were prepared by using spleen cells from mice immunized with a conjugate of keyhole limpet hemocyanin with monoclonal IgE. Serum titers varied from approximately 40 to 1000 micrograms/ml. The anti-IgE mAb were isolated by affinity chromatography on columns containing immobilized monoclonal IgE. The mAb are specific for isotypic determinants of IgE and do not react with other immunoglobulin isotypes. One of the mAb, which has a high affinity for IgE (Ka = 4.7 X 10(8) M-1), should be useful for studies of regulation of IgE. The applicability of the mAb to quantitative assays for IgE was demonstrated.     

Purification of immunoreactive radiolabeled monoclonal antibodies with anti-idiotypic monoclonal antibodies

A method is described to purify immunoreactive monoclonal antibodies from radiolabeled monoclonal antibody preparations. The method is based on incubation of radiolabeled monoclonal antibodies with insolubilized anti-idiotypic monoclonal antibodies to idiotopes within the antigen-combining site of monoclonal antibodies to be purified and elution of bound monoclonal antibodies with a low pH buffer. The immunoreactive fraction of the purified monoclonal antibodies was at least 82%; the yield was at least 73%. The purification procedure did not cause any detectable change in the affinity constant of the eluted monoclonal antibodies. The method is simple and rapid; the requirement for anti-idiotypic monoclonal antibodies to idiotopes within the antigen-combining site of the antibodies to be purified is not likely to represent a major limitation in the broad application of the present method, since the hybridoma technology has greatly facilitated the development of anti-idiotypic monoclonal antibodies.