Epithelial Basal Lamina Research Articles

Laminin-332 γ2 Monomeric Chain Promotes Adhesion and Migration of Hepatocellular Carcinoma Cells.

Extracellular matrix (ECM) has a well-recognized impact on the progression of solid tumors, including hepatocellular carcinoma (HCC). Laminin 332 (Ln332) is a ECM molecule of epithelial basal lamina, composed of three polypeptide chains (α3, β3, and γ2), that is usually poorly expressed in the normal liver but is detected at high levels in HCC. This macromolecule was shown to promote the proliferation, epithelial-to-mesenchymal transition (EMT), and drug resistance of HCC cells. The monomeric γ2 chain is up-regulated and localized preferentially at the invasive edge of metastatic intrahepatic HCC nodules, suggesting its potential involvement in the acquisition of invasive properties of HCC cells. HCC cells were tested in in vitro adhesion, scattering, and transwell migration assays in response to fibronectin and the Ln332 and Ln332 γ2 chains, and the activation status of major signaling pathways involved was evaluated. Here, we show that the Ln332 γ2 chain promotes HCC the cell adhesion, migration, and scattering of HCC cells that express the Ln332 receptor α3β1 integrin, proving to be a causal factor of the EMT program achievement. Moreover, we found that efficient HCC cell adhesion and migration on γ2 require the activation of the small cytosolic GTPase Rac1 and ERKs signaling. These data suggest that the γ2 chain, independently from the full-length Ln332, can contribute to the pro-invasive potential of aggressive HCC cell subpopulations.

Formation of free oocysts in Anopheles mosquitoes injected with Plasmodium ookinetes.

Malaria needs new strategies for its control. Plasmodium spp., the causative agent of malaria, is transmitted by mosquitoes. These parasites develop into oocysts and sporozoites in the body of the mosquitoes. A deeper understanding of oocysts that produce the infectious form of the parasite, sporozoites, can facilitate the development of novel countermeasures. However, the isolation of Plasmodium oocysts is challenging as these are formed between midgut epithelial cells and basal lamina after gametocytes enter the mosquito's body through blood feeding. Further research on oocysts has been impeded by issues related to oocyst isolation. Therefore, in this study, we injected Plasmodium into mosquitoes-an artificial and unique method-and aimed to clarify how oocysts were formed in mosquitoes after Plasmodium injection and whether free oocysts were formed from the mosquito tissue. Plasmodium berghei (ANKA strain) ookinetes cultured in vitro were injected into the thoracic body cavity (hemocoel) of female and male Anopheles stephensi mosquitoes. Oocysts were formed in the body of female and male mosquitoes at 14 days post injection. In addition, oocysts formed as a result of injection developed into sporozoites, which were infectious to mice. These findings suggest that P. berghei can complete its developmental stage in mosquitoes by injection. Some of the oocysts formed were free from mosquito tissue, and it was possible to collect oocysts with minimal contamination of mosquito tissue. These free oocysts can be used for investigating oocyst proteins and metabolism.

Renal adaptation in relation to insectivorous feeding habit in the greater mouse-eared bat, Myotis myotis (Chiroptera: Vespertilionidae).

We examined the histological structure of the kidneys of Myotis myotis to better understand their structural adaptations to dietary habits. M. myotis is an insectivorous bat species that belongs to the family Vespertilionidae. The kidneys of M. myotis are unilobular, bean-shaped, and surrounded by a renal capsule. The two parts are distinguished by a thin cortex and a thicker medulla. Renal corpuscles consist of the glomerulus and Bowman's capsule. The proximal tubule consists of cubic cells with a well-developed brush border, whereas the distal tubule is lined with a simple cubic epithelium without a brush border. The Henle's loop, located in the medullary region, was composed of flat cells. The microvilli of proximal tubule epithelial cells and basal lamina of proximal and distal tubule epithelial cells were periodic acid Schiff (PAS)-positive. The PAS-positive reaction of the microvilli of proximal tubular epithelial cells and basal lamina of proximal and distal tubule epithelial cells is due to the presence of glycogen, which may be used as an energy substrate during absorption. The presence of acidic glycoconjugates in the papilla was demonstrated by Alcian blue (pH2.5)-PAS staining. According to the result of silver impregnation staining, it was determined that reticular fibers form a dense fibrillary network in the kidney parenchymal tissue. Reticular fibers are responsible for demarcating and supporting the borders of cells by forming a thin network of fibrils beneath the basal lamina of the cells. Structural features in the kidney, such as a thin cortex and thicker medulla, long conical papilla, and division of the thick medulla into inner and outer regions, of M. myotis may be an adaptation to produce concentrated urine, thereby reducing water loss associated with insectivorous feeding habits.

Diet-related changes of basal lamina fenestrations in the villous epithelium of the rat small intestine: Statistical analysis on scanning electron microscopy.

The epithelial basal lamina of the small intestine has numerous fenestrations for intraepithelial migration of leukocytes. We have reported dynamic changes of fenestrations in dietary conditions. To investigate this phenomenon, we performed statistical analyses using scanning electron microscopy images of the epithelial basal lamina of rat intestinal villi after removal of the villous epithelium by osmium maceration. We examined structural changes in the number and size of fenestrations in the rat jejunum and ileum under fasted and fed states for 24 h. Our findings revealed that, in the jejunum, the number of free cells migrating into the epithelium through fenestrations increased from 2 h after feeding, resulting in an increase in the fenestration size of intestinal villi; the number of free cells then tended to decrease at 6 h after feeding, and the fenestration size also gradually decreased. By contrast, the increase in the fenestration size by feeding was not statistically significant in the ileum. These findings indicate that the number of migrating cells increases in the upper part of the small intestine under dietary conditions, which may influence the absorption efficiency of nutrients including lipids, as well as the induction of nutrient-induced inflammation.

Observations on nascent matrix structures in embryonic cornea: Important in cell interactions, or merely vestiges of the lens surface?

Here we present some new observations on early stages in chick corneal development obtained by remining of datasets obtained via serial block face scanning electron microscopy. We focus on matrix cords, proteoglycan-rich structures of apparent ectodermal origin, emerging from the epithelial basal lamina, which extend proximally into the growing collagenous matrix destined to become the corneal stroma. Cords have no known function. In their earliest manifestation, we describe how they appear to run continuously from epithelium to the lens, in contact with both tissues and may therefore be simply vestigial structures, remaining from the earlier detachment of the lens from its parent ectoderm. However, neural crest cells migrating to form the corneal endothelial monolayer appear to form close associations with cords via elaborate pseudopodial extensions. Presumptive endothelium and keratocytes, in the subsequent wave of neural crest cell influx, may conceivably utilise cords, as well as utilising collagenous fibrils of the interstitial matrix, as substrate cues in cell guidance, attachment and migration. The possibility also exists that cords fulfil a functional role in corneal morphogenesis via mechanotransduction through cell matrix interactions.

Serial block-face scanning electron microscopy reveals neuronal-epithelial cell fusion in the mouse cornea.

The cornea is the most highly innervated tissue in the body. It is generally accepted that corneal stromal nerves penetrate the epithelial basal lamina giving rise to intra-epithelial nerves. During the course of a study wherein we imaged corneal nerves in mice, we observed a novel neuronal-epithelial cell interaction whereby nerves approaching the epithelium in the cornea fused with basal epithelial cells, such that their plasma membranes were continuous and the neuronal axoplasm freely abutted the epithelial cytoplasm. In this study we sought to determine the frequency, distribution, and morphological profile of neuronal-epithelial cell fusion events within the cornea. Serial electron microscopy images were obtained from the anterior stroma in the paralimbus and central cornea of 8–10 week old C57BL/6J mice. We found evidence of a novel alternative behavior involving a neuronal-epithelial interaction whereby 42.8% of central corneal nerve bundles approaching the epithelium contain axons that fuse with basal epithelial cells. The average surface-to-volume ratio of a penetrating nerve was 3.32, while the average fusing nerve was smaller at 1.39 (p ≤ 0.0001). Despite this, both neuronal-epithelial cell interactions involve similarly sized discontinuities in the basal lamina. In order to verify the plasma membrane continuity between fused neurons and epithelial cells we used the lipophilic membrane tracer DiI. The majority of corneal nerves were labeled with DiI after application to the trigeminal ganglion and, consistent with our ultrastructural observations, fusion sites recognized as DiI-labeled basal epithelial cells were located at points of stromal nerve termination. These studies provide evidence that neuronal-epithelial cell fusion is a cell-cell interaction that occurs primarily in the central cornea, and fusing nerve bundles are morphologically distinct from penetrating nerve bundles. This is, to our knowledge, the first description of neuronal-epithelial cell fusion in the literature adding a new level of complexity to the current understanding of corneal innervation.

Cell-independent matrix configuration in early corneal development

Mechanisms controlling the spatial configuration of the remarkably ordered collagen-rich extracellular matrix of the transparent cornea remain incompletely understood. We previously described the assembly of the emerging corneal matrix in the mid and late stages of embryogenesis and concluded that collagen fibril organisation was driven by cell-directed mechanisms. Here, the early stages of corneal morphogenesis were examined by serial block face scanning electron microscopy of embryonic chick corneas starting at embryonic day three (E3), followed by a Fourier transform analysis of three-dimensional datasets and theoretical considerations of factors that influence matrix formation. Eyes developing normally and eyes that had the lens surgically removed at E3 were studied. Uniformly thin collagen fibrils are deposited by surface ectoderm-derived corneal epithelium in the primary stroma of the developing chick cornea and form an acellular matrix with a striking micro-lamellar orthogonal arrangement. Fourier transform analysis supported this observation and indicated that adjacent micro-lamellae display a clockwise rotation of fibril orientation, depth-wise below the epithelium. We present a model which attempts to explain how, in the absence of cells in the primary stroma, collagen organisation might be influenced by cell-independent, intrinsic mechanisms, such as fibril axial charge derived from associated proteoglycans. On a supra-lamellar scale, fine cords of non-collagenous filamentous matrix were detected over large tissue volumes. These extend into the developing cornea from the epithelial basal lamina and appear to associate with the neural crest cells that migrate inwardly to form, first the corneal endothelium and then keratocytes which synthesise the mature, secondary corneal stroma. In a small number of experimental specimens, matrix cords were present even when periocular neural crest cell migration and corneal morphogenesis had been perturbed following removal of the lens at E3.

Sparkling Reflective Stacks of Purine Crystals in the Nudibranch Flabellina iodinea.

Although pigments contribute to much of the brilliant purple and orange coloration of the aeolid nudibranch Flabellina iodinea, the optical appearance of the animal was found to be augmented by dynamically sparkling, brightly reflective material in cells located throughout its epidermis. Electron microscopy revealed that specialized cells most abundant near the epithelial basal lamina contain numerous multilayer stacks of crystals, each within a fragile membrane capsule. High-resolution light microscopy of tissue sections showed that these crystalline stacks intermittently reflect light, with a temporally dynamic, sparkling appearance, suggesting that they are free to move-a phenomenon also observed in the live, intact whole animal and in the purified crystal stacks as well. Thin-layer chromatography and ultraviolet spectrometry show that the crystals isolated from all epithelial tissues are identical in composition, with guanine being the major component and its derivative, hypoxanthine, a minor component, regardless of the tissue's pigmentary color. Electron diffraction of the crystals purified separately from the orange and purple tissues exhibits nearly identical lattice parameters that closely match those measured for guanine crystals, which are widely distributed in other biophotonic systems ranging from marine invertebrates to terrestrial vertebrates. Heterogeneity of the thickness and spacing of the crystals within their stacks accounts for their broadband silvery reflectance. The optical appearance of the epidermis of this nudibranch thus results from the interaction of incident light with mobile stacks of purine crystals, augmenting the effects of its pigmentary colors.

Dynamics of basal lamina fenestrations in the rat intestinal villous epithelium in response to dietary conditions.

The epithelial basal lamina of the small intestine forms a felt-like sheet at the base of the epithelium. Previous studies have shown that the basal lamina has numerous fenestrations, which are produced by leukocytes penetrating through the basal lamina. In this study, we aimed to directly visualize fenestrations of the rat basal lamina in intestinal villi by scanning electron microscopy (SEM) after removal of the villous epithelium by osmium maceration and ultrasonic treatment. Structural changes in fenestrations were then investigated in relation to dietary conditions. SEM of these tissues revealed the presence of fenestrations in the villous epithelial basal lamina in all segments of the small intestine, although the number was the highest in the jejunum. The present study also showed that the number and size of fenestrations increased after feeding in the jejunum, whereas changes were unclear in the ileum. These findings suggested that the basal lamina fenestrations were changed through the dynamics of migrating leukocytes in dietary conditions and may also be related to the regulation of nutrient absorption, particularly as lipids are transported from the intercellular space of the epithelium to the lamina propria.

Knockdown of laminin α5 stimulates intestinal cell differentiation

Interactions between cells and the extracellular matrix regulate a wide range of cell processes such as proliferation and differentiation. Laminins are major components of the basement membrane that actively participate in most biological functions via their interactions with a variety of specific cell receptors. The α5-containing laminins (LAMA5) are one of the three main types of laminins identified at the epithelial basal lamina in the adult intestine. The aim of the present study was to investigate the role of α5-containing laminins on intestinal cell proliferation and differentiation. Using an shRNA targeting approach, the effects of knocking down the expression of LAMA5 were investigated in the enterocytic-like Caco-2/15 cell line, a well-characterized model for intestinal cell differentiation. Surprisingly, the abolition of the laminin α5 chain resulted in a drastic increase in the differentiation marker sucrase-isomaltase which was correctly expressed at the apical pole of the cells as observed by indirect immunofluorescence. Transient increases of dipeptidylpeptidase IV, villin, CDX2, HNF-1α, HNF-4α and transepithelial resistance as well as an apparent redistribution of the junctional components ZO-1 and E-cadherin were also observed at early stages of differentiation but no specific effect was observed on cell proliferation as evaluated by BrdU incorporation. Taken together, these data suggest that α5-containing laminins repress intestinal differentiation in its early stages.

Spdef Null Mice Lack Conjunctival Goblet Cells and Provide a Model of Dry Eye

Goblet cell numbers decrease within the conjunctival epithelium in drying and cicatrizing ocular surface diseases. Factors regulating goblet cell differentiation in conjunctival epithelium are unknown. Recent data indicate that the transcription factor SAM-pointed domain epithelial-specific transcription factor (Spdef) is essential for goblet cell differentiation in tracheobronchial and gastrointestinal epithelium of mice. Using Spdef(-/-) mice, we determined that Spdef is required for conjunctival goblet cell differentiation and that Spdef(-/-) mice, which lack conjunctival goblet cells, have significantly increased corneal surface fluorescein staining and tear volume, a phenotype consistent with dry eye. Microarray analysis of conjunctival epithelium in Spdef(-/-) mice revealed down-regulation of goblet cell-specific genes (Muc5ac, Tff1, Gcnt3). Up-regulated genes included epithelial cell differentiation/keratinization genes (Sprr2h, Tgm1) and proinflammatory genes (Il1-α, Il-1β, Tnf-α), all of which are up-regulated in dry eye. Interestingly, four Wnt pathway genes were down-regulated. SPDEF expression was significantly decreased in the conjunctival epithelium of Sjögren syndrome patients with dry eye and decreased goblet cell mucin expression. These data demonstrate that Spdef is required for conjunctival goblet cell differentiation and down-regulation of SPDEF may play a role in human dry eye with goblet cell loss. Spdef(-/-) mice have an ocular surface phenotype similar to that in moderate dry eye, providing a new, more convenient model for the disease.

Is the lung a target organ in diabetes?

BACKGROUND The lung is not considered a target organ in diabetes mellitus. In English language literature there are many papers showing the opposite. DISCUSSION Many studies demonstrated a thickened alveolar epithelial and pulmonary capillary basal lamina and a reduced lung elasticity, others showed that these histopathological alterations develop into functional abnormalities such as reduced lung volumes, pulmonary diffusion capacity and elastic recoil. The diabetes related pulmonary disease has not an impact on normal life in otherwise “healthy” patients, but it is possible that in specific pathologic (such as heart, kidney and lung comorbidities) or paraphysiologic (such as physical exercise, smoking or exposure to high altitude) conditions, diabetes could contribute to the clinical manifestation of a restricitive lung disease. On the other hand, diabetic patients have an increased propensity to acquire infections. The prevalence of pulmonary tuberculosis is reported to be four times than in healthy subjects; there is a predilection for the lower lobes and the disease is more aggressive in poorly controlled diabetes mellitus.

Decellularized Ear Tissues as Scaffolds for Stem Cell Differentiation

Permanent sensorineural hearing loss is a major medical problem and is due to the loss of hair cells and subsequently spiral ganglion neurons in the cochlea. Since these cells lack the capacity of renewal in mammals, their regeneration would be an optimal solution to reverse hearing loss. In other tissues, decellularized extracellular matrix (ECM) has been used as a mechanical and biochemical scaffold for the induction of stem and other cells toward a target tissue phenotype. Such induced cells have been used for tissue and organ transplants in preclinical animal and human clinical applications. This paper reports for the first time the decellularization of the cochlea and identification of remaining laminin and collagen type IV as a first step in preparing an ECM scaffold for directing stem cells toward an auditory lineage. Fresh ear tissues were removed from euthanized mice, a rat and a human and processed for decellularization using two different detergent extraction methods. Cochleas were imaged with scanning thin-sheet laser imaging microscopy (sTSLIM) and brightfield microscopy. Detergent treatment of fresh tissue removed all cells as evidenced by lack of H&E and DNA staining of the membranous labyrinth while preserving components of the ECM. The organ of Corti was completely removed, as were spiral ganglion neurons, which appeared as hollow sheaths and tubes of basal lamina (BL) material. Cells of the stria vascularis were removed and its only vestige left was its laterally linking network of capillary BL that appeared to "float" in the endolymphatic space. Laminin and type IV collagen were detected in the ECM after decellularization and were localized in vascular, neural and epithelial BL. Further work is necessary to attempt to seed neural and other stem cells into the decellularized ECM to hopefully induce differentiation and subsequent in vivo engraftment into damaged cochleas.

Improved Corneal Wound Healing through Modulation of Gap Junction Communication Using Connexin43-Specific Antisense Oligodeoxynucleotides

Gap junctions play a major role in corneal wound healing. This study used reproducible models of corneal wound healing to evaluate the effect of a gap junction channel modulator, connexin43 (Cx43) antisense oligodeoxynucleotides (AsODN), on corneal healing dynamics. A mechanical scrape wound model was used to evaluate Cx43 AsODN penetration and initial wound reepithelialization 12 hours postsurgery. Thereafter, detailed analyses of corneal edema, inflammation, and healing were performed in an excimer laser surface ablation model. In vivo confocal microscopy determined clinical parameters (edema, haze) and cellular changes (stromal hypercellularity, reepithelialization), whereas histology and immunohistochemistry were used to quantify stromal edema, inflammation, and reepithelialization. Cx43 AsODN penetrated through the hydrophilic stroma where the epithelium had been removed and accumulated in the basal epithelium close to the wound edge. Twelve hours after scrape wounding, Cx43 AsODN-treated eyes showed a significant reduction in wound area compared with the vehicle alone (1.59±0.37 and 2.29±0.58 mm2, respectively, P<0.01). After excimer laser ablation, stromal edema and inflammation were reduced, with endothelial structures being clearly visible, and reepithelialization rates were again increased in Cx43 AsODN-treated eyes. Histologic analysis confirmed reduced edema in the central wound site and at the periphery of treated corneas (P<0.05), whereas immunohistochemistry showed lower Cx43 levels (P<0.05), reduced myofibroblast activation, and improved epithelial basal lamina deposition in antisense-treated wounds (P<0.01). Application of Cx43 AsODN to the cornea reduces stromal edema and inflammation, promoting faster wound closure and a more uniform repair of the epithelial basal lamina after laser ablation.

Collagen VI is a basement membrane component that regulates epithelial cell–fibronectin interactions

Collagen VI is a heterotrimer composed of three α chains (α1, α2, α3) widely expressed throughout various interstitial matrices. Collagen VI is also found near the basement membranes of many tissues where it serves as an anchoring meshwork. The aim of this study was to investigate the distribution and role of collagen VI at the epithelial–stromal interface in the intestine. Results showed that collagen VI is a bona fide epithelial basal lamina component and constitutes the major collagen type of epithelial origin in this organ. In vitro, collagen VI co-distributes with fibronectin. Targeted knockdown of collagen VI expression in intestinal epithelial cells was used to investigate its function. Depletion of collagen VI from the matrix led to a significant increase in cell spreading and fibrillar adhesion formation coinciding with an upregulation of fibronectin expression, deposition and organization as well as activation of myosin light chain phosphorylation by the myosin light chain kinase and Rho kinase dependent mechanisms. Plating cells deficient for collagen VI on collagen VI rescued the phenotype. Taken together, these data demonstrate that collagen VI is an important basal lamina component involved in the regulation of epithelial cell behavior most notably as a regulator of epithelial cell–fibronectin interactions.

Pancreas Transplantation Prevents Morphologic and Ultrastructural Changes in Pulmonary Parenchyma of Alloxan-Induced Diabetic Rats

Purpose The aim of this study was to evaluate whether pancreas transplantation (PT) is a suitable method for controlling histopathologic changes in lungs of alloxan-induced diabetic rats. Methods Sixty inbred male Lewis rats were randomly assigned to 3 experimental groups: NC, 20 nondiabetic control rats; DC, 20 untreated diabetic control rats; and PT, 20 diabetic rats that received syngeneic PT from normal donor Lewis rats. Each group was further divided into 2 subgroups of 10 rats each, which were killed after 4 and 12 weeks of follow-up. Clinical and laboratory parameters, fresh and fixed lung weights, and fixed lung volumes were recorded for all rats. Total number of alveoli, alveolar perimeter, alveolar surface area, and alveolar epithelial (AE) and endothelial capillary (EC) basal laminae thickening were randomly measured in 5 rats from each subgroup by using an image analyzer. For light microscopy, 250 alveoli were analyzed in each subgroup. For electron microscopy, 50 electron micrographs were examined for each subgroup. Results The DC rats showed elevated blood glucose and glycosylated hemoglobin levels, with insulin blood levels significantly lower than the NC rats ( P < .001). Fresh and fixed lung weights and fixed volumes were significantly reduced in these rats, although their proportions to body weight were increased at 12 weeks ( P < .01). The total number of alveoli in diabetic rats was higher than in control rats, whereas alveolar perimeter and surface area were significantly diminished ( P < .01). AE and EC basal laminae were significantly thicker in DC than in NC ( P < .01). Successful PT corrected all clinical and metabolic changes in diabetic rats, with sustained normoglycemia throughout the study. Morphologic and morphometric changes observed in diabetic lungs were completely prevented in PT rats from 4 weeks after transplant. Conclusion We conclude that PT can control morphologic and ultrastructural changes in pulmonary parenchyma, suggesting a promising perspective for preventing other chronic diabetic lesions.

A morphological study of pulmonary macrophages in bleomycin-injected murine models

Pulmonary changes induced in a murine model by intraperitoneal injections of bleomycin were morphologically studied by light and electron microscopy. The number of pulmonary macrophages and the distribution of fibronectin in these cells were evaluated by acid phosphatase and affinity staining using anti-fibronectin horseradish peroxidase conjugates. The onset of acute inflammation occurred 4 days after intraperitoneal injection of bleomycin and reached its peak on the 14th day post-injection. The inflammatory reaction then gradually decreased. Areas of subpleural fibrosis was observed on day 42. On day 14, the ratio of the macrophage in bleomycin-treated mice to that in control mice was 5:1. Many activated and foamy macrophages were observed at that time. These findings indicate that macrophage turnover activity is remarkably increased during the acute inflammatory phase. Fibronectin was detected in the cytoplasm of macrophages on day 14, 21, and 28. It was also detected in both alveolar capillary and epithelial basal lamina as well as in interstitial collagen fibers. On day 42, fibronectin staining was strongly positive in areas of subpleural fibrosis. These results suggest that fibronectin released from pulmonary macrophages plays a role in the process of pulmonary fibrosis.

The Disruption of the Epithelial Mesenchymal Trophic Unit in COPD

ABSTRACTProgression of COPD is associated with a measurable increase in small airway wall thickness resulting from a repair and remodeling process that involves fibroblasts of the epithelial mesenchymal trophic unit (EMTU). The present study was designed to examine the organization of fibroblasts within the lamina propria of small airways with respect to their contacts with the epithelium and with each other in persons with COPD. Transmission electron microcopy (TEM) and three-dimensional (3D) reconstructions of serial TEM sections were used to estimate the frequency and determine the nature of the contacts between the epithelium and fibroblasts within the EMTU in small airways from 5 controls (smokers with normal lung function), from 6 persons with mild (GOLD-1) and 5 with moderate (GOLD-2) COPD. In airways from control lungs fibroblasts make frequent contact with cytoplasmic extensions of epithelial cells through apertures in the epithelial basal lamina, but the frequency of these fibroblast-epithelial contacts is reduced in both mild and moderate COPD compared to controls (p < 0.01). The 3D reconstructions showed that the cytoplasmic extensions of lamina propria fibroblasts form a reticulum with fibroblast-fibroblast contacts in an airway from a control subject but this reticulum may be reorganized in airways of COPD patients. Development of COPD is associated with significant disruption of the EMTU due to a reduction of contacts between fibroblasts and the epithelium.

Epithelial Dendritic Cell Distribution in Normal and Inflamed Human Cornea: In Vivo Confocal Microscopy Study

To evaluate dendritic cell (DCs) density, distribution, and morphology in central corneal and limbal epithelium in normal subjects and patients with immune-mediated corneal inflammation using in vivo confocal microscopy (IVCM). Comparative case-controlled, observational confocal microscopy study. A total of 135 eyes of 135 patients were investigated. Group 1 (normal eyes) included 45 eyes of 45 healthy volunteers, group 2 photorefractive keratectomy (PRK-treated eyes) included 45 myopic eyes of 45 patients treated with PRK, and group 3 (inflamed eyes) comprised 45 eyes of 45 patients affected by immune-mediated corneal inflammation. The central cornea and limbus were examined for epithelial dendritic-shaped cells using laser scanning IVCM. DCs density was calculated using image analysis software. Cells with a branching dendritic morphology were visualized in the basal epithelial layer, basal lamina, and subbasal nerve plexus, in the central cornea, and in the basal layer and basal membrane of the limbal epithelium. The limbal epithelium demonstrated DCs in 93.3%, 89%, and 97.7% of eyes in group 1, 2, and 3, respectively (P = ns). Central epithelial DCs were observed in 20.0% and 13.3% of eyes in group 1 and 2 (P = ns), while in 93.3% of eyes in group 3 (P < .001). DCs were found to be significantly higher at the limbus compared with central cornea in each group (P < .001). Cell densities observed in group 3 were significantly greater than groups 1 and 2, at both locations (P < .05). Laser scanning IVCM is a useful method for evaluating epithelial DCs distribution at the limbus and central cornea in both healthy and diseased eyes.

Laminin α 3 Forms a Complex with β3 and γ3 Chains That Serves as the Ligand for α 6β1-Integrin at the Apical Ectoplasmic Specialization in Adult Rat Testes

Apical ectoplasmic specialization (ES) is a testis-specific hybrid cell/cell actin-based adherens junction and cell/matrix focal contact anchoring junction type restricted to the interface between Sertoli cells and developing spermatids. Recent studies have shown that laminin gamma3, restricted to elongating spermatids, is a putative binding partner of alpha 6beta 1-integrin localized in Sertoli cells at the apical ES. However, the identity of the alpha and beta chains, which constitute a functional laminin ligand with the gamma3 chain at the apical ES, is not known. Using reverse transcription-PCR and immunoblotting to survey all laminin chains in cells of the seminiferous epithelium, it was noted that alpha 2, alpha 3, beta1, beta2, beta3, and gamma3 chains were found in germ cells, whereas alpha 1, alpha 2, alpha 4, alpha 5, beta1, beta2, gamma1, gamma2, and gamma3 chains were found in Sertoli cells, implying that alpha 3 and beta3 are the plausible laminin chains restricted to germ cells that may be the bona fide partners of gamma3. To verify this postulate, recombinant proteins based on domain G of alpha 3 and domain I of beta3 and gamma3 chains were produced and used to obtain the corresponding specific polyclonal antibodies. Additional studies have demonstrated that the laminin alpha 3, beta3, and gamma3 chains indeed are restricted to germ cells at the apical ES, co-localizing with each other and with beta1-integrin. Furthermore, co-immunoprecipitation studies have confirmed the interactions among laminin alpha 3, beta3, and gamma3, as well as beta1-integrin. When the functional laminin ligand at the apical ES was disrupted via blocking antibodies, such as using anti-laminin alpha 3 or gamma3 IgG, this treatment perturbed adhesion between Sertoli and germ cells (mostly spermatids), leading to germ cell loss from the epithelium. More important, a transient disruption of the blood-testis barrier was also detected.