Superficial Cell Layers Research Articles

Light and electron microscopic analyses of the high deformability of adhesive toe pads in White's tree frog, Litoria caerulea.

White's tree frog (Litoria caerulea) has large, adhesive toe pads that are among the softest of all known biological structures. To explore the morphological basis for the physical properties of the toe pads, the internal microstructure of the toe pads in L. caerulea was examined using both light and transmission electron microscopy. Three design elements that are distinct from other areas of skin were observed. First, the keratinocytes comprising the adhesive surface of the toe pad all contained keratin filament bundles (tonofibrils) exhibiting structural anisotropy. Specifically, the curved conformation of the hierarchical (branching) tonofibrils was characterized by the formation of anastomoses consisting of tonofibrils beneath the adhesive cell surface and stem keratin filament bundles concentrated in the lower-middle part of the dorsal-side of adhesive cells. Second, the cytoplasm of keratinocytes in the most superficial cell layer contained glycoproteins (stained by periodic acid/Schiff reagent) that are considered to confer high viscoelasticity. Third, the dermis contained large lymph spaces interspersed with elastic fibers and collagen fibers, which were relatively sparsely distributed compared to the dorsal skin of the toe pads. The profiles of these structures were easily deformed by the slight application of pressure. These findings reaffirmed that the unique internal architecture of the toe pads in L. caerulea contributed to their remarkable softness and high deformability, which in turn increased the contact area and provided improved adaptability to the local topography of natural surfaces. J. Morphol. 277:1509-1516, 2016. © 2016 Wiley Periodicals, Inc.

Orthokeratinized Odontogenic Cyst with an Associated Keratocystic Odontogenic Tumor Component and Ghost Cell Keratinization and Calcifications in a Patient with Gardner Syndrome

Gardner syndrome (GS) is caused by mutations in the APC and besides adenomatous colorectal polyps includes such manifestations as osteomas, epidermoid cysts (ECs) and occasionally multiple pilomatricomas. More than 50% of ECs in patients with GS exhibit pilomatricoma-like ghost cell keratinization. The latter may be explained by the fact that the development of both GS and pilomatricoma is driven by activation of the Wnt/β-catenin signaling pathway. A 62-year-old, Caucasian male with history of GS presented with a unilocular, mixed radiopaque/radiolucent mandibular lesion causing divergence and external root resorption of involved teeth. Histopathologically, the lesion was composed of two cystic components, an orthokeratinized odontogenic cyst (OOC) and a smaller one with characteristics of keratocystic odontogenic tumor (KCOT) featuring, focally, ghost cells and an epithelial morule-like structure. Dystrophic calcifications essentially similar to those seen in pilomatricomas were observed in the fibrous connective tissue wall. The KCOT and OOC epithelia revealed strong and diffuse cytokeratin (AE1/AE3) and β-catenin immunoreactivity. CD10 positive immunostaining was seen in the keratin and superficial spinous cell layers in both OOC and KCOT. The intraepithelial and mural ghost cells showed a cytokeratin (+), β-catenin and CD10 (-) immunophenotype. The diagnosis of OOC with ghost cell calcifications in association with KCOT was rendered. The patient was lost to follow-up. Although a coincidental co-existence cannot be excluded, ghost cell calcifications mimicking pilomatricoma-like changes in an unusual odontogenic cyst combining OOC and KCOT features as seen in this patient with GS may be explained by the common molecular mechanisms underlying the pathogenesis of cutaneous pilomatricomas and GS.

The global mechanical properties and multi-scale failure mechanics of heterogeneous human stratum corneum.

The outermost layer of skin, or stratum corneum, regulates water loss and protects underlying living tissue from environmental pathogens and insults. With cracking, chapping or the formation of exudative lesions, this functionality is lost. While stratum corneum exhibits well defined global mechanical properties, macroscopic mechanical testing techniques used to measure them ignore the structural heterogeneity of the tissue and cannot provide any mechanistic insight into tissue fracture. As such, a mechanistic understanding of failure in this soft tissue is lacking. This insight is critical to predicting fracture risk associated with age or disease. In this study, we first quantify previously unreported global mechanical properties of isolated stratum corneum including the Poisson's ratio and mechanical toughness. African American breast stratum corneum is used for all assessments. We show these parameters are highly dependent on the ambient humidity to which samples are equilibrated. A multi-scale investigation assessing the influence of structural heterogeneities on the microscale nucleation and propagation of cracks is then performed. At the mesoscale, spatially resolved equivalent strain fields within uniaxially stretched stratum corneum samples exhibit a striking heterogeneity, with localized peaks correlating closely with crack nucleation sites. Subsequent crack propagation pathways follow inherent topographical features in the tissue and lengthen with increased tissue hydration. At the microscale, intact corneocytes and polygonal shaped voids at crack interfaces highlight that cracks propagate in superficial cell layers primarily along intercellular junctions. Cellular fracture does occur however, but is uncommon. Human stratum corneum protects the body against harmful environmental pathogens and insults. Upon mechanical failure, this barrier function is lost. Previous studies characterizing the mechanics of stratum corneum have used macroscopic testing equipment designed for homogenous materials. Such measurements ignore the tissue's rich topography and heterogeneous structure, and cannot describe the underlying mechanistic process of tissue failure. For the first time, we establish a mechanistic insight into the failure mechanics of soft heterogeneous tissues by investigating how cracks nucleate and propagate in stratum corneum. We further quantify previously unreported values of the tissue's Poisson's ratio and toughness, and their dramatic variation with ambient humidity. To date, skin models examining drug delivery, wound healing, and ageing continue to estimate these parameters.

Invagination of Ectodermal Placodes Is Driven by Cell Intercalation-Mediated Contraction of the Suprabasal Tissue Canopy.

Ectodermal organs such as teeth, hair follicles, and mammary glands begin their development as placodes. These are local epithelial thickenings that invaginate into mesenchymal space. There is currently little mechanistic understanding of the cellular processes driving the early morphogenesis of these organs and of why they lead to invagination rather than simple tissue thickening. Here, we show that placode invagination depends on horizontal contraction of superficial layers of cells that form a shrinking and thickening canopy over underlying epithelial cells. This contraction occurs by cell intercalation and is mechanically coupled to the basal layer by peripheral basal cells that extend apically and centripetally while remaining attached to the basal lamina. This process is topologically analogous to well-studied apical constriction mechanisms, but very different from them both in scale and molecular mechanism. Mechanical cell–cell coupling is propagated through the tissue via E-cadherin junctions, which in turn depend on tissue-wide tension. We further present evidence that this mechanism is conserved among different ectodermal organs and is, therefore, a novel and fundamental morphogenetic motif widespread in embryonic development.

Hypoxia-inducible factor-2α induces expression of type X collagen and matrix metalloproteinases 13 in osteoarthritic meniscal cells.

To evaluate whether Hypoxia-inducible factor-2α (HIF-2α) regulates expression of endochondral ossification-related molecules in human OA meniscus. Expressions of HIF-2α, type X collagen (COL10), matrix metalloproteinase (MMP)-13, and vascular endothelial growth factor (VEGF) in non-OA and OA menisci were analyzed by real-time RT-PCR and immunohistochemistry (IHC). Meniscal cells from OA patients were treated with interleukin-1β (IL-1β) and gene expression was analyzed. After knockdown of HIF-2α in OA meniscal cells, COL10 and MMP-13 expression were analyzed by RT-PCR, western blotting, immunofluorescence and ELISA. Histological analysis demonstrated weak staining of the superficial layer and large round cells in OA meniscus. RT-PCR analysis showed that HIF-2α, COL10, MMP-13, and VEGF mRNA expressions were higher in OA than non-OA meniscal cells. IHC showed a coordinated staining pattern of HIF-2α, COL10, and MMP-13 in OA meniscus. IL-1β treatment increased HIF-2α, COL10, and MMP-13 expressions in OA meniscal cells, and knockdown of HIF-2α suppressed IL-1β-mediated increase in COL10 and MMP-13 expression. These results suggested that HIF-2α may cause meniscal matrix degradation by transactivation of MMP-13. HIF-2α may be a therapeutic target for modulating matrix degradation in both articular cartilage and meniscus during knee OA progression.

Assessment of histological changes in antemortem gingival tissues fixed at various time intervals: A method of estimation of postmortem interval

Introduction:Conventional methods to estimate the time of death are adequate, but a histological method is yet unavailable to assess postmortem interval (PMI). The autolytic changes that occur in an unfixed antemortem gingival tissue which reflects histologically at an early stage are similar to changes that occur in postmortem tissue. These histological changes can be used and applied in a postmortem tissue as a method to assess PMI.Aims:The aim of the study is to assess the histological changes in a gingival tissue left unfixed for various time intervals and to correlate the findings with duration.Materials and Methods:Sixty gingival tissues obtained from patients following therapeutic extractions, impactions, gingivectomy and crown lengthening procedures were used. Each tissue obtained was divided into two pieces and labeled as “A”, the control group and “ B” the study group. Tissues labeled “A” were fixed in 10% formalin immediately and tissues labeled“B” were placed in closed containers and fixed after 15, 30, 45 min, 1, 2, and 4 h time interval. Of the sixty tissues in the study group “ B”, ten tissues were used for each time interval under investigation. All the fixed tissues were processed, stained, assessed, and analyzed statistically using Pearson correlation and regression analysis.Results:Histological changes appear at 15 min in an unfixed antemortem tissue. At 2 h interval, all layers with few cells in basal cell layer are involved. At 4 h interval, loss of stratification and complete homogenization of cells in the superficial layers with prominent changes in basal layer is evident. There was a positive correlation (<1.0) between the time interval and the appearance of the histological changes.Conclusion:Histological changes such as complete homogenization of cells in superficial layers and loss of epithelial architecture at 4 h in unfixed antemortem tissue may be used as a criterion to estimate PMI, after further studies on postmortem tissues.

HISTOLOGICAL FEATURES AND MUC1 DISTRIBUTION IN THE PALPEBRAL CONJUNCTIVA OF THE DROMEDARY CAMEL (CAMELUS DROMEDARIUS)

The conjunctiva is important adnexa of the eye that maintains the stability of the tear film. The objective of current study is to describe the histological features of palpebral conjunctiva of the dromedary, emphasizing the distribution of Muc1 using indirect tissue immunofluorescence technique. Conjunctival samples of ten adult, apparently healthy dromedary camels of both sexes were collected, fixed, embedded in paraffin and stained with H&E for general histological structure, Masson´s trichrome for fibrous tissue, Periodic acid–Schiff (PAS) for goblet cells and immunofluorescence for the distribution of Muc1. The palpebral conjunctiva of the dromedary camel is lined with stratified squamous to stratified columnar epithelium. Large numbers of goblet cells were identified associated with conjunctival epithelial cells. The lamina propria consisted of loose superficial cell layer and a deeper tarsal plate. Within the lamina propria, there were lymphoid follicles, numerous tarsal glands and palpebral seromucoid glands. The palpebral conjunctiva and associated palpebral glands were stained positive for PAS and Muc1. In conclusions, the eye of the dromedary camel is protected with a palpebral conjunctival tissue that is richly populated with PAS positive cells and numerous Muc1-producing cells.

Engineering cartilaginous grafts using chondrocyte-laden hydrogels supported by a superficial layer of stem cells.

During postnatal joint development, progenitor cells that reside in the superficial region of articular cartilage first drive the rapid growth of the tissue and later help direct the formation of mature hyaline cartilage. These developmental processes may provide directions for the optimal structuring of co-cultured chondrocytes (CCs) and multipotent stromal/stem cells (MSCs) required for engineering cartilaginous tissues. The objective of this study was to engineer cartilage grafts by recapitulating aspects of joint development where a population of superficial progenitor cells drives the development of the tissue. To this end, MSCs were either self-assembled on top of CC-laden agarose gels (structured co-culture) or were mixed with CCs before being embedded in an agarose hydrogel (mixed co-culture). Porcine infrapatellar fat pad-derived stem cells (FPSCs) and bone marrow-derived MSCs (BMSCs) were used as sources of progenitor cells. The DNA, sGAG and collagen content of a mixed co-culture of FPSCs and CCs was found to be lower than the combined content of two control hydrogels seeded with CCs and FPSCs only. In contrast, a mixed co-culture of BMSCs and CCs led to increased proliferation and sGAG and collagen accumulation. Of note was the finding that a structured co-culture, at the appropriate cell density, led to greater sGAG accumulation than a mixed co-culture for both MSC sources. In conclusion, assembling MSCs onto CC-laden hydrogels dramatically enhances the development of the engineered tissue, with the superficial layer of progenitor cells driving CC proliferation and cartilage ECM production, mimicking certain aspects of developing cartilage. Copyright © 2015 John Wiley & Sons, Ltd.

Immunohistochemistry and in situ hybridization for the diagnosis and classification of squamous lesions of the anogenital region.

Distinguishing anogenital squamous intraepithelial lesions from benign conditions and mimics may be problematic. Immunohistochemistry for surrogate markers of HPV infection, such as Ki-67, p16, and ProEx™ C, may aid the diagnosis in equivocal cases. The main diagnostic pitfall in the diagnosis of LSIL is the occurrence of "pseudokoilocytes" in benign squamous mucosa, which may lead to overdiagnosis. When interpreted correctly, Ki-67 is a sensitive and specific marker for dysplasia in mature squamous epithelium and is therefore useful for confirmation of LSIL and condyloma. A Ki-67 positive result is defined as the presence of a cluster of at least two strongly stained epithelial nuclei in the upper two-thirds of the epithelial thickness. With such a definition, there is almost complete concordance between consensus diagnosis of LSIL/condyloma confirmed by detection of HPV DNA and positive Ki-67. A related proliferation marker, ProEx™ C, has similar staining patterns and utility for the diagnosis of low grade dysplasia. The differential diagnosis of HSIL includes atypical immature squamous metaplasia and atrophy. A marker with high sensitivity and specificity for the detection of HSIL in cervical, vulvar, and anal mucosa is p16. A 2-tier scoring system is used to evaluate p16 staining. No staining or a discontinuous, patchy nuclear and cytoplasmic staining pattern is considered as a negative result. A positive result is defined as diffuse and strong staining of cells of the basal and parabasal layers of the squamous epithelium, with or without staining of superficial cell layers. New markers that are undergoing evaluation for their clinical utility include stathmin-1, phosphorylated S6, and SOX2. Confirmation of the diagnosis of dysplasia by HPV detection in tissue sections using HPV capsid protein immunohistochemistry, HPV DNA or HPV RNA in situ hybridization offers lower sensitivity as compared to immunohistochemistry for surrogate markers and therefore has more limited utility in this context.

Physiology of the pleural space.

The pleural cavity is created between the 4(th) and 7(th) week of embryologic development. These embryonic components of visceral and parietal pleurae develop different anatomic characteristics with regard to vascular, lymphatic, and nervous supply. There are two layers: a superficial mesothelial cell layer facing the pleural space and an underlying connective tissue layer. The pleura might present inflammatory response and maintenance of the pleural fluid is observed. The latter function is especially important in the mechanical coupling of the lung and chest wall. Fluid is filtered into the pleural space according to the net hydrostatic oncotic pressure gradient. It flows downward along a vertical pressure gradient, presumably determined by hydrostatic pressure and resistance to viscous flow. There also may be a net movement of fluid from the costal pleura to the mediastinal and interlobar regions. In these areas, pleural fluid is resorbed primarily through lymphatic stomata on the parietal pleural surface. In the current review we will present the physiology of the pleural space in a step by step manner.

Adipose Derived Tissue Engineered Heart Valve

Abstract Introduction: A major challenge associated with heart valve tissue engineering is the in vitro creation of mature tissue structures compliant with native valve functionality. Various cell types have been investigated for heart valve tissue engineering. In addition to prenatal, umbilical cord- and vascular-derived cells, mesenchymal stem cells (MSCs) have gained large interest for tissue engineering purposes, because of their broad differentiation potential. However, bone marrow derived MSCs require a highly invasive harvesting procedure and decline in both cell number and differentiation potential proportionally with the donor’s age. In contrast, adipose derived stem cells (ADSCs) represent an interesting alternative. The ease of repeated access to subcutaneous adipose tissue as well as the less invasive donation procedures provide clear advantages. Therefore, this study investigated the suitability of ADSCs as alternative cell source for tissue engineered heart valves (TEHVs). Methods: Human ADSCs were seeded on TEHV-scaffolds (n=11) made of nonwoven polyglycolic acid coated with poly-4-hydroxybutyrate. TEHVs were cultivated in diastolic-pulse-duplicator-bioreactor systems and subsequently seeded with a superficial layer of ADSC-derived endothelial cells. Quantitative assessment of extracellular matrix composition of the TEHV-leaflets was performed with biochemical analyses for sulphated glycosaminoglycans, hydroxyproline and DNA content. Microstructural evaluation was performed on representative samples of the TEHVleaflets by (immuno-)histochemistry and scanning electron microscopy. The mechanical properties of the ADSC derived TEHV-leaflets were characterized by biaxial tensile tests. Results: ADSC-derived TEHV-leaflets showed a homogenous vital cell distribution throughout the whole leaflet structure that consisted of large amounts of glycosaminoglycans and collagen and was endothelialized. Furthermore, the mechanically stable matrix of the ADSC-derived TEHVs showed a stiffness range in the right order of magnitude for heart valve applications. Conclusion: Human ADSCs represent a promising alternative autologous mesenchymal cell source for TEHVs that is of large clinical relevance due to their easy accessibility, efficient proliferation and excellent tissue formation capacities.

Oral Lesions Induced by Chronic Khat Use Consist Essentially of Thickened Hyperkeratinized Epithelium.

Objectives. The habit of khat chewing is prevalent in many Middle Eastern and African cultures and has been associated with various adverse conditions in humans. This study aimed to describe histological changes induced by chronic khat chewing on the buccal mucosa. Methods. Biopsies of the buccal mucosa from 14 chronic khat chewers, 20 chronic khat chewers who also smoked tobacco, and 8 nonchewers were compared for epithelial thickness, degree and type of keratinization, and connective tissue changes. Results. Tissues from khat chewers depicted abnormal keratinization of the superficial cell layer and showed increased epithelial thickness affecting all layers. Epithelial thickness in control samples was 205 ± 26 μm whereas thickness in khat chewers and khat chewers who smoked tobacco was significantly higher measuring 330 ± 35 μm and 335 ± 19 μm, respectively. Tissues from khat chewers also showed increased intracellular edema, increased melanin pigment deposits, and increased number of rete pegs most of which were thin and deep. Conclusions. These results show that oral lesions induced by chronic chewing of khat in the buccal mucosa present with white and brown discoloration due to increased epithelial thickness, increased keratinization, and melanin deposition.

Landolt ring-shaped epithelial keratopathy: a novel clinical entity of the cornea.

Landolt ring-shaped epithelial keratopathy is a newly identified disease with vesicular changes of unknown origin in the epithelial cells. Eleven Japanese patients with specific epithelial lesions that resembled a Landolt ring in the corneal epithelium were assessed. The main symptoms of Landolt ring-shaped epithelial keratopathy are foreign-body sensation and photophobia. Slitlamp examination indicates that Landolt ring-shaped lesions located only in the corneal epithelium are randomly distributed and occur unilaterally, bilaterally, or asymmetrically. Small lesions are sometimes connected to each other to form a large Landolt ring in a fractal pattern. Confocal microscopy reveals that the Landolt ring lesions are vesicular changes in the epithelial cells from the basal cell layer to the superficial cell layer without inflammation. The lesions form for weeks to months with sporadic exacerbations and natural remissions with or without treatments. The outcomes do not include epithelial disorders in the affected corneas or visual disruptions. We describe a new disease entity of unknown origin referred to as Landolt ring-shaped epithelial keratopathy, which is usually bilateral and characterized by specific Landolt ring-shaped focal epithelial lesions with vesicular changes only in the epithelial cells. The disorder has an insidious onset and self-limiting nature despite treatment and should be included in the differential diagnosis of corneal epithelial disorders.

Morphological Studies on the Vagina and Vestibule of Dromedary Camels (Camelus dromedarius)

*** *** SUMMARY: The camel (Camelus dromedarius) is an important multipurpose livestock species and its meat represents about 10% of the red meat consumption in Egypt. The reproductive efficiency of camel under natural condition is generally considered to be low. Sound knowledge about the tubular genital organs of this species might facilitate the application of new reproductive meth odology. Our study was therefore conducted to investigate the morphology of mucosal surface of vagina and vestibule on camels using ligh t, scanning and transmission electron microscopy. The mucosal surface of vagina consisted of stratified columnar epithelium with m ucous secreting cells (goblet-like cells). SEM of vagina revealed the presence of longitudinal primary and secondary folds and small transverse folds. The columnar epithelium showed marked cell boundary and its apical surface was studded by a lot of microvilli. TEM confi rmed the presence of microvilli at apical surfaces and showed some secretory granules in the supranuclear region of the columnar epi thelium. The vestibule of dromedary camel was lined by stratified squamous keratinized epithelium. Basal lamina and stratum granulosum o f this epithelium showed strong PAS positive reaction. SEM of vestibule revealed the presence of small longitudinal and fine transvers e folds with a lot of mucous debris. However TEM of vestibule showed the stratified squamous keratinized epithelium with basal layer of cuboidal cells and superficial layers of squamous cells.

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Background: Homeostatic maintenance and repair of the bladder urothelium has been attributed to proliferation of keratin 5‐expressing basal cells (K5‐BC) with subsequent differentiation into superficial cells. Recent evidence, however, suggests that the intermediate cell layer harbors a population of progenitor cells. We use label‐retaining cell (LRC) methodology in conjunction with a clinically relevant model of uropathogenic Escherichia coli (UPEC)‐induced injury to characterize urothelial ontogeny during development and in response to diffuse urothelial injury. Results: In the developing urothelium, proliferating cells were dispersed throughout the K5‐BC and intermediate cells layers, becoming progressively concentrated in the K5‐BC layer with age. When 5‐bromo‐2‐deoxyuridine (BrdU) was administered during urothelial development, LRCs in the adult were found within the K5‐BC, intermediate, and superficial cell layers, the location dependent upon time of labeling. UPEC inoculation resulted in loss of the superficial cell layer followed by robust proliferation of K5‐BCs and intermediate cells. LRCs within the K5‐BC and intermediate cell layers proliferated in response to injury. Conclusions: Urothelial development and regeneration following injury relies on proliferation of K5‐BC and intermediate cells. The existence and proliferation of LRCs within both the K5‐BC and intermediate cell layers suggests the presence of two populations of urothelial progenitor cells. Developmental Dynamics 243::988–998, 2014. © 2014 Wiley Periodicals, Inc.

Transit amplification in the amniote cerebellum evolved via a heterochronic shift in NeuroD1 expression

The cerebellum has evolved elaborate foliation in the amniote lineage as a consequence of extensive Atoh1-mediated transit amplification in an external germinal layer (EGL) comprising granule cell precursors. To explore the evolutionary origin of this layer, we have examined the molecular geography of cerebellar development throughout the life cycle of Xenopus laevis. At metamorphic stages Xenopus displays a superficial granule cell layer that is not proliferative and expresses both Atoh1 and NeuroD1, a marker of postmitotic cerebellar granule cells. Premature misexpression of NeuroD1 in chick partially recapitulates the amphibian condition by suppressing transit amplification. However, unlike in the amphibian, granule cells fail to enter the EGL. Furthermore, misexpression of NeuroD1 once the EGL is established both triggers radial migration and downregulates Atoh1. These results show that the evolution of transit amplification in the EGL required adaptation of NeuroD1, both in the timing of its expression and in its regulatory function, with respect to Atoh1.

A population of progenitor cells in the basal and intermediate layers of the murine bladder urothelium contributes to urothelial development and regeneration.

Homeostatic maintenance and repair of the bladder urothelium has been attributed to proliferation of keratin 5-expressing basal cells (K5-BC) with subsequent differentiation into superficial cells. Recent evidence, however, suggests that the intermediate cell layer harbors a population of progenitor cells. We use label-retaining cell (LRC) methodology in conjunction with a clinically relevant model of uropathogenic Escherichia coli (UPEC)-induced injury to characterize urothelial ontogeny during development and in response to diffuse urothelial injury. In the developing urothelium, proliferating cells were dispersed throughout the K5-BC and intermediate cells layers, becoming progressively concentrated in the K5-BC layer with age. When 5-bromo-2-deoxyuridine (BrdU) was administered during urothelial development, LRCs in the adult were found within the K5-BC, intermediate, and superficial cell layers, the location dependent upon time of labeling. UPEC inoculation resulted in loss of the superficial cell layer followed by robust proliferation of K5-BCs and intermediate cells. LRCs within the K5-BC and intermediate cell layers proliferated in response to injury. Urothelial development and regeneration following injury relies on proliferation of K5-BC and intermediate cells. The existence and proliferation of LRCs within both the K5-BC and intermediate cell layers suggests the presence of two populations of urothelial progenitor cells.

Forced Resurgence and Targeting of Intracellular Uropathogenic Escherichia coli Reservoirs

Intracellular quiescent reservoirs of uropathogenic Escherichia coli (UPEC), which can seed the bladder mucosa during the acute phase of a urinary tract infection (UTI), are protected from antibiotic treatments and are extremely difficult to eliminate. These reservoirs are a potential source for recurrent UTIs that affect millions annually. Here, using murine infection models and the bladder cell exfoliant chitosan, we demonstrate that intracellular UPEC populations shift within the stratified layers of the urothelium during the course of a UTI. Following invasion of the terminally differentiated superficial layer of epithelial cells that line the bladder lumen, UPEC can multiply and disseminate, eventually establishing reservoirs within underlying immature host cells. If given access, UPEC can invade the superficial and immature bladder cells equally well. As infected immature host cells differentiate and migrate towards the apical surface of the bladder, UPEC can reinitiate growth and discharge into the bladder lumen. By inducing the exfoliation of the superficial layers of the urothelium, chitosan stimulates rapid regenerative processes and the reactivation and efflux of quiescent intracellular UPEC reservoirs. When combined with antibiotics, chitosan treatment significantly reduces bacterial loads within the bladder and may therefore be of therapeutic value to individuals with chronic, recurrent UTIs.

Cytokeratin localization in toe pads of the anuran amphibian Philautus annandalii (Boulenger, 1906)

We have examined cytokeratin distribution and their nature in toe pads of the Himalayan tree-frog Philautus annandalii. Toe pads are expanded tips of digits and show modifications of their ventral epidermis for adhesion. The toe pad epidermal cells, being organized into 3-4 rows, possess keratin bundles, especially in surface nanostructures that are involved in adhesion. Immunohistochemical localization using a pan-cytokeratin antibody revealed that cytokeratin immunoreactivity is the strongest in the mid- to basal cell rows of the epidermis, which parallels our previous ultrastructural observation of dense keratin bundles present in this part of the epidermis. The remainder of the epidermis (i.e., the superficial cell layer) showed little immunoreactivity. Immunoblot analysis revealed that toe-pads possessed keratins prominently in the molecular mass of 50 kDa. Possible presence of keratin 5 in toe pad epidermis has been correlated with its usual distribution pattern in mammalian epidermis.

A Pou5f1/Oct4 dependent Klf2a, Klf2b, and Klf17 regulatory sub-network contributes to EVL and ectoderm development during zebrafish embryogenesis

In mammalian ES cells, the transcription factors Klf4 and Klf2 contribute to maintenance of pluripotency and self-renewal and are regulated by Pou5f1/Oct4. In the early zebrafish embryo Pou5f1/Oct4 is necessary for expression of three Klf2/4 family members, klf2a, klf2b and klf17 (previously klf4b), similar to the regulation reported for mammalian ES cells. In this study, we analyzed blastula and gastrula stage Klf regulatory networks and their influence on zebrafish embryonic patterning. We show that Pou5f1 acts in combination with region-specific factors to activate klf2a, klf2b, and klf17 in the superficial cell layer of the embryo. In addition, Pou5f1 acts together with the BMP signaling pathway to activate and maintain expression of klf2a and klf2b in a ventral ectodermal domain. We used microarray expression profiles of klf2a, klf2b and klf17 knockdown and overexpression embryos to identify Klf target genes, which reveals that Klfs participate in specification of the extraembryonic enveloping layer (EVL). We discuss mechanistic implications of simultaneous activation of transcriptional targets by ubiquitous, like Pou5f1, and region-specific inducers, emerging as a common regulatory motif in early development.