Characterization of porcine CD19 and anti-CD19 monoclonal antibodies

CD19 is an important pan B cell marker and co-stimulatory protein in humans and mice. Efforts to further characterize B cell ontogeny in swine have been hampered by the lack of monoclonal antibodies (mAb) to valuable surface markers like Vpre-B, CD19, CD34 and CD43. We report here on the complete nucleotide and deduced amino acid sequence of porcine CD19, the cross-reactivity of anti-human CD19 monoclonals and efforts to prepare anti-porcine CD19 mAb to bacterially-expressed products. Porcine CD19 is highly homologous to those in the few other species studied, i.e. human, mouse and guinea pig, but only in certain domains. Among the 14 CD19 exons, homology approaches 90% to human CD19 in exons 6, 9, 11 and 12 and is approximately 80% with other species in this region. The highly homologous C-terminal cytoplasmic region contains nine tyrosines including the YEND/E motif that binds the SH2 domain of Fyn. Two different porcine CD19 isoforms that differ in their 3' UTRs were identified just as in human CD19. Thus, the signaling properties of CD19 may be similar to those in humans. On the other hand, only 60% sequence similarity was seen in exons 1-5 that encode the N-terminal extracellullar region that is involved in ligand binding and is the target of CD19-specific mAb. This probably explains why only 1 of the 17 anti-human CD19 mAb tested recognized swine B cells. Furthermore, when the extracellular domains of CD19 were expressed in E. coli, mAbs to the bacterially-expressed product did not recognize CD19 on porcine B cells suggesting that carbohydrate-dependent conformation may determine antigenicity.     

Effect of the number of high-fat and low-fat cues on food choice

This study examined the effects on food choice of increasing the number of healthy items available (fruit) and decreasing the number of unhealthy items available (candy bars). A similar choice, involving nonfood items, was also examined. Two hundred eighty-nine men and women were randomly assigned to 1 of 4 experimental groups: (a) control group, (b) increased number of fruits, (c) decreased number of candy bars, and (d) combination. Between 30% and 40% of participants chose fruit regardless of the amount of fruit and candy presented: there was no effect of increasing fruit or decreasing candy bars. However, restrained participants and current dieters were more likely to choose fruit. In contrast, both stimulus control techniques were effective in increasing the percentage of participants choosing a nonfood item. These results suggest that stimulus control may not be sufficient to modify food choice: other powerful factors affect eating behavior, and these must be considered.     

Control of established experimental allergic encephalomyelitis by inhibition of tumor necrosis factor (TNF) activity within the central nervous system using monoclonal antibodies and TNF receptor-immunoglobulin fusion proteins

Tumor necrosis factor (TNF) activity was inhibited during the development of actively-induced, chronic relapsing experimental allergic encephalomyelitis (CREAE) in Biozzi AB/H mice, using a mouse TNF-specific (TN3.19.12) antibody and bivalent human p55 and p75 TNF receptor-immunoglobulin (TNFR-Ig) fusion proteins. The development of disease could be inhibited when repeated doses of antibody were administered prior to the anticipated onset. It has now also been shown that a therapeutic effect is evident even when antibody is administered after the onset of clinical signs, further indicating an important role for TNF in pathogenic effector mechanisms in CREAE. Although biologically-active TNF was not detected in the circulation, TNF-α was detected in lesions within the central nervous system (CNS). This suggested that the CNS may be the main site for TNF-specific immunomodulation and was supported by the observation that intracranial injection was significantly more potent than that administered systemically, for both antibody and TNFR-Ig fusion proteins. The fusion proteins were as effective as antibody at doses 10—100-fold lower than that used for antibody, reflecting their higher neutralizing capacity in vitro. Although treatment was not curative and relapse inevitably occurred in this model if treatment was not sustained, the data indicate that anti-TNF immunotherapy, especially within the CNS, can inhibit CREAE and may, therefore, be useful in the control of human neuroimmunological diseases.     

Large deletions of the MECP2 gene detected by gene dosage analysis in patients with Rett syndrome

The original article to which this Erratum refers was published in Human Mutation 23:234–244 Human Mutation(2004) 23(3) 234–244     

A sandwich ELISA for properdin in clinical specimens

We have developed a symmetrical sandwich ELISA for measuring human properdin (P) in serum by using the globulin fraction from a commercial antiserum as the capture antibody adsorbed on the plastic. The detecting reagent was a glutaraldehyde conjugate of this Ig fraction with alkaline phosphatase. Two types of inhibition were observed in this study. First, inhibition was observed when greater than 2.5 micrograms/ml of the globulin fraction was used to coat the plates. A second type of inhibition was observed for serum dilutions less than 1/400; it was independent of the concentration of capture Ab and did not occur when purified P was assayed. The data generated with this assay could be fitted in log-log mode by a quadratic equation. The coefficient of the linear term in this equation was found to be the same for serum and purified P, within the limits of experimental error. The results for different samples run on the same plate were expressed in terms of the relative concentration of each sample required to produce an OD405 = 0.2. A sample of pooled normal human serum was run on each plate as a reference; it was assigned a titer of 100 ELISA units/ml (EU/ml). The titers of the unknown samples were expressed in terms of EU/ml by reference to this standard. For purified P, the assay could readily detect 10 ng/ml. By comparing purified P with our reference serum pool, we found that 1 EU equals 0.57 microgram. Day-to-day variation for a group of nine normal sera showed a mean difference of -0.85 EU/ml, SD 5.85 EU/ml. The mean titer for these normal sera was 78.9 EU/ml, SD 15.7 EU/ml. In three recovery experiments in which purified P was mixed with pooled normal serum, the recoveries ranged from 96 to 117%. We conclude that the sandwich ELISA constitutes an adequate immunochemical assay for human P in serum specimens.     

Fine structure of the adrenocortical homologue in the north American eel and modifications following seawater adaptation

The ultrastructure of the adrenocortical homologue (AH) of the north American eel (Anguilla rostrata) was studied from freshwater and long-term (1.5 years) seawater (SW) adpated animals. The AH tissue situated in the wall of cardinal veins is surrounded by a thin layer of collagenous capsule; in the region away from the vein wall, parenchymal cells are separated by interstitial lacunae containing collagen bundles, capillaries, chromaffin cells and nerve fibers often applied closely to the surface of AH cells. The free surface of the cell near the vein wall, capillaries or interstitial space extends numerous slender microplicae. The cytoplasm is characterized by the presence of mitochondria with tubular cristae, a network of smooth endoplasmic reticulum, a few cisternae of rough surfaced endoplasmic reticulum, well developed Golgi apparatus, coated vesicles, centrioles, cilium, filaments, microtubules, dense bodies of variable nature and a scarcity of liposomes. The cell nuclei possess invaginated cytoplasmic pseudo-inclusions. Electron histochemical reaction for free cholesterol revealed the occurrence of needle-shaped crystals mainly associated with surface microplicae and smooth endoplasmic reticulum, which seems to be the major organelle for storage or synthesis of this steroid precursor. SW animals indicated ultrastructural signs of stimulated steroid synthesis and secretion, i.e., high degree of pseudo-follicle formation, increased electron density of mitochondria, greater abundance of lysosomal dense bodies, hyperactivity of Golgi apparatus and dilation of smooth endoplasmic reticulum tubules. Some SW fishes showed extensive deposition of osmiophilic inclusions in the mitochondria and stacks of elongated cup-shaped mitochondria. Chronic seawater acclimation enhances AH activity as judged by ultrastructural criteria with ultimate mitochondrial degeneration resulting possibly from prolonged cortisol hypersecretion; the latter may be linked with physiologic re-adjustment of ionic transfer mechanism in hyperosmotic medium.