T cell developmental defects in 'viable motheaten' mice deficient in SHP-1 protein-tyrosine phosphatase. Developmental defects are corrected in vitro in the presence of normal hematopoietic-origin stromal cells and in vivo by exogenous IL-7

Defects in the gene that encodes SHP-1 protein tyrosine phosphatase result in multiple hematopoietic abnormalities and generalized autoimmunity in viable motheaten (me(v)) mice. These mice also exhibit early thymic involution and abnormalities in T cell development. Here, we describe the use of fetal thymic organ culture (FTOC) and bone marrow adoptive transfer to study the effects of SHP-1 deficiency on thymocyte development. Chimeric FTOC established with normal bone marrow placed onto deoxyguanosine-treated fetal thymic lobes or onto scid fetal thymic lobes generated T cells. Bone marrow from SHP-1-deficient me(v)/ me(v) mice generated decreased numbers of T cells in chimeric FTOC established using deoxyguanosine-treated thymi but generated normal numbers in chimeric FTOC established using scid thymi. However, scid fetal thymi seeded with me(v)/ me(v) bone marrow also exhibited morphological abnormalities and contained elevated numbers of macrophages. Addition of IL-7 to me(v)/ me(v) bone marrow-seeded scid FTOC led to increased cell numbers, particularly of macrophages. Intrathymic injection of IL-7 partially restored the ability of progenitor cells in me(v)/ me(v) bone marrow to populate the thymus of adoptive recipients. We conclude that abnormal T cell development in me(v)/ me(v) mice may in part be due to defects in the ability of bone marrow-derived accessory cells to provide bioavailable IL-7 to developing thymocytes.     

Propagation of action potentials from the soma to individual dendrite of cultured rat amacrine cells is regulated by local GABA input

Retinal amacrine cells are interneurons that make lateral and vertical connections in the inner plexiform layer of the retina. Amacrine cells do not possess a long axon, and this morphological feature is the origin of their naming. Their dendrites function as both presynaptic and postsynaptic sites. Half of all amacrine cells are GABAergic inhibitory neurons that mediate lateral inhibition, and their light-evoked response consists of graded voltage changes and regenerative action potentials. There is evidence that the amount of neurotransmitter release from presynaptic sites is increased by spike propagation into the dendrite. Thus understanding of how action potentials propagate in dendrites is important to elucidating the extent and strength of lateral inhibition. In the present study, we used the dual whole cell patch-clamp technique on the soma and the dendrite of cultured rat amacrine cells and directly demonstrated that the action potentials propagate into the dendrites. The action potential in the dendrite was TTX sensitive and was affected by the local membrane potential of the dendrite. Propagation of the action potential was suppressed by local application of GABA to the dendrite. Dual dendrite whole cell patch-clamp recordings showed that GABA suppresses the propagation of action potentials in one dendrite of an amacrine cell, while the action potentials propagate in the other dendrites. It is likely that the action potentials in the dendrites are susceptible to various external factors resulting in the nonuniform propagation of the action potential from the soma of an amacrine cell.     

Expression of epithelial adhesion proteins and integrins in chronic inflammation.

Epithelial cell behavior in chronic inflammation is poorly characterized. During inflammation of tooth-supporting structures (periodontal disease), increased proliferation of epithelial cells into the inflamed connective tissue stroma is commonly seen. In some areas ulceration and degeneration take place. We studied alterations in the expression of adhesion molecules and integrins during chronic periodontal inflammation. In inflamed tissue, laminin-1 and type IV collagen were still present in the basement membrane and surrounding blood vessels, but they were also found extravascularly in inflamed connective tissue stroma. Type VII collagen and laminin-5 (also known as kalinin, epiligrin, or nicein) were poorly preserved in the basement membrane zone, but both were found in unusual streak-like distributions in the subepithelial connective tissue stroma in inflamed tissue. Both fibronectin and tenascin were substantially decreased in chronically inflamed connective tissue, showing only punctate staining at the basement membrane zone. Integrins of the beta 1 family showed two distinct staining patterns in epithelial cells during chronic inflammation; focal losses of beta 1 integrins (alpha 2 beta 1 and alpha 3 beta 1) were found in most areas, while in other areas the entire pocket epithelium was found to be strongly positive for beta 1 integrins. No members of the alpha v integrin family were found in any epithelia studied. Expression of the alpha 6 beta 4 integrin was high in basal cells of healthy tissue, but weak in epithelium associated with chronic inflammation. Chronic inflammation therefore involves alterations in both adhesion proteins and integrins expressed by epithelial cells. Basement membrane components found at abnormal sites in stroma in chronic inflammation might serve as new adhesive ligands for various cell types in inflamed stroma.     

Intracellular pH measurement in frog muscle by means of 31P-nuclear magnetic resonance.

The 31P-NMR technique was used for the monitoring of intracellular pH and studying its heterogeneity in the femoral biceps muscle of Rana catesbiana under anaerobic conditions. The value of intracellular pH of fresh muscle calculated from the chemical shift of intracellular inorganic phosphate (P1) was 7.3 on average and the line width of P1 was about 0.2 ppm. As the line width determined by the relaxation mechanism was 0.099 ppm, the P1 signal in fresh muscle was concluded to consist of overlapped narrow components, which indicated the heterogeneity of muscular pH (about 0.2 pH unit). Living muscle showed gradual acidification due to glycolysis and the decrease in heterogeneity. When glycolysis was suppressed by iodoacetic acid, slight alkalization due to the breakdown of creatine phosphate was observed. When the Lohmann reaction was suppressed by 2, 4-dinitro-1-fluorobenzene, rapid acidification accompanied by the appearance of a new acidic component was observed with the onset of ATP decrease. This new component was not detected in the muscle pretreated with glycerol to disrupt the transverse tubules. Therefore, it is likely that this new acidic component originates in the intracellular compartment, and not in the cellular difference.     

IgE antibody responses induced by transplantation of the nematode Nippostrongylus brasiliensis in rats: a possible role of nematode excretory-secretory product in IgE production.

In order to examine the effective site of sensitization for IgE responses, we transplanted 2000 adult-stage worms of the nematode Nippostrongylus brasiliensis into the duodenum or the peritoneal cavity of naive rats. Total serum IgE began to increase 1 week after the nematode inoculations and reached a peak at week 2. Living worms inoculated into the duodenum induced the highest serum IgE, this being 800 times the level in control animals. Intraperitoneal inoculations of living and dead worms resulted in increases of the serum IgE levels to 120 and 13 times the control level, respectively. The intraduodenal inoculation of living adult worms also induced a significant increase in specific IgE against the excretory-secretory (ES) product of adult N. brasiliensis 1 week later than the rise in total IgE, whereas intraperitoneal inoculations did not induce such an increase. These results suggest that sensitization through the intestinal mucosa with adult N. brasiliensis might be important for the effective induction of both specific and non-specific IgE responses. Since these findings also indicated that factors secreted by living worms play an important role in the induction of total IgE response, the ES product was injected to naive rats for 6 consecutive days (total 2.7-4.4 mg). Intraperitoneal injection of the ES product alone induced a 14.7-fold increase in total IgE without any specific IgE response. This indicates that some constituents of the ES product have the potential to trigger a non-specific IgE response.     

Detection of antibody to Staphylococcus aureus teichoic acid by enzyme-linked immunosorbent assay.

A sensitive, specific, and rapid enzyme-linked immunosorbent assay has been developed for the detection of immunoglobulin G to Staphylococcus aureus teichoic acid in human sera. Detection of S. aureus teichoic acid antibody is at least 800 times more sensitive than a double diffusion in gel assay, and positive titers of 1:25,600 and greater were observed with this assay. Results with the enzyme-linked immunosorbent assay can be obtained within 3.5 h by using antigen-coated cuvettes. Quantitation of S. aureus teichoic acid antibody by this enzyme-linked immunosorbent assay may be useful in the initial as well as the follow-up diagnosis of serious S. aureus infections.     

Mucopolysaccharidosis type II (Hunter disease): Identification and characterization of eight point mutations in the iduronate-2-sulfatase gene in Japanese patients

Mucopolysaccharidosis type II (Hunter disease) is a lysosomal storage disorder caused by a deficiency of the enzyme iduronate-2-sulfatase. Varied clinical phenotypes of this disease have been described. To identify mutations in individual patients and to examine possible correlations between mutations and clinical phenotypes, we analyzed the iduronate-2-sulfatase gene in Japanese patients with different clinical phenotypes. Five missense mutations, S333L (severe), R468Q (severe), R468L (severe), W337R (intermediate), R48P (mild), and three nonsense mutations, W345X (severe), R443X (intermediate), Q531X (mild), were identified by the RT-PCR method. Transient expression in the enzyme-deficient fibroblasts revealed that all five missense mutant enzymes were synthesized as the normal-size precursor (73 kD), and the nonsense mutant enzymes were synthesized as truncated ones (W345X:54 kD, R443X:59 kD, and Q531X:69 kD), although stable mature enzymes (45–56 kD) were not detected by Western blot analysis. Further more, expression of the eight mutant cDNAs resulted in severe reductions of iduronate-2-sulfatase enzyme activity in comparison with a normal cDNA. © 1995 Wiley-Liss, Inc.