The effect on the ear of late closure of the cleft hard palate

Thirty-three patients with cleft palate are reviewed. The late effects on the ear of delayed closure of the hard palate after early soft palate repair are compared with the results of simultaneous early closure of both hard and soft palate clefts. Delayed closure of the hard palate does not affect the incidence of otitis media, the number of insertions of ventilation tubes or the findings on otoscopy. The prevalence of sensorineural hearing loss is significantly raised by late closure of the hard palate. The reason for this is unknown.     

Freeze-fracture morphology and quantification of human bronchial epithelial tight junctions.

A comprehensive investigation of the morphology of human airway epithelial tight junctions was carried out by freeze-fracture electron microscopy using quantitative methods designed to analyze a range of junctional characteristics. Extrapulmonary bronchi that appeared grossly normal were taken at sites distant from tumor in lungs resected for pulmonary carcinoma. The absence of cellular atypia in the samples was confirmed by histology. Airway levels I (main bronchus; n = 7 subjects) and II (lobar bronchus; n = 5 subjects) were compared with respect to junctional depth, strand number, and junctional complexity. Junctional complexity was assessed by frequency of strand interconnection and numbers of strands per interconnection. Comparisons between airway levels I and II for these parameters showed that there were no significant differences in strand number or junctional complexity between the two airway levels. However, junctional depth was slightly but significantly reduced at level II compared with level I (P less than 0.01). The arrangement of strands varied considerably from one junction to the next, irrespective of the cell types involved. "Parallel" and "network" patterns of junctions were observed; the existence of gradations between these two patterns indicated that they represent opposite extremes of a single junctional form rather than distinct categories of junction. These results have allowed us to establish a data pool for normal human bronchi from which the structure of epithelial cell junctions in bronchial diseases can be compared.     

Antigen-induced tolerance by intrathymic modulation of self-recognizing inhibitory receptors

CD1d-restricted invariant natural killer T cell (iNKT cells) have a limited T cell receptor (TCR) repertoire and share characteristics common to T cells and natural killer cells. While intrathymic selection facilitates the production of T cells carrying self major histocompatibility complex-restricted TCRs, natural killer cells carry an appropriate repertoire of self major histocompatibility complex-recognizing receptors to avoid self-reactivity. Here we show that chronic exposure to specific glycolipid antigen resulted in iNKT cell disappearance and thymus-dependent repopulation of iNKT cells with increased expression of inhibitory Ly-49 molecules that resulted in impaired responsiveness. Thymic selection of peripheral Ly-49-expressing iNKT cell repertoire inhibited cytokine production and other functions in vivo. These observations emphasize the acquisition of self-recognizing inhibitory receptors on NKT cells as a previously unknown mechanism of thymic tolerance after chronic antigen exposure.     

Characterization of lymphokine fibronectin from guinea pig lymphoid cell culture supernatants.

Fibronectins (FN) in guinea pig lymphoid cell culture supernatants have been studied using a panel of polyclonal and monoclonal anti-FN antibodies to clarify their relationship with macrophage agglutination factor (MAggF), an inflammatory lymphokine sharing many properties with this family of high molecular weight glycoproteins. MAggF contained cellular FN epitopes, and was reversibly bound by antibodies specific for cellular FN. Enzyme-linked immunoassay and inhibition of MAggF activity by monoclonal anti-plasma FN antibodies revealed immunoreactive FN in guinea pig lymphoid cell culture supernatants to share three epitopes with plasma FN and to lack a fourth epitope present in plasma FN. Immunoreactive FN in gelatin-affinity purified lymph node cell culture supernatants was polydisperse; MAggF activity (Mr 410 kD) was associated with only 13% of total immunoreactive FN. Although a low molecular weight FN fragment (Mr 67 kD) was associated with MAggF activity in salt-fractionated peritoneal exudate culture supernatants, it was not possible to generate MAggF activity by limited proteolysis of MAggF-inactive, high molecular weight FN in lymph node cell culture supernatants. We conclude that MAggF is a cellular FN containing a number of epitopes in common with plasma FN and suggest it may be a unique species of cellular FN produced by T lymphocytes involved in initiating delayed hypersensitivity reactions.     

Modulation of Borrelia burgdorferi stringent response and gene expression during extracellular growth with tick cells

Borrelia burgdorferi N40 multiplied extracellularly when it was cocultured with tick cells in L15BS medium, a medium which by itself did not support B. burgdorferi N40 growth. Growth of B. burgdorferi N40 in the presence of tick cells was associated with decreased production of (p)ppGpp, the stringent response global regulator, a fourfold decrease in relA/spoT mRNA, an eightfold net decrease in bmpD mRNA, and a fourfold increase in rpsL-bmpD mRNA compared to growth of B. burgdorferi in BSK-H medium. As a result, the polycistronic rpsL-bmpD mRNA level increased from 3 to 100% of the total bmpD message. These observations demonstrate that there are reciprocal interactions between B. burgdorferi and tick cells in vitro and indicate that the starvation-associated stringent response mediated by (p)ppGpp present in B. burgdorferi growing in BSK-H medium is ameliorated in B. burgdorferi growing in coculture with tick cell lines. These results suggest that this system can provide a useful model for identifying genes controlling interactions of B. burgdorferi with tick cells in vitro when it is coupled with genetic methods to isolate and complement B. burgdorferi mutants.     

Role of B70/B7-2 in CD4+ T-cell immune responses induced by dendritic cells.

Dendritic cells (DC) are potent antigen-presenting cells (APC). However, the molecular basis underlying this activity remains incompletely understood. To address this question, we generated murine monoclonal antibodies (mAb) against human peripheral blood-derived DC. One such antibody, designated IT209, stained differentiated DC and adherent monocytes, but failed to stain freshly isolated peripheral blood mononuclear cells (PBMC). The antigen recognized by IT209 was identified as B70 (B7-2; also recently identified as CD86). Using this mAb we studied the role of B70 in CD4+ T-cell activation by DC in vitro. IT209 partly inhibited the proliferative response of CD4+ T cells to allogeneic DC and to recall antigens, such as tetanus toxoid (TT) and purified protein derivative (PPD) of tuberculin, presented by autologous DC. More importantly, the mAb had a potent inhibitory effect on the primary response of CD4+ T cells to autologous DC pulsed with human immunodeficiency virus (HIV) gp160 or keyhole limpet haemocyanin (KLH). Adherent monocytes, despite their expression of B70, failed to induce T-cell responses to these antigens. IT209-mediated inhibition of CD4+ T-cell responses was equivalent to that produced by anti-CD25 mAb, whereas an anti-CD80 mAb was only marginally inhibitory and did not augment the effect of IT209. These findings indicate that the B70 antigen plays an important role in DC-dependent CD4+ T-cell activation, particularly in the induction of primary CD4+ T-cell responses to soluble antigens. However, since activated monocytes, despite their expression of B70, failed to prime naive T cells to these antigens, our results suggest that additional molecules contribute to the functions of DC in CD4+ T-cell activation.     

Delivery of a hammerhead ribozyme specifically down-regulates the production of fibrillin-1 by cultured dermal fibroblasts

The hammerhead ribozyme is a small catalytic RNA molecule. Potential hammerhead ribozymes that possess a catalytic domain and flanking sequence complementary to a target mRNA can cleave in trans at a putative cleavage site within the target molecule. We have investigated the potential of hammerhead ribozymes to down-regulate the product of the fibrillin-1 gene (FBN1). Fibrillin is a 347 kDa glycoprotein that is a major constituent of the elastin-associated microfibrils. Mutations in the FBN1 gene are responsible for Marfan syndrome (MFS), a common systemic disorder of the connective tissue. Many FBN1 mutations responsible for MFS appear to act in a dominant-negative fashion, raising the possibility that reduction of the amount of product from the mutant FBN1 allele might be a valid therapeutic approach for MFS. A trans-acting hammerhead ribozyme (FBN1-RZ1) targeted to the 5' end of the human FBN1 mRNA has been designed and synthesized, and shown to cleave its target efficiently in vitro. FBN1-RZ1 cleavage is magnesium dependent and efficient at both 37 and 50 degrees C. Delivery of the FBN1-RZ1 ribozyme into cultured dermal fibroblasts, by receptor- mediated endocytosis of a ribozyme-transferrin-polylysine complex, specifically reduces both cellular FBN1 mRNA and the deposition of fibrillin in the extracellular matrix. These results suggest that the use of hammerhead ribozymes is a valid approach to the study of fibrillin gene expression and possibly to the development of a therapeutic approach to MFS.