Uncoupling of Natural IgE Production and CD23 Surface Expression Levels

CD23, the low affinity receptor for immunoglobulin E (IgE), has been proposed to play a critical role in the regulation of IgE production, based on altered IgE levels in CD23-deficient mice and transgenic mouse models, as well as in mouse strains with mutations in the CD23 gene, e.g. 129 substrains. Here, we have investigated a mouse line termed LxT1 that expresses reduced CD23 surface levels on B cells, and its influence on natural IgE production. Extensive phenotypic analysis showed that CD23 surface expression was reduced in LxT1 compared to the control, without affecting B cell development in general. This CD23^low surface level in LxT1 mice is not as a result of reduced CD23 mRNA expression levels or intracellular accumulation, but linked to a recessive locus, a 129-derived region spanning 28 Mb on chromosome 8, which includes the CD23 gene. Sequence analysis confirmed five mutations within the CD23 coding region in LxT1 mice, the same as those present in New Zealand Black (NZB) and 129 mice. However, this CD23^low phenotype was not observed in all 129 substrains despite carrying these same CD23 mutations in the coding region. Moreover, serum IgE levels in LxT1 mice are as low as those in the C57BL/6 (B6) strain, and much lower than those in 129 substrains. These data indicate that the CD23 surface level and serum IgE level are uncoupled and that neither is directly regulated by the mutations within the CD23 coding region. This study suggests that caution should be taken when interpreting the immunological data derived from mice with different genetic background, especially if the gene of interest is thought to influence CD23 surface expression or serum IgE level.     

Development of a Genus-Specific Antigen Capture ELISA for Orthopoxviruses – Target Selection and Optimized Screening

Orthopoxvirus species like cowpox, vaccinia and monkeypox virus cause zoonotic infections in humans worldwide. Infections often occur in rural areas lacking proper diagnostic infrastructure as exemplified by monkeypox, which is endemic in Western and Central Africa. While PCR detection requires demanding equipment and is restricted to genome detection, the evidence of virus particles can complement or replace PCR. Therefore, an easily distributable and manageable antigen capture enzyme-linked immunosorbent assay (ELISA) for the detection of orthopoxviruses was developed to facilitate particle detection. By comparing the virus particle binding properties of polyclonal antibodies developed against surface-exposed attachment or fusion proteins, the surface protein A27 was found to be a well-bound, highly immunogenic and exposed target for antibodies aiming at virus particle detection. Subsequently, eight monoclonal anti-A27 antibodies were generated and characterized by peptide epitope mapping and surface plasmon resonance measurements. All antibodies were found to bind with high affinity to two epitopes at the heparin binding site of A27, toward either the N- or C-terminal of the crucial KKEP-segment of A27. Two antibodies recognizing different epitopes were implemented in an antigen capture ELISA. Validation showed robust detection of virus particles from 11 different orthopoxvirus isolates pathogenic to humans, with the exception of MVA, which is apathogenic to humans. Most orthopoxviruses could be detected reliably for viral loads above 1 × 10³ PFU/mL. To our knowledge, this is the first solely monoclonal and therefore reproducible antibody-based antigen capture ELISA able to detect all human pathogenic orthopoxviruses including monkeypox virus, except variola virus which was not included. Therefore, the newly developed antibody-based assay represents important progress towards feasible particle detection of this important genus of viruses.     

Changes throughout a Genetic Network Mask the Contribution of Hox Gene Evolution

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Which Obesity Index Best Explains Prevalence Differences in Type 2 Diabetes Mellitus?

Objective: Obesity drives the diabetes epidemic. However, it is not known which obesity index best explains variations in type 2 diabetes mellitus prevalence across populations. Research Methods and Procedures: We analyzed three cross‐sectional studies from San Antonio, TX, (Mexican‐Americans and non‐Hispanic whites, n = 2839), Mexico City (n = 2233), and Spain (n = 2161) (age range, 35 to 64 years). We used the area under the receiver operating characteristic curve (AUC) to assess performance for identifying diabetic subjects and logistic regression analysis to examine differences in diabetes prevalence. Results: AUCs for waist circumference and BMI were similar in white subjects, but the AUC for waist circumference was greater in Mexican‐origin subjects (Mexican men, 0.594 vs. 0.549, p = 0.008; and women, 0.605 vs. 0.557, p = 0.002; Mexican‐American men, 0.648 vs. 0.600, p < 0.001; and women, 0.744 vs. 0.693, p < 0.001). The AUC for waist‐to‐height ratio tended to be greater than that for waist circumference, but statistical significance was demonstrated only in Mexican women (0.628 vs. 0.613, p = 0.044), Mexican‐American women (0.774 vs. 0.758, p < 0.001), and Spanish women (0.734 vs. 0.715, p = 0.039). No obesity index was consistently superior to the others for explaining differences in diabetes prevalence among populations. Conclusions: In white and Mexican‐origin men, waist circumference may be the preferred marker for identifying diabetic subjects on account of its simplicity; in women, waist‐to‐height ratio may be better. Differences in diabetes prevalence among these populations cannot be attributed to a single measure of obesity.     

Cutting edge: detection of antigen-specific CD4+ T cells by HLA-DR1 oligomers is dependent on the T cell activation state

Class I MHC tetramers have proven to be invaluable tools for following and deciphering the CD8(+) T cell response, but the development of similar reagents for detection of CD4(+) T cells based on class II MHC proteins has been more difficult. We evaluated fluorescent streptavidin-based oligomers of HLA-DR1 for use as reagents to analyze Ag-specific human CD4(+) T cells. Staining was blocked at low temperatures and by drugs that disrupt microfilament formation and endocytosis. Cell-associated MHC oligomers were resistant to a surface stripping protocol and were observed by microscopy in intracellular compartments. This behavior indicates that detection of CD4(+) T cells using class II MHC oligomers can depend on an active cellular process in which T cells cluster and/or endocytose their Ag receptors. T cells of identical specificity but in different activation states varied greatly in their ability to be detected by class II MHC oligomers.     

Function and cytochemical characteristics of postural limb muscles of the rhesus monkey: A telemetered EMG and immunofluorescence study

Using telemetered electromyography and immunocytochemical fibre typing (of both fresh frozen and preserved specimens), the present paper demonstrates clearly that at the elbow, knee, and ankle joints, the rhesus monkey (Macaca mulatta) is endowed with one extensor-muscle head specialized for posture. These postural heads are distinguished by (a) recruitment at low levels to maintain joint position against the effect of gravity, (b) recruitment near maximum levels during walking, and (c) high content and relatively large size of slow, fatigue-resistant (type I) muscle fibres. The nonpostural heads of the investigated muscles were recruited at levels correlated to the strenuousness of the effort and are notable by the small percentage and size of slow muscle fibres. The postmortem stability of the structural properties of myosin makes immunocytochemical fibre typing suitable for the study of preserved cadavers.