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(1/36125) Interleukin-8 receptor modulates IgE production and B-cell expansion and trafficking in allergen-induced pulmonary inflammation.

We examined the role of the interleukin-8 (IL-8) receptor in a murine model of allergen-induced pulmonary inflammation using mice with a targeted deletion of the murine IL-8 receptor homologue (IL-8r-/-). Wild-type (Wt) and IL-8r-/- mice were systemically immunized to ovalbumin (OVA) and were exposed with either single or multiple challenge of aerosolized phosphate-buffered saline (OVA/PBS) or OVA (OVA/OVA). Analysis of cells recovered from bronchoalveolar lavage (BAL) revealed a diminished recruitment of neutrophils to the airway lumen after single challenge in IL-8r-/- mice compared with Wt mice, whereas multiply challenged IL-8r-/- mice had increased B cells and fewer neutrophils compared with Wt mice. Both Wt and IL-8r-/- OVA/OVA mice recruited similar numbers of eosinophils to the BAL fluid and exhibited comparable degrees of pulmonary inflammation histologically. Both total and OVA-specific IgE levels were greater in multiply challenged IL-8r-/- OVA/OVA mice than in Wt mice. Both the IL-8r-/- OVA/OVA and OVA/PBS mice were significantly less responsive to methacholine than their respective Wt groups, but both Wt and IL-8r mice showed similar degrees of enhancement after multiple allergen challenge. The data demonstrate that the IL-8r modulates IgE production, airway responsiveness, and the composition of the cells (B cells and neutrophils) recruited to the airway lumen in response to antigen.  (+info)

(2/36125) Cystic fibrosis transmembrane conductance regulator-mediated corneal epithelial cell ingestion of Pseudomonas aeruginosa is a key component in the pathogenesis of experimental murine keratitis.

Previous findings indicate that the cystic fibrosis transmembrane conductance regulator (CFTR) is a ligand for Pseudomonas aeruginosa ingestion into respiratory epithelial cells. In experimental murine keratitis, P. aeruginosa enters corneal epithelial cells. We determined the importance of CFTR-mediated uptake of P. aeruginosa by corneal cells in experimental eye infections. Entry of noncytotoxic (exoU) P. aeruginosa into human and rabbit corneal cell cultures was inhibited with monoclonal antibodies and peptides specific to CFTR amino acids 108 to 117. Immunofluorescence microscopy and flow cytometry demonstrated CFTR in the intact murine corneal epithelium, and electron microscopy showed that CFTR binds to P. aeruginosa following corneal cell ingestion. In experimental murine eye infections, multiple additions of 5 nM CFTR peptide 103-117 to inocula of either cytotoxic (exoU+) or noncytotoxic P. aeruginosa resulted in large reductions in bacteria in the eye and markedly lessened eye pathology. Compared with wild-type C57BL/6 mice, heterozygous DeltaF508 Cftr mice infected with P. aeruginosa had an approximately 10-fold reduction in bacterial levels in the eye and consequent reductions in eye pathology. Homozygous DeltaF508 Cftr mice were nearly completely resistant to P. aeruginosa corneal infection. CFTR-mediated internalization of P. aeruginosa by buried corneal epithelial cells is critical to the pathogenesis of experimental eye infection, while in the lung, P. aeruginosa uptake by surface epithelial cells enhances P. aeruginosa clearance from this tissue.  (+info)

(3/36125) Altered trafficking of lysosomal proteins in Hermansky-Pudlak syndrome due to mutations in the beta 3A subunit of the AP-3 adaptor.

Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defective lysosome-related organelles. Here, we report the identification of two HPS patients with mutations in the beta 3A subunit of the heterotetrameric AP-3 complex. The patients' fibroblasts exhibit drastically reduced levels of AP-3 due to enhanced degradation of mutant beta 3A. The AP-3 deficiency results in increased surface expression of the lysosomal membrane proteins CD63, lamp-1, and lamp-2, but not of nonlysosomal proteins. These differential effects are consistent with the preferential interaction of the AP-3 mu 3A subunit with tyrosine-based signals involved in lysosomal targeting. Our results suggest that AP-3 functions in protein sorting to lysosomes and provide an example of a human disease in which altered trafficking of integral membrane proteins is due to mutations in a component of the sorting machinery.  (+info)

(4/36125) Enhanced myocardial glucose use in patients with a deficiency in long-chain fatty acid transport (CD36 deficiency).

CD36 is a multifunctional, 88 kDa glycoprotein that is expressed on platelets and monocytes/macrophages. CD36 also has high homology with the long-chain fatty acid (LFA) transporter in the myocardium. Although platelet and monocyte CD36 levels can indicate a CD36 deficiency, they cannot predict specific clinical manifestations in the myocardium of a given person. We examined the hypothesis that a deficiency in LFA transport augments myocardial glucose uptake in patients with a type I CD36 deficiency. METHODS: Seven fasting patients with a type I CD36 deficiency and 9 controls were assessed by cardiac radionuclide imaging using beta-methyl-p-iodophenyl-pentadecanoic acid (BMIPP) as a LFA tracer and by PET with 18F-fluorodeoxyglucose (FDG). RESULTS: None of the patients with a CD36 deficiency showed myocardial uptake of BMIPP. The percentage dose uptake of BMIPP in these subjects was significantly lower than that in normal controls (1.31+/-0.24 versus 2.90+/-0.2; P < 0.005). PET studies revealed that myocardial FDG accumulation was substantially increased in patients with a CD36 deficiency. Quantitative analysis showed that the percentage dose uptake of FDG in patients with a CD36 deficiency was significantly higher than that in normal controls (1.28+/-0.35 versus 0.43+/-0.22; P< 0.01). CONCLUSION: CD36 functions as a major myocardial LFA transporter and its absence may cause a compensatory upregulation of myocardial glucose uptake.  (+info)

(5/36125) Proliferation and differentiation of rat theca-interstitial cells: comparison of effects induced by platelet-derived growth factor and insulin-like growth factor-I.

This study was designed to evaluate mechanisms regulating proliferation of steroidogenically active and steroidogenically inactive theca-interstitial (T-I) cells, and, specifically, to evaluate the effects of platelet-derived growth factor (PDGF) and insulin-like growth factor-I (IGF-I). T-I cells obtained from immature Sprague-Dawley rats were cultured in chemically defined media. Proliferation was assayed by thymidine incorporation and cell counting. Steroidogenically active cells were identified by the presence of 3beta-hydroxysteroid dehydrogenase activity. Flow cytometry facilitated separation of dividing cells (in S and G2/M phases of the cell cycle) from nondividing cells (in G0 and G1 phases of the cell cycle). PDGF alone (0.1-1 nM) produced a dose-dependent increase in DNA synthesis by up to 136%. IGF-I alone (10 nM) increased DNA synthesis by 56%. In the presence of both IGF-I (10 nM) and PDGF (0.1-1 nM), DNA synthesis increased by 108-214%. PDGF (1 nM) increased the total number of T-I cells by 43%; this effect was due to an increase in the number of steroidogenically inactive cells (47%). In contrast, the stimulatory effect of IGF-I (10 nM) was predominantly due to an increase in the number of steroidogenically active cells (163%). Separation of dividing cells from nondividing cells was accomplished with the aid of flow cytometry. In the absence of growth factors, the proportion of steroidogenically active cells was 35% lower among proliferating than resting cells. PDGF (1 nM) decreased the proportion of steroidogenically active cells among both proliferating and resting cells (by 43% and 16%, respectively). In contrast, IGF-I (10 nM) increased the proportion of steroidogenically active cells among proliferating cells by 56%. These findings indicate that differentiated/steroidogenically active cells divide; furthermore, PDGF and IGF-I may selectively stimulate proliferation of individual subpopulations of T-I cells, thereby providing a mechanism for development of structural and steroidogenically active components of the T-I compartment.  (+info)

(6/36125) Use of RhD fusion protein expressed on K562 cell surface in the study of molecular basis for D antigenic epitopes.

The human D antigens, one of the most clinically important blood groups, are presented by RhD protein with a putative 12 transmembrane topology. To understand the molecular basis for the complex antigenic profile of RhD protein, we expressed a series of RhD fusion proteins using different portions of Duffy protein as a tag in erythroleukemic K562 cells. Because the reactivity of monoclonal anti-RhD antibody, LOR15C9, depends mainly on the sequence coded by exon 7 of RhD, we altered DNA sequence corresponding to the amino acid residues 323-331(A) and 350-354(B) in the exon 7. The mutation in region B resulted in a severe reduction in LOR15C9 binding by flow cytometry analysis, suggesting that region B may play an important role in constituting antigen epitopes recognized by LOR15C9. On the other hand, a slight decrease in the antibody binding was observed for the region A mutant, suggesting that the intracellularly located region A may elicit a long distance effect on the formation of exofacial antigen epitopes. In addition, using various monoclonal antibodies against RhD, we compared the antigenic profile of expressed RhD fusion protein with that of endogenous RhD in K562 cells as well as in erythrocytes.  (+info)

(7/36125) Phenotypic and functional characterisation of myofibroblasts, macrophages, and lymphocytes migrating out of the human gastric lamina propria following the loss of epithelial cells.

BACKGROUND: The basement membrane of human colonic mucosa contains numerous discrete pores. We have recently shown that following loss of the surface epithelium, many cells migrate out of the colonic lamina propria via basement membrane pores. AIMS: To characterise cells migrating out via basement membrane pores of the human gastric lamina propria, following loss of the surface epithelium. METHODS: Fresh human gastric mucosal samples were completely denuded of epithelial cells and placed in culture. Tissue samples were studied by electron microscopy (EM) and cells by EM, FACS analysis, immunohistochemistry, and reverse transcription polymerase chain reaction (RT-PCR). RESULTS: EM showed numerous discrete pores (0. 65-8.29 microm in diameter) in the subepithelial basement membrane. During culture of mucosal samples denuded of epithelial cells, lymphocytes, macrophages, and myofibroblasts migrated out of the lamina propria via the basement membrane pores. The lymphocytes were predominantly CD45RO+ and CD69+ T cells. Macrophages were shown to express cyclooxygenase (COX) 1 and 2 enzymes. Myofibroblasts were established in culture and, despite prolonged culture and passage, retained their phenotype. They expressed mRNA and protein for COX 1 and 2 enzymes and their release of prostaglandin E2 was inhibited by selective COX 1 and 2 inhibitors. CONCLUSIONS: Lamina propria cells migrating out of cultured denuded gastric mucosal samples have been characterised phenotypically and functionally. Such cells would be suitable for studies of their interactions with epithelial cells and also with Helicobacter pylori and its products.  (+info)

(8/36125) Adenovirus mediated p53 tumour suppressor gene therapy for human gastric cancer cells in vitro and in vivo.

BACKGROUND/AIMS: Gastric cancer is one of the most prevalent forms of cancer in East Asia. Point mutation of the p53 gene has been reported in more than 60% of cases of gastric cancer and can lead to genetic instability and uncontrolled cell proliferation. The purpose of this investigation was to evaluate the potential of p53 gene therapy for gastric cancer. METHODS: The responses of human gastric cancer cell lines, MKN1, MKN7, MKN28, MKN45, and TMK-1, to recombinant adenoviruses encoding wild type p53 (AdCAp53) were analysed in vitro. The efficacy of the AdCAp53 treatment for MKN1 and MKN45 subcutaneous tumours in nude mice was assessed in vivo. RESULTS: p53-specific growth inhibition was observed in vitro in two of four gastric cancer cell lines with mutated p53, but not in the wild type p53 cell line. The mechanism of the killing of gastric cancer cells by AdCAp53 was found, by flow cytometric analysis and detection of DNA fragmentation, to be apoptosis. In vivo studies showed that the growth of subcutaneous tumours of p53 mutant MKN1 cells was significantly inhibited by direct injection of AdCAp53, but no significant growth inhibition was detected in the growth of p53 wild type MKN45 tumours. CONCLUSIONS: Adenovirus mediated reintroduction of wild type p53 is a potential clinical utility in gene therapy for gastric cancers.  (+info)