Characterization of epithelial chemoattractants for human intestinal intraepithelial lymphocytes

BACKGROUND & AIMS: Although homing of intraepithelial lymphocytes (IEL) into intestinal epithelia seems to be guided by signals from epithelia, little is known concerning functional epithelial-derived chemoattractants for IEL. METHODS: Epithelial chemoattractants for IEL were analyzed using chemotaxis chamber system, enzyme-linked immunosorbent assay, and in situ hybridization using human epithelial lines and IEL lines. RESULTS: Epithelial-conditioned media induced IEL chemotaxis, and this activity was markedly enhanced by prestimulation of epithelia with interferon-(IFN)-gamma. This chemotaxis (stimulation +) was significantly inhibited by neutralizing antibodies to IFN-gamma inducible protein-10 (IP-10) or to monokine induced by IFN-gamma (MIG). Furthermore, while high amounts of IP-10 and MIG were detected in epithelial-conditioned media after IFN-gamma stimulation, equivalent concentrations of recombinant IP-10 and MIG reproduced IEL chemotaxis. Production of IP-10 and MIG in fresh epithelial cells was supported by in situ hybridization and enzyme-linked immunosorbent assay. Lastly, fresh human IEL constitutively expressed CXCR-3 (the common receptor for IP-10 and MIG), and fresh IEL also exhibited chemotaxis to by rIP-10, rMIG, and epithelial-conditioned media. CONCLUSIONS: Epithelial cells produce chemoattractants for IEL, and such chemokine production is regulated by proinflammatory cytokines such as IFN-gamma. IP-10 and MIG may serve as potentially important epithelial chemokines for IEL, especially under inflammatory conditions.     

Recovery of the ventilatory and upper airway muscles and exercise performance after type A botulism

We studied six patients with type A botulism to determine the degree of initial respiratory compromise and to quantitate the time course and extent of recovery of the ventilatory and upper airway muscles and exercise performance. The VM weakness was identified in all patients early after botulism. Upper airway muscle weakness was also common, requiring intubation for airway protection in one patient. Recovery of VM and upper airway muscle strength occurred in all patients, predominantly over the first 12 weeks but continued up to one year in several. A similar time course of improvement was noted for exercise performance. Ventilatory limitation was an unusual cause for exercise limitation. By 12 months, lung function, VM and upper airway muscle strength and exercise performance had returned to normal in all but one patient. We conclude that VM and upper airway muscle weakness occurs in most patients with clinically significant type A botulism.     

Characterization of the effects of cultured vascular cells on the activation of blood coagulation

The coagulant properties of intact bovine vascular cells (aortic endothelial and smooth muscle cells) and human vascular cells (cutaneous and foreskin microvascular cells, umbilical venous endothelium) grown in vitro were studied. Compared to nonvascular cells (fibroblasts, corneal endothelial cells, fetal lung or intestinal mucosal cells), vascular cells had little procoagulant activity. Radioimmunologic measurement of thrombin in recalcified plasma demonstrated markedly lower concentrations of thrombin in the presence of vascular endothelial and smooth muscle cells compared to corneal endothelial and fetal lung cells. The low thrombin concentrations were not a consequence of thrombin binding to the vascular cells nor were they due to accelerated thrombin inactivation by antithrombin-III or alpha 2-macroglobulin. Neither vascular cells nor the nonvascular cells promoted contact activation of plasma as measured by a sensitive specific assay for kallikrein. Studies with intact cell monolayers and purified factors VIIa and X indicated that while nonvascular cells express tissue factor activity, vascular cells do not exhibit this property. These data suggest that the nonthrombogenic nature of intact vascular cells is due to their failure to initiate contact activation and to express tissue factor activity. In addition, the primary difference in coagulant potential between vascular cells and nonvascular cells is the lack of tissue factor expression by the vascular cells.     

delta-Aminolevulinate dehydratase in human erythroleukemia cells: an immunologically distinct enzyme

Physicochemical and immunologic properties of delta-aminolevulinate (ALA) dehydratase in human K562 erythroleukemia cells were examined. ALA dehydratase activity was found to increase in K562 cells after treatment with butyric acid or selenium oxide. Enzyme activity in untreated K562 cells was comparable to that in normal adult erythrocytes but was increased three- to six-fold in K562 cells treated with 1.2 mmol/L butyric acid or 0.03 mmol/L selenium oxide. The Michaelis-Menten constant (Km), the inhibitor constant (Ki), and elution profile by diethylaminoethyl (DEAE) cellulose chromatography were similar for ALA dehydratase from K562 cells and normal human adult and human fetal erythrocytes. However, ALA dehydratase from K562 cells did not react with a monospecific rabbit antibody against ALA dehydratase purified from normal adult erythrocytes, although the antibody reacted with the enzyme from normal adult and fetal red cells. These findings indicate that ALA dehydratase in K562 cells is immunologically distinct from the normal enzyme.     

A blocking primer increases specificity in environmental DNA detection of bull trout (Salvelinus confluentus)

Environmental DNA (eDNA) is increasingly applied as a highly sensitive way to detect aquatic animals non-invasively. However, distinguishing closely related taxa can be particularly challenging. Previous studies of ancient DNA and genetic diet analysis have used blocking primers to enrich target template in the presence of abundant, non-target DNA. Here we apply a blocking primer to increase the specificity of a TaqMan assay for eDNA detection of rare and endangered bull trout (Salvelinus confluentus) in the presence of the closely related (Salvelinus namaycush). We found that addition of a blocking primer substantially increased assay specificity without compromising sensitivity or quantification ability.