Epithelial sheet movement: effects of tunicamycin on migration and glycoprotein synthesis

Corneas with central epithelial wounds, 3 mm in diameter, were organ cultured in the presence of tunicamycin (TM) (1 microgram/ml), an antibiotic that inhibits glycosylation of asparagine-linked glycoproteins. Compared with control corneas, which healed in 22 hr, corneas cultured in the presence of TM for the entire culture time or for only the first 6 hr displayed a progressively slower epithelial healing rate that essentially dropped to zero by 24 hr of culture time. At 24 hr, approximately 75% of the wound was covered. After repeated washings with TM-free culture media (6X, 10 min each), this effect could consistently be reversed in corneas exposed to TM for 6 hr. Incorporation of [3H]glucosamine into trichloroacetic acid-precipitable proteins of migrating epithelial sheets was reduced to 14% that of controls after 12 hr of culture with TM, whereas [14C]leucine incorporation was not significantly affected. The decreased glycosylation was reflected on the cell surface after 12 and 20 hr culture in the presence of TM: apical cell membranes of the first six cells of the leading edge of the migrating sheet bound significantly fewer ferritin-concanavalin A particles per micrometer of membrane than did controls. These results indicate that synthesis of asparagine-linked glycoproteins is required for continued migration of corneal epithelial sheets. The asparagine-linked glycoproteins that are required for migration probably include cell-surface glycoproteins.     

PET-CT在非完全实性肺结节中的应用价值

肺癌在中国恶性肿瘤的发病率位居第一,随着低剂量薄层CT在肺癌筛查中的广泛应用,临床发现更多表现为非完全实性结节的肺腺癌,目前众多研究使CT影像学特征和肺腺癌病理的关系得到更进一步的认知,虽然CT能对部分非完全实性结节做出定性和定位诊断,但仍有部分非完全实性结节诊断困难,PET-CT结合了病灶的代谢信息和精确的定位信息,从而提高对肺部结节诊断的敏感性、特异性、准确性,综合多个文献PET-CT在非完全实性结节中的诊断分期价值较CT无明显提升,却在评估预后和制定合适手术方案上可以起到一定的作用,本文就PET-CT在SSN中的应用价值进行阐述。     

Basement membrane components in healing rabbit corneal epithelial wounds: immunofluorescence and ultrastructural studies

The nature of the substrate that supports epithelial migration in vivo is of interest, particularly with respect to mechanisms of wound healing. Immunofluorescence and electron microscopy were used to search for common substrate components in prototype rabbit corneal wounds: epithelial scrape wounds, in which the corneal or conjunctival epithelium migrated over the denuded lamina densa of the corneal basement membrane (CBM), and superficial keratectomy, in which the corneal epithelium migrated over a bare stroma without CBM. The corneal epithelium moved rapidly over the CBM or stroma to cover the defect within 2-3 d, whereas the conjunctival epithelium required 1-2 wk. In all wounds, fibronectin and fibrin/fibrinogen were deposited onto the bare surface within 8 h after wounding and persisted under the migrating epithelium until migration was complete. Bullous pemphigoid antigen (BPA), a normal component of the CBM, was removed with the epithelium upon scrape wounding and reappeared in the CBM after migration was completed. In contrast, the conjunctival epithelium had a continuous subepithelial band of BPA out to the migrating tip. Laminin, also a normal component of the CBM, was not removed in the scrape wounds, indicating that the region of least resistance to shear stress was between the BPA and laminin layers. Laminin was removed by superficial keratectomy and was not detectable under the leading edge of the migrating cells. Laminin and BPA were restored in the CBM by 2-4 wk. Type IV collagen could not be detected in normal CBM, but was conspicuously present in conjunctival basement membrane and in blood vessels. Focal bands of type IV collagen did appear in the newly synthesized CBM 2-4 wk after keratectomy. These results argue that BPA, laminin, and type IV collagen are not essential for the migration of corneal epithelium during wound healing and support the hypothesis that fibronectin and fibrin/fibrinogen are the common, perhaps the essential, components of the provisional matrix that serves as a substrate until the permanent attachment components are regenerated.     

Hemidesmosome formation in vitro

Intact, viable sheets of adult rabbit corneal epithelium, 9 mm in diameter, were prepared by the Dispase II method (Gipson, I. K., and S. M. Grill, 1982, Invest. Ophthalmol. Vis. Sci. 23:269-273). The sheets, freed of the basal lamina, retained their desmosomes and stratified epithelial characteristics, but lacked hemidesmosomes (HD). Epithelial sheets were placed on fresh segments of corneal stroma with denuded basal laminae and incubated in serum-free media for 1, 3, 6, 18, or 24 h. Tissue was processed for electron microscopy, and the number of HD/micron membrane, the number of HDs with anchoring fibrils directly across the lamina densa from them, and the number of anchoring fibrils not associated with HDs were counted. After 6 h in culture, the number of newly formed HD was 82% of controls (normal rabbit corneas), and by 24 h the number had reached 95% of controls. At all time periods studied, 80-86% of HDs had anchoring fibrils directly across the lamina densa from them. Anchoring fibrils not associated with HDs decreased with culture time. These data indicate that the sites where anchoring fibrils insert into the lamina densa may be nucleation sites for new HD formation. Corneal epithelial sheets placed on two other ocular basal laminae, Descemet's membrane and lens capsule, had not formed HDs after 24 h in culture. These two laminae do not have anchoring fibrils associated with them. Rabbit epithelial sheets placed on the denuded epithelial basal lamina of rat and human corneas formed new HDs. Thus, at least in these mammalian species, HD formation may involve some of the same molecular components.     

Lectin binding to cell surfaces: comparisons between normal and migrating corneal epithelium

Comparisons were made between cell surfaces of normal and migrating corneal epithelium of the rat by localizing and/or quantifying concanavalin A (Con A) and wheat germ agglutinin (WGA) binding. Our results indicate that apical cell surfaces of the leading edge of a migrating sheet of epithelium differ from those of normal epithelium and that the various cell layers within the stratified normal epithelium have different lectin-binding characteristics. Three methods of monitoring lectin binding to cell surfaces were employed. Based on ferritin-conjugated Con A, ferritin-conjugated WGA, and [3H]Con A binding, apical cell membranes of migrating epithelia bind more Con A and WGA than do apical membranes of superficial cells of normal stratified epithelia. With both fluorescein isothiocyanate (FITC)-Con A and -WGA, membranes of all the cells of the leading edge of the migrating sheet fluoresce intensely. FITC-Con A binding of normal stratified epithelium is relatively uniform through all cell layers with no discernible staining of the apical membrane of superficial cells. With FITC-WGA, however, fluorescence is present only on basal cell layers but not on superficial cells. These data demonstrate that apical cell surface sugars on a sheet of epithelium migrating to cover a wound differ from the apical cell surface sugars of normal epithelium. As indicated by FITC-WGA binding, cells of the migrating sheet have cell surface characteristics similar to basal cells of normal epithelia. Perhaps, upon wounding, the leading edge of the migrating sheet is derived from the basal cell population of the normal stratified epithelium, or perhaps there is an alteration in cell surface glycoproteins as the cells become migratory.     

Enhancement of vinculin synthesis by migrating stratified squamous epithelium

A 110-115-kD protein is present at levels 27-fold higher in migratory epithelium in the rat cornea than in stationary epithelium. This protein represents 2.7% of the total protein in migratory epithelium 6-h postabrasion wound and 0.1% of the total protein in stationary epithelium. Our findings demonstrate that this 110-115-kD protein is vinculin. In Western blots comparing proteins from migratory and control epithelium, antibody against vinculin cross-reacted with the 110-115-kD protein. Using immunoslot blots, vinculin was determined to be present at maximal levels 6 h postabrasion wound, at levels 22- and 8-fold higher than control at 18 and 48 h, respectively, returning to control levels 72 h postwounding. Vinculin was also localized by indirect immunohistochemistry in migrating corneal epithelium. 3-mm scrape wounds were allowed to heal in vivo for 20 h. In flat mounts of these whole wounded corneas, vinculin was localized as punctate spots in the leading edge of migrating epithelium. In cryostat sections, vinculin was localized as punctate spots along the basal cell membranes of the migrating sheet adjacent to the basement membrane and in patches between cells as well as diffusely throughout the cell. Only very diffuse localization with occasional punctate spots between adjacent superficial cells was present in stationary epithelium. The increased synthesis of vinculin during migration and the localization of vinculin at the leading edge of migratory epithelium suggest that vinculin may be involved in cell-cell and cell-substrate adhesion as the sheet of epithelium migrates to cover a wound.     

Somatoform disorders among patients attending walk-in clinics in Trinidad: prevalence and association with depression and anxiety

Objectives Somatoform disorders are common in international primary care settings, but have been little studied in the developing world. The objective of this study was to determine the prevalence of severe undifferentiated somatoform disorder, and its relationship to depression and anxiety, among patients attending walk-in clinics in Trinidad.Methods The study participants, who were all aged 18 years or older and attending walk-in clinics at 16 randomly selected health centres, were surveyed between May and August 2007 using the PRIME-MD questionnaire.Results There were 594 participants (the response rate was 92%), of whom 72.7% were female. Their ages ranged from 18 to 93 years, and 54.5% were over 50 years of age. In total, 37.2% were married and 25.9% were single. Indo-Trinidadians represented 43.1% and Afro-Trinidadians represented 36% of the study sample; 56.5% of the participants reported that their income was less than US$ 400 per month, and 65.7% were unemployed. At walk-in clinics in Trinidad, the estimated prevalence of severe undifferentiated somatoform disorder was 10.3% (95% CI: 7.86–12.74), that of hypochondriasis was 28.5% (95% CI: 24.9–32.1), and that of body dysmorphic disorder was 15.8% (95% CI: 11.9–18.7). Severe undifferentiated somatoform disorder was statistically significantly associated with gender and ethnicity but not with age, level of education, employment status or income. Chi-square testing found significant associations between the presence of severe undifferentiated somatoform disorder and both depression and anxiety (P < 0.05), between hypochondriasis and both anxiety and depression (P < 0.05), and between body dysmorphic disorder and depression (P < 0.05) but not anxiety. Regression analysis suggested that the demographic features that predicted severe undifferentiated somatoform disorder were being female or Indo-Trinidadian.Conclusions Walk-in clinics in Trinidad that serve older patients on a lower income have a high proportion of patients with somatoform disorders as measured by the PRIME-MD scale. These patients exhibit many features of anxiety and depression. These findings have implications for medical training and service delivery.     

Analysis of the K+ Current Profile of Mature Rat Oligodendrocytes in situ

Previous studies have reported that mature oligodendrocytes (OLGs) in vitro display various voltage-dependent K+ currents while in situ OLGs show only voltage-independent K+ currents. Given this discrepancy and the lack of information on myelinating OLG ion channel expression in situ, we characterized mature OLG currents in myelinating corpus callosum slices from 17 to 36-day old rats. OLGs were recorded in cell-attached and whole-cell patch-clamp configurations, displayed morphology typical of mature OLGs, and stained positive for myelin basic protein. OLGs displayed large voltage-independent currents that decayed during the voltage pulse and small voltage-activated outward currents. The latter were blocked by TEA, activated between -40 and -50 mV, and decayed slowly. The former were composed of large voltage-independent, time-dependent Ba2+ (1 mM)-sensitive currents, and voltage-dependent Cs+ (5 mM) and Ba2+ (100 mM)-sensitive currents that reversed near the K+ equilibrium potential and inactivated at hyperpolarized potentials, identifying them as inwardly rectifying K+ currents. Inwardly rectifying single-channel K+currents could be recorded in the cell-attached configuration. The estimated single-channel slope conductance was 30 pS. The steady-state open probability was voltage-dependent and declined from 0.9 to 0.5 between -80 and -150 mV. Overall, mature OLGs in situ possess time- and also voltage-dependent K+ currents, which may facilitate clearance of K+ released during axonal firing.     

Expression of human complement receptor type 2 (CD21) in mice during early B cell development results in a reduction in mature B cells and hypogammaglobulinemia

Complement receptor (CR) type 2 (CR2/CD21) is normally expressed only during the immature and mature stages of B cell development. In association with CD19, CR2 plays an important role in enhancing mature B cell responses to foreign Ag. We used a murine Vlambda2 promoter/Vlambda2-4 enhancer minigene to develop transgenic mice that initiate expression of human CR2 (hCR2) during the CD43(+)CD25(-) late pro-B cell stage of development. We found peripheral blood B cell numbers reduced by 60% in mice expressing high levels of hCR2 and by 15% in mice with intermediate receptor expression. Splenic B cell populations were altered with an expansion of marginal zone cells, and basal serum IgG levels as well as T-dependent immune responses were also significantly decreased in transgenic mice. Mice expressing the highest levels of hCR2 demonstrated in the bone marrow a slight increase in B220(int)CD43(+)CD25(-) B cells in association with a substantial decrease in immature and mature B cells, indicative of a developmental block in the pro-B cell stage. These data demonstrate that stage-specific expression of CR2 is necessary for normal B cell development, as premature receptor expression substantially alters this process. Alterations in B cell development are most likely due to engagement of pre-B cell receptor-mediated or other regulatory pathways by hCR2 in a CD19- and possibly C3 ligand-dependent manner.