Novel OCTN2 mutations: No genotype–phenotype correlations: Early carnitine therapy prevents cardiomyopathy

Primary systemic carnitine deficiency or carnitine uptake defect (OMIM 212140) is a potentially lethal, autosomal recessive disorder characterized by progressive infantile‐onset cardiomyopathy, weakness, and recurrent hypoglycemic hypoketotic encephalopathy, which is highly responsive to L ‐carnitine therapy. Molecular analysis of the SLC22A5 (OCTN2) gene, encoding the high‐affinity carnitine transporter, was done in 11 affected individuals by direct nucleotide sequencing of polymerase chain reaction products from all 10 exons. Carnitine uptake (at Km of 5 μM) in cultured skin fibroblasts ranged from 1% to 20% of normal controls. Eleven mutations (delF23, N32S, and one 11‐bp duplication in exon 1; R169W in exon 3; a donor splice mutation [IVS3+1 G > A] in intron 3; frameshift mutations in exons 5 and 6; Y401X in exon 7; T440M, T468R and S470F in exon 8) are described. There was no correlation between residual uptake and severity of clinical presentation, suggesting that the wide phenotypic variability is likely related to exogenous stressors exacerbating carnitine deficiency. Most importantly, strict compliance with carnitine from birth appears to prevent the phenotype. © 2002 Wiley‐Liss, Inc.     

Factors affecting the determination of threshold doses for allergenic foods: how much is too much?

BACKGROUND: Ingestion of small amounts of an offending food can elicit adverse reactions in individuals with IgE-mediated food allergies. The threshold dose for provocation of such reactions is often considered to be zero. However, because of various practical limitations in food production and processing, foods may occasionally contain trace residues of the offending food. Are these very low, residual quantities hazardous to allergic consumers? How much of the offending food is too much? Very little quantitative information exists to allow any risk assessments to be conducted by the food industry. OBJECTIVE: We sought to determine whether the quality and quantity of existing clinical data on threshold doses for commonly allergenic foods were sufficient to allow consensus to be reached on establishment of threshold doses for specific foods. METHODS: In September 1999, 12 clinical allergists and other interested parties were invited to participate in a roundtable conference to share existing data on threshold doses and to discuss clinical approaches that would allow the acquisition of that information. RESULTS: Considerable data were identified in clinical files relating to the threshold doses for peanut, cows' milk, and egg; limited data were available for other foods, such as fish and mustard. CONCLUSIONS: Because these data were often obtained by means of different protocols, the estimation of a threshold dose was very difficult. Development of a standardized protocol for clinical experiments to allow determination of the threshold dose is needed.     

The Cbl family of ubiquitin ligases: critical negative regulators of tyrosine kinase signaling in the immune system

The Cbl family of proteins are evolutionarily conserved negative regulators of activated tyrosine kinase-coupled receptors. Antigen receptors are prominent targets of negative regulation by the Cbl family members, Cbl and Cbl-b, which proteins function as ubiquitin ligases. Cbl and Cbl-b contain substrate recognition domains that interact specifically with activated protein tyrosine kinases of the Src and Syk/ZAP-70 families. Cbl-mediated ubiquitination of these kinases leads to their degradation, resulting in attenuation of receptor signals. Cbl may also control activation-induced monoubiquitination of antigen receptors, thus facilitating their delivery to lysosomes for subsequent degradation. Finally, the interactions of Cbl proteins with downstream targets of tyrosine kinases, such as PI-3-kinase and Vav, could provide an additional mechanism to attenuate receptor signaling. By targeting multiple components of antigen receptor signaling for degradation, the Cbl protein family provides a critical mechanism to ensure an appropriate immune response. The hyperresponsiveness of Cbl(-/-) and Cbl-b(-/-) lymphocytes and the autoimmune phenotype of Cbl-b(-/-) mice lend strong support for this proposal. The ability to control early receptor signals through regulated protein degradation provides a novel paradigm of immunoregulation.     

Interlaboratory validation of a CD71-based flow cytometric method (Microflow) for the scoring of micronucleated reticulocytes in mouse peripheral blood

An interlaboratory study was performed to validate an anti-CD71/flow cytometry-based technique for enumerating micronucleated reticulocytes (MN-RETs) in mouse peripheral blood. These experiments were designed to address International Workshop on Genotoxicity Test Procedures validation criteria by evaluating the degree of correspondence between MN-RET measurements generated by flow cytometry (FCM) with those obtained using traditional microscopy-based methods. In addition to these cross-methods data, flow cytometric MN-RET measurements for each blood sample were performed at two separate sites in order to evaluate the reproducibility of data between laboratories. In these studies, groups of male CD-1 mice were treated with vehicle (saline or vegetable oil), a negative control (saline or vegetable oil), or four dose levels of five known genotoxicants (clastogens: cyclophosphamide, benzo[a]pyrene, 5-fluorouracil, methotrexate; aneugen: vincristine sulfate). Exposure occurred on 3 consecutive days via intraperitoneal injection, and blood samples were obtained approximately 24 hr after the final treatment. MN-RET frequencies were determined for each sample based on the analysis of 2,000 (microscopy) and 20,000 (FCM) reticulocytes. Regardless of the method utilized, each genotoxic agent was observed to cause statistically significant increases in the frequency of MN-RETs, and each response occurred in a dose-dependent manner. Spearman's correlation coefficient (rs) for FCM versus microscopy-based MN-RET measurements (nine experiments, 252 paired measurements) was 0.740, indicating a high degree of correspondence between methods. The rs value for all flow cytometric MN-RET measurements performed at the two independent sites was 0.857 (n = 248), suggesting that the automated method is highly transferable between laboratories. Additionally, the flow cytometric system offered advantages relative to microscopy-based scoring, including a greater number of cells analyzed, much faster analysis times, and a greater degree of objectivity. Collectively, data presented in this report suggest that the overall performance of mouse peripheral blood micronucleus tests is enhanced by the use of the flow cytometric scoring procedure.     

Adoptive immunotherapy for posttransplantation viral infections.

Viral diseases are a major cause of morbidity and mortality after hemopoietic stem cell transplantation. Because viral complications in these patients are clearly associated with the lack of recovery of virus-specific cellular immune responses, reconstitution of the host with in vitro expanded cytotoxic T lymphocytes is a potential approach to prevent and treat these diseases. Initial clinical studies of cytomegalovirus and Epstein-Barr virus in human stem cell transplant patients have shown that adoptively transferred donor-derived virus-specific T cells may restore protective immunity and control established infections. Preclinical studies are evaluating this approach for other viruses while strategies for generating T cells specific for multiple viruses to provide broader protection are being evaluated in clinical trials. The use of genetically modified T cells or the use of newer suicide genes may result in improved safety and efficacy.     

GABRD encoding a protein for extra- or peri-synaptic GABAA receptors is a susceptibility locus for generalized epilepsies

A major challenge in understanding complex idiopathic generalized epilepsies has been the characterization of their underlying molecular genetic basis. Here, we report that genetic variation within the GABRD gene, which encodes the GABAA receptor delta subunit, affects GABA current amplitude consistent with a model of polygenic susceptibility to epilepsy in humans. We have found a GABRD Glu177Ala variant which is heterozygously associated with generalized epilepsy with febrile seizures plus. We also report an Arg220His allele in GABRD which is present in the general population. Compared with wild-type receptors, alpha1beta2Sdelta GABAA receptors containing delta Glu177Ala or Arg220His have decreased GABAA receptor current amplitudes. As GABAA receptors mediate neuronal inhibition, the reduced receptor current associated with both variants is likely to be associated with increased neuronal excitability. Since delta subunit-containing receptors localize to extra- or peri-synaptic membranes and are thought to be involved in tonic inhibition, our results suggest that alteration of this process may contribute to the common generalized epilepsies.     

Light scattering from aqueous solutions of poly(acrylamide-co-maleic anhydride)

Light scattering measurements from aqueous solutions for two samples of poly(acrylamide-co-maleic anhydride) in the presence of salt are conducted. One of the samples exhibits a negative initial slope and a minimum in the plot of the conventional reciprocal scattered intensity function of sin² (θ/2). The explanation for this anomaly is a large optical anisotropy of the segment. A correction for this effect of segmental anisotropy is made. The behaviour of the other sample of this copolymer is typical for a polyelectrolyte. By analysing the disymmetry of the scattered light, the influence of the salt concentration on the polyion is evidenced by the determination of the macromolecular dimensions. Light scattering data allow the evaluation of the Mark-Kuhn-Houwink exponent a for various degrees of dissociation of the polyion; a correlation between the variation of a and the shape of the macromolecule is observed.     

Comparative efficacy,speed, and adverse effects of three PTSD treatments: exposure therapy,EMDR, and relaxation training

The authors examined the efficacy, speed, and incidence of symptom worsening for 3 treatments of posttraumatic stress disorder (PTSD): prolonged exposure, relaxation training, or eye movement desensitization and reprocessing (EMDR; N = 60). Treaments did not differ in attrition, in the incidence of symptom worsening, or in their effects on numbing and hyperarousal symptoms. Compared with EMDR and relaxation training, exposure therapy (a) produced significantly larger reductions in avoidance and reexperiencing symptoms, (b) tended to be faster at reducing avoidance, and (c) tended to yield a greater proportion of participants who no longer met criteria for PTSD after treatment. EMDR and relaxation did not differ from one another in speed or efficacy.