Connective Tissue Compartment Research Articles

Expression of Wnt signaling proteins in rare congenital bladder disorders

Introduction and AimsCongenital bladder anomalies are rare and are a leading cause of end stage renal failure in children. The Wnt signaling pathway, important during embryonic development, has been implicated in the pathogenesis of these conditions through regulation of gene expression, including essential transcription factors. We investigated the expression of four Wnt transcriptional targets, namely, Pygopus 1 (Pygo1), FRA1, TCF7L1 and Connexin 43 (Cx43) in three rare congenital bladder disorders: bladder exstrophy (BE), neurogenic bladder (NGB) and posterior urethral valves (PUV). MethodsBladder tissue samples were collected from patients at the Great Ormond Street Hospital for Sick Children, London, UK, with control (normally-functioning bladder, N=9), BE (N=15), NGB (N=6) and PUV (N=5). Histological analysis was performed using the van Gieson stain to differentiate smooth muscle (SM) and connective tissue (CT) compartments. An unbiased, automated, semi-quantitative immunofluorescence analysis was performed to measure the labelling intensity of four Wnt-related proteins in tissue from these four groups. Results and DiscussionThere was a significant (p<0.05) increase in the expression of Pygo1 in the smooth muscle of all anomalies examined and also in the connective tissue in PUV compared to control. Cx43 also showed overexpression in the smooth muscle across all conditions; however, there was a reduced expression in NGB and an increase in PUV in connective tissue. TCF7L1 showed a significant decrease in both tissue compartments for NGB, whereas FRA1 expression remained unchanged across all anomalies. We also measured colocalization of Wnt-related proteins. TCF7L1 exhibited increased colocalization with Pygo1 and FRA1 in exstrophy compared to control. These results suggest a complex dysregulation of the Wnt pathway in congenital bladder disorders. ConclusionWnt signaling-related proteins show dysregulation in congenital bladder disorders compared to control tissue. Understanding these mechanisms should help towards non-invasive early diagnosis, drug target discovery and development of treatment strategies for these conditions.Summary tableSummary of Wnt-related protein expression and colocalization in bladder exstrophy (BE), neurogenic bladder (NGB) and posterior urethral valves (PUV) disorder compared to normally-functioning control bladder. The table shows the levels of expression as significantly up (↑) down (↓) or no change (-) of Pygopus 1 (Pygo1), Connexin 43 (Cx43), FRA1 and TCF7L1 expression in smooth muscle and connective tissue compartments in BE, NGB and PUV relative to control bladder tissue samples. The table also shows colocalization of six protein pairs of Pygo1 and FRA1; Pygo1 and Cx43; Pygo1 and TCF7L1; FRA1 and Cx43; FRA1 and TCF7L1 and Cx43 and TCF7L1 in bladder disorders compared to control.Summary tableProtein Expression (Smooth muscle)Pygo1Cx43FRA1TCF7L1BE↑↑--NGB↑↑-↓PUV↑↑--Protein Expression (Connective tissue)BE----NGB-↓-↓PUV↑↑--ColocalizationPygo1/FRA1Pygo1/Cx43Pygo1/TCF7L1FRA1/Cx43FRA1/TCF7L1Cx43/TCF7L1Exstrophy--↑-↑-NGB------PUV------

FOXO1 regulates wound-healing responses in human gingival fibroblasts.

Forkhead box-O 1 (FOXO1) is a transcription factor actively involved in oral wound healing at the epithelial barrier. However, less is known regarding the role of FOXO1 during the tissue repair response in the connective tissue compartment. This study explored the involvement of FOXO1 in the modulation of fibroblast activity related to wound healing. Primary cultures of human gingival fibroblasts were obtained from four healthy young donors. Myofibroblastic differentiation, collagen gel contraction, cell migration, cell spreading, and integrin activation were evaluated in the presence or absence of a FOXO1 inhibitor (AS1842856). Variations in mRNA and proteins of interest were evaluated through qRT-PCR and western blot, respectively. Distribution of actin, α-smooth muscle actin, and β1 integrin was evaluated using immunofluorescence. FOXO1 and TGF-β1 expression in gingival wound healing was assessed by immunohistochemistry in gingival wounds performed in C57BL/6 mice. Images were analyzed using ImageJ/Fiji. ANOVA or Kruskal-Wallis test followed by Tukey's or Dunn's post-hoc test was performed. All data are expressed as mean ± SD. p < .05 was considered statistically significant. FOXO1 inhibition caused a decrease in the expression of the myofibroblastic marker α-SMA along with a reduction in fibronectin, type I collagen, TGF-β1, and β1 integrin mRNA level. The FOXO1 inhibitor also caused decreases in cell migration, cell spreading, collagen gel contraction, and β1 integrin activation. FOXO1 and TGF-β1 were prominently expressed in gingival wounds in fibroblastic cells located at the wound bed. The present study indicates that FOXO1 plays an important role in the modulation of several wound-healing functions in gingival fibroblast. Moreover, our findings reveal an important regulatory role for FOXO1 on the differentiation of gingival myofibroblasts, the regulation of cell migration, and collagen contraction, all these functions being critical during tissue repair and fibrosis.

Qualitative and Quantitative Characteristics of the Passive Compartment Underneath the Myocardial Bridge Comprising the Blood Vessel Adventitia and the Perivascular Connective Tissue

Myocardial bridges are inborn anomalies frequently found in authopsies. Although tipically clinically silent, they are occasionally associated with severe clinical manifestations, e.g. myocardial ischemia or even sudden death. The pathophysiology and risk factors for these manifestations have not yet been completely elucidated. The connective tissue underneath the bridge has been considered as one of the factors the symptoms depend on. Thus, the aim of this research was to determine the histological characteristics of the connective tissue lying underneath the myocardial bridge and to contribute to a better understanding of the protective effects this passive compartment might have in prevention of severe clinical manifestations of myocardial bridging. The study was carried out on twenty hearts with myocardial bridges. Length of the bridge was determined using a precise electronic caliper. Sections of the myocardial bridges with the underlying connective tissue were obtained and prepared for qualitative and quantitative analysis. The connective tissue underneath the bridges was composed of adipose tissue and loose connective tissue in different ratios. The tissue underneath thin bridges was predominantly composed of adipose tissue, while loose connective tissue was the dominant component under thick bridges. The myocardial bridges had an average thickness of 0,98 ± 0.44 mm and an average length of 15,25±5,65 mm. We found a strong positive correlation between the myocardial bridge thickness and length (r = 0,860, p = 0,0001). The thickness of the passive connective tissue compartment under the myocardial bridges was 0,58±0,22 mm, and there was no correlation between this parameter and the myocardial bridge thickness (r = -0,011; p = 0,963). In the clinical evaluation of patients with these anomalies it is necessary to take into account independently the myocardial bridge thickness and length on one side and the thickness of the connective tissue lying underneath it on the other.

Telocytes and lymphatic endothelial cells: Two immunophenotypically distinct and spatially close cell entities

Telocytes (TCs) have recently emerged as a peculiar type of stromal cells located in both perivascular and interstitial compartments of multiple anatomical sites in humans, other mammals and vertebrates. Pioneer electron microscopy studies have ultrastructurally defined TCs as “stromal cells with telopodes” (i.e. very long and thin cell processes with a moniliform morphology conferred by the irregular alternation of slender segments and small, bead-like, dilated portions), whereupon it has become apparent that TCs largely correspond to the CD34+ stromal/interstitial cells detectable by immunohistochemical assays. Besides CD34, TCs are also characterized by the expression of platelet-derived growth factor receptor (PDGFR)α. Interestingly, recent works recommended that lymphatic endothelial cell (LEC) markers should be routinely assessed to discriminate with certainty TCs from LECs, because these two cell types may exhibit similar morphological traits, especially when initial lymphatics are sectioned longitudinally and appear as vascular profiles with no obvious lumen. Furthermore, it has been argued that lymphatic microvessels immunostained for the small mucin-type transmembrane glycoprotein podoplanin (PDPN), which is widely used as lymphatic endothelial marker, can be easily misidentified as TCs. Nevertheless, surprisingly these assumptions were not based on double tissue immunostaining for TC and LEC markers. Therefore, the present morphological study was undertaken to precisely investigate the mutual spatial organization and putative relationships of TCs and lymphatic vessels in tissues from different human organs. For this purpose, we carried out a series of double immunofluorescence analyses simultaneously detecting the CD34 or PDGFRα antigen and a marker of LECs, either PDPN or lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1). In the connective tissue compartment of different organs, TCs were CD34+/PDGFRα+/PDPN−/LYVE-1− while LECs were CD34−/PDGFRα−/PDPN+/LYVE-1+, thus representing two definitely distinct, though spatially close, cell entities. The arrangement of telopodes to intimately surround the abluminal side of LECs suggests a possible role of TCs in the regulation of lymphatic capillary functionality, which is worth investigating further.

P71-T Distal digital phalanx pulp ultrasound images of individual fingers and toes

Background Ultrasound can visualise digital pulp structures and provides precise measurements of the connective tissue compartments as well as recently described fluid filled sacculi which are accountable for distending digital pulp under normal circumstances and skin wrinkling after water immersion. Material and method A High Frequency (22 MHz) Ultrasound and Power Doppler imaging of the distal fingers and toes phalanx of a non-dominant hand and foot were carried out in a normal subject in both transverse and longitudinal planes. The US images were stored for offline quantitative and qualitative analysis. Results Based on the various digital pulp US echogenicity the following layers were depicted form ventral do dorsal direction in the fingers and toes: epidermis, dermis; vascularisation; hyper-echogenic areas (connective tissue compartments); hypo-echogenic areas (fluid filled sacculi) and distal phalanx bone. All fingers and toes showed similar distribution of the digital pulp structures whilst the number and area of the hyper-echogenic and hypo-ecogenic spaces varied for the individual fingers and toes. Conclusion The examples of individual fingers and toes distal phalanx ultrasound imaging provided can be used to determine and standardise the technique of digital pulp structures ultrasound assessment in normal physiological as well as in pathological conditions.

Role of Fibroblast Populations in Periodontal Wound Healing and Tissue Remodeling.

After injury to periodontal tissues, a sequentially phased healing response is initiated that enables wound closure and partial restoration of tissue structure and function. Wound closure in periodontal tissues involves the tightly regulated coordination of resident cells in epithelial and connective tissue compartments. Multiple cell populations in these compartments synergize their metabolic activities to reestablish a mucosal seal that involves the underlying periodontal connective tissues and the attachment of these tissues to the tooth surface. The formation of an impermeable seal around the circumference of the tooth is of particular significance in oral health since colonization of tooth surfaces by pathogenic biofilms promotes inflammation, which can contribute to periodontal tissue degradation and tooth loss. The reformation of periodontal tissue structures in the healing response centrally involves fibroblasts, which synthesize and organize the collagen fibers that link alveolar bone and gingiva to the cementum covering the tooth root. The synthesis and remodeling of nascent collagen matrices are of fundamental importance for the reestablishment of a functional periodontium and are mediated by diverse, multi-functional fibroblast populations that reside within the connective tissues of gingiva and periodontal ligament. Notably, after gingival wounding, a fibroblast sub-type (myofibroblast) arises, which is centrally involved in collagen synthesis and fibrillar remodeling. While myofibroblasts are not usually seen in healthy, mature connective tissues, their formation is enhanced by wound-healing cytokines. The formation of myofibroblasts is also modulated by the stiffness of the extracellular matrix, which is mechanosensed by resident precursor cells in the gingival connective tissue microenvironment. Here, we consider the cellular origins and the factors that control the differentiation and matrix remodeling functions of periodontal fibroblasts. An improved understanding of the regulation and function of periodontal fibroblasts will be critical for the development of new therapies to optimize the restoration of periodontal structure and function after wounding.

Fluid Containing Structures in the tips of the fingers and toes delineated by Ultrasound Imaging before and after Induced Skin Wrinkling

This Ultrasound study identified spaces within the pulp of distal phalanx of the fingers and toes that halve in area after Water Induced Skin Wrinkling. These spaces appeared as fluid filled sacculi between connective tissue compartments accountable for distending digital pulp under normal circumstances and skin wrinkling after water immersion. Whilst studying conditions related to sympathetic nerve function with WISW ultrasound imaging is a valuable adjunct to the visual assessment.

Contrast Enhanced Imaging as an Alternative to Dissection in Tree Shrew (Tupaiidae: Scandentia) Chewing Musculature

A burgeoning technique in the field of comparative biology involves staining specimens with iodine, followed by CT scanning. Along with providing excellent 3D visualization of soft tissue by giving it a higher attenuation, iodine staining enables digital isolation of muscles and the potential for measuring individual muscle fascicles. It is well suited for visualizing muscles of mastication, especially the medial and lateral pterygoid muscles; located internally, they are often difficult to dissect in small mammals. Here, we test the reliability of this method as an alternative to traditional dissection for obtaining accurate volume and muscle fascicle length measurements. This was accomplished by digitally isolating the masticatory muscles in three tree shrew cadaver specimens that had been subjected to iodine staining. These specimens represent the largest (Tupaia tana), smallest (T. minor), and average (T. belangeri) sizes of tree shrew. All specimens were frozen from death. After fixing with 10% buffered formalin, specimens were stained in a 3% solution of I2KI (T. tana; T. minor) or 5% solution of I2KI (T. belangeri) for 14 days. The heads were microCT scanned (at resolutions of 44.5 μm, 26.5 μm, and 32.2 μm respectively), enabling their muscles to be digitally isolated. Fascicles were visible only in T. minor due to a higher resolution, and were not measurable in any of the specimens. Larger bundles of fascicles contained within connective tissue compartments were identified and used to delineate separate muscles layers (eg. superficial vs deep masseter). These results indicate that while muscle fascicles are not measureable using these scans, they may be measurable if resolution could be increased. Isolating the muscles digitally provided more accurate measures of volume as compared to traditional dissection, while also preserving their orientation. Future work will focus on using these data to digitally map tree shrew muscles of mastication onto fossil primate mandibles as an analogue for studying early primate chewing systems.Support or Funding InformationThis work was supported through an American Association of Anatomists Short‐term Visiting Scholarship to H. Kristjanson

Photoprotection by pistachio bioactives in a 3-dimensional human skin equivalent tissue model

Reactive oxygen species (ROS) generated during ultraviolet (UV) light exposure can induce skin damage and aging. Antioxidants can provide protection against oxidative injury to skin via “quenching” ROS. Using a validated 3-dimensional (3D) human skin equivalent (HSE) tissue model that closely mimics human skin, we examined whether pistachio antioxidants could protect HSE against UVA-induced damage. Lutein and γ-tocopherol are the predominant lipophilic antioxidants in pistachios; treatment with these compounds prior to UVA exposure protected against morphological changes to the epithelial and connective tissue compartments of HSE. Pistachio antioxidants preserved overall skin thickness and organization, as well as fibroblast morphology, in HSE exposed to UVA irradiation. However, this protection was not substantiated by the analysis of the proliferation of keratinocytes and apoptosis of fibroblasts. Additional studies are warranted to elucidate the basis of these discordant results and extend research into the potential role of pistachio bioactives promoting skin health.

Live Imaging of Axolotl Digit Regeneration Reveals Spatiotemporal Choreography of Diverse Connective Tissue Progenitor Pools

SummaryConnective tissues—skeleton, dermis, pericytes, fascia—are a key cell source for regenerating the patterned skeleton during axolotl appendage regeneration. This complexity has made it difficult to identify the cells that regenerate skeletal tissue. Inability to identify these cells has impeded a mechanistic understanding of blastema formation. By tracing cells during digit tip regeneration using brainbow transgenic axolotls, we show that cells from each connective tissue compartment have distinct spatial and temporal profiles of proliferation, migration, and differentiation. Chondrocytes proliferate but do not migrate into the regenerate. In contrast, pericytes proliferate, then migrate into the blastema and give rise solely to pericytes. Periskeletal cells and fibroblasts contribute the bulk of digit blastema cells and acquire diverse fates according to successive waves of migration that choreograph their proximal-distal and tissue contributions. We further show that platelet-derived growth factor signaling is a potent inducer of fibroblast migration, which is required to form the blastema.

Biosynthesis of glycosaminoglycans: associated disorders and biochemical tests.

Glycosaminoglycans (GAG) are long, unbranched heteropolymers with repeating disaccharide units that make up the carbohydrate moiety of proteoglycans. Six distinct classes of GAGs are recognized. Their synthesis follows one of three biosynthetic pathways, depending on the type of oligosaccharide linker they contain. Chondroitin sulfate, dermatan sulfate, heparan sulfate, and heparin sulfate contain a common tetrasaccharide linker that is O-linked to specific serine residues in core proteins. Keratan sulfate can contain three different linkers, either N-linked to asparagine or O-linked to serine/threonine residues in core proteins. Finally, hyaluronic acid does not contain a linker and is not covalently attached to a core protein. Most inborn errors of GAG biosynthesis are reported in small numbers of patients. To date, in 20 diseases, convincing evidence for pathogenicity has been presented for mutations in a total of 16 genes encoding glycosyltransferases, sulfotransferases, epimerases or transporters. GAG synthesis defects should be suspected in patients with a combination of characteristic clinical features in more than one connective tissue compartment: bone and cartilage (short long bones with or without scoliosis), ligaments (joint laxity/dislocations), and subepithelial (skin, sclerae). Some produce distinct clinical syndromes. The commonest laboratory tests used for this group of diseases are analysis of GAGs, enzyme assays, and molecular testing. In principle, GAG analysis has potential as a general first-line diagnostic test for GAG biosynthesis disorders.

DYNAMICS OF ENDOTHELIN LEVELS IN MUSCLE REGENERATION AFTER MECHANICAL TRAUMA

Objectives. Muscle regeneration after trauma is a complex phenomenon involving several cellular processes, such as angiogenesis, inflammation, fibrosis, activation of satellite cells and their differentiation into myocytes and myotubes. Although many studies explored these mechanisms in the last years, there is still an unmet need to find new therapy targets, especially regarding some cellular molecules involved in muscular recovery after mechanical or pathological injury. In the present study we investigated the dynamics of endothelin-1 (ET-1), an important factor that has been shown to be involved in all stages of tissue regeneration, but which is poorly investigated in skeletal muscle. Materials and methods. We used an experimental animal model of acute mechanical trauma on mouse gastrocnemius muscle. ET-1 levels were investigated at different time-points after muscle injury by in situ immunofluorescence, xMAP assay on tissue and serum samples, and Western Blot analysis. Results. By xMAP assay, tissue ET-1 levels increased significantly up to the 5th day after trauma, correlated with serum levels. xMAP assay was confirmed by Western blot analysis which showed a significant increase in the level of ET-1 towards the end of the first week after trauma. This corresponds with the inflammatory stage of the regeneration process, followed by angiogenesis and satellite cell activation. In situ immunostaining showed a multiplication of interstitial cells expressing ET-1 in the first week after muscle injury. Two cellular subtypes were detected in the connective tissue – one is represented by blood-derived CD45 positive cells and the other by local interstitial cells. Such cells were detected in all connective tissue compartments, in close association with CD56 positive satellite cells, myoblasts and myotubes and most of them co-express sca-1. Conclusions. The present study demonstrated that ET-1 is synthesized mostly by mesenchymal progenitors and their number greatly increases after mechanical trauma in muscle interstitium. Based on ET-1 expression and their close association with activated satellite cells, such cells could have a paracrine influence not only over angiogenesis but also over fiber regeneration. ET-1 appears as an important molecule working in conjunction with other various signalling pathways especially during first stages of the regeneration process after acute mechanical injury. ET-1 and its receptors could become therapeutic targets, especially for inflammatory myopathies and muscular dystrophies with significant pathological fibrosis.

Photoprotective Effects of Pistachio Antioxidants in a 3‐Dimensional Human Skin Equivalent Tissue Model

Reactive oxygen species (ROS) generated principally by irradiation from ultraviolet light (UV) exposure mediate many pathways leading to skin damage and skin aging. Antioxidants provide protection against oxidative injury to the skin through “quenching” ROS. Pistachios contain several antioxidants, including β‐carotene, lutein, anthocyanins, and γ‐tocopherol. Using a validated 3‐dimensional human skin cell equivalent (HSE) tissue, we examined whether pistachio antioxidants could protect the skin against 35 J/cm2 UVA‐induced damage following their uptake from cell culture media (a model reflecting systemic distribution). Lutein and γ‐tocopherol are the predominant lipophilic antioxidants in pistachios. Treatment with 0.1 µM lutein or 1 µM γ‐tocopherol for 48 h prior to UVA exposure appears to exert protection against UVA‐induced morphological changes to both the epithelial and connective tissue compartments of HSE. Overall skin thickness and organization, as well as fibroblast morphology, appears preserved in HSE treated for 48 h with a pistachio methanol extract containing 0.14 µM lutein, 0.59 µM γ‐tocopherol &amp; 0.37 µM cyanidin‐3‐galactose prior to exposure to UVA. However, this protection from individual antioxidants and the pistachio extract was based primarily on the assessment of skin architecture and was not substantiated by tests of the proliferation of keratinocytes and apoptosis of fibroblasts. Additional studies are warranted to elucidate the basis of these discordant results and extend research into the potential protection by pistachios against UVA‐induced morphological changes in human skin.

Roles of Proteoglycans and Glycosaminoglycans in Wound Healing and Fibrosis.

A wound is a type of injury that damages living tissues. In this review, we will be referring mainly to healing responses in the organs including skin and the lungs. Fibrosis is a process of dysregulated extracellular matrix (ECM) production that leads to a dense and functionally abnormal connective tissue compartment (dermis). In tissues such as the skin, the repair of the dermis after wounding requires not only the fibroblasts that produce the ECM molecules, but also the overlying epithelial layer (keratinocytes), the endothelial cells, and smooth muscle cells of the blood vessel and white blood cells such as neutrophils and macrophages, which together orchestrate the cytokine-mediated signaling and paracrine interactions that are required to regulate the proper extent and timing of the repair process. This review will focus on the importance of extracellular molecules in the microenvironment, primarily the proteoglycans and glycosaminoglycan hyaluronan, and their roles in wound healing. First, we will briefly summarize the physiological, cellular, and biochemical elements of wound healing, including the importance of cytokine cross-talk between cell types. Second, we will discuss the role of proteoglycans and hyaluronan in regulating these processes. Finally, approaches that utilize these concepts as potential therapies for fibrosis are discussed.

Ductular reaction in alcoholic steatohepatitis, nonalcoholic steatohepatitis and hepatitis C virus infection (immunohistochemical study)

Background and aim. Alcoholic steatohepatitis (ASH), nonalcoholic steatohepatitis (NASH) and hepatitis C virus infection (HCV) are the most frequent causes of liver cirrhosis in the Western world. The course of the disease depends on the balance in fibrogenesis, angiogenesis and regeneration. But if fibrosis was studied intensively during the last decades, mechanisms and signs of reparative regeneration in these diseases remain unknown. Ductular reaction is one of the morphologic signs of regeneration, but it was examined mostly in diseases associated with cholestasis. The aim of our work is to study ductular reaction associated with expression of immunohistochemical marker CK19 in alcoholic steatohepatitis, nonalcoholic steatohepatitis and hepatitis C virus infection.Material and methods. 45 autopsies with diagnosed alcoholic steatohepatitis, nonalcoholic steatohepatitis and viral hepatitis C were enrolled in this study. Diagnosis of ASH was based on the data of alcohol abuse and morphologic signs of alcoholic disease – cardiomyopathy, chronic pancreatitis, alcoholic encephalopathy and typical liver changes. Viral genesis was proved by serological study (RNA HCV) and morphologic signs of HCV (METAVIR criteria). Diagnosis of NASH was verified by the features of metabolic syndrome and hepatic changes (Brunt criteria). The employed measures of reparative regeneration were CK19-positive cells with “biliary phenotype”, evaluated in the liver tissue (area of ductular reaction). Marker quantification was performed by assessing the ratio of stained tissue to the total area of the liver section using image analysis. Histotopographic features of ductular reaction and cellular phenotype were also studied. “STATISTICA FOR WINDOWS6.0” was used to analyze the data. The data were expressed as mean value ± standard deviation (M±s). Differences between groups were analyzed using ANOVA analysis (LSD test). P &lt; 0,05 was considered significant.Results. Evaluation of CK19-positive cells area in the liver section showed the highest degree in HCV group (10,17±0,23). Differences of this marker from the markers revealed in NASH (1,17±0,25) and ASH (1,99±0,4) were statistically significant. In addition to quantitative parameters localization of ductular reaction and cellular phenotype were analyzed. Diffuse spreading of the cells with “biliary phenotype” within the thickness of connective tissue septas was found in NASH and ASH. Most ductules were laid by homogenous cuboidal cells and contained distinct lumens. Probably these findings may be a result of biliary epithelial cell proliferation from the preexisting ductules. In HCV CK19-positive cells were revealed mostly in the site of marginal plate between the lobular parenchyma and connective tissue septa. In histologic examination cellular population with “biliary phenotype” was more heterogenous and some ductules had no lumens. Genesis of such lesions may be associated with activation and proliferation of immature precursors – intermediate hepato-biliary cellsConclusions. Severe ductular reaction associated with marginal localization of CK19-positive cells between the parenchymal and connective tissue compartments is revealed in hepatitis C virus infection. In chronic steatohepatitis (alcoholic and nonalcoholic) diffuse spreading of the cells with “biliary phenotype” within the thickness of connective tissue septas is found. Quantitative and qualitative differences in morphologic signs of ductular reaction revealed in our examination confirm the variable role of new-formed ductules in the pathogenesis of different forms of chronic hepatitis which may be the aim for future researches.

The role of oral flora in the development of chemotherapy‐induced oral mucositis

Chemotherapy-induced mucositis is considered to be a major oncological problem, caused by the cytotoxic effects of cancer chemotherapy. In the last 10 years, there have been significant advances in the understanding of mucositis pathobiology. At the basic level, it is now well-understood that it is not just an epithelial process, but rather a complex interaction between epithelial and connective tissue compartments. There is also potential interaction between the oral microenvironment and the development of mucositis. Changes occur in the resident oral flora (commensal) throughout cancer treatment, and it is conceivable that these organisms and changes that occur may have an influence on the development of mucosal toxicity associated with cancer treatment. The aim of this review was to examine the potential contributions of oral microflora in the pathobiology of mucositis and identify pathways and interactions that could be targeted for therapeutic management of mucositis.

Derived sperm morphology in the interstitial sea cucumberRhabdomolgus ruber, with observations on oogenesis and spawning behavior

Some life history features of the interstitial sea cucumber Rhabdomolgus ruber are described from intertidal specimens collected from the northern coast of Maine. Histological studies suggest that the population consists of hermaphrodites with gametogenesis being initiated in April and reproduction beginning in May and continuing through the summer months. Sexually mature adults possess a single, blind-ended gonadal tubule that functions as an ovotestis by producing both eggs and sperm. The ovotestis wall consists of an outer peritoneum composed of flagellated epithelial cells and muscles; an inner germinal epithelium of germ and somatic cells; and a middle connective tissue (hemal) compartment bounded by the basal laminas of the peritoneum and germinal epithelium. During the reproductive season, the gonadal tubule contains all stages of oocyte development. Vitellogenesis appears to involve the biosynthetic activities of the Golgi complex and rough endoplasmic reticulum. A few specimens had transitional ovotestes with mature sperm in the gonad lumen and asynchronously developing oocytes and a small number of spermatocytes within the germinal epithelium. The mature spermatozoon is an ent-aquasperm with ultrastructural features significantly different from those described from other echinoderm classes including a highly elongated acrosome, a large periacrosomal region between the acrosome and nucleus, numerous unfused mitochondria in the midpiece, and a cytoplasmic sleeve or collar extending posteriorly along the proximal portion of the flagellum. The sperm head reaches 11.5 μm in length (combined midpiece, nucleus, periacrosomal region, acrosome), making it the longest yet reported from the Holothuroidea and among the longest in the Echinodermata. Some elements of this derived morphology could be attributed to fertilization biology, but others may have phylogenetic significance. Spawning behavior was observed in which two individuals appeared to pseudocopulate by intertwining their oral tentacles for several minutes before one of them abruptly secreted an egg mass containing three eggs.

The anus as a second mouth: anal suspension feeding by an oral deposit‐feeding sea cucumber

AbstractRespiratory trees of holothurians are blind‐ended evaginations of the posterior digestive system that are rhythmically inflated with seawater via the anus, and are considered to have respiratory and excretory functions. We tested the assimilatory capability of the respiratory tree epithelium by exposing adults of the oral deposit‐feeding aspidochirotid sea cucumber Parastichopus californicus to (1) 14C‐labeled unicellular algae and (2) iron‐containing macromolecules, and then following the distribution of the labels in various body regions. The abundance of the 14C (nmol 14C g wet weight−1) varied among body regions. The majority of the label was associated with the respiratory trees. The next greatest concentration was in the rete mirabile of the hemal system. The amount of 14C within samples of the anterior digestive system, posterior digestive system, and body wall muscle at each sampling time was lower than that of the respiratory trees. The presence and abundance of the 14C label in samples of the hemal system and the physical association between the respiratory tree and the rete mirabile suggest initial uptake by the respiratory tree and subsequent transfer to the hemal system. The iron label from the protein ferritin and the polysaccharide iron dextran entered cells of the respiratory tree luminal epithelium. The presence of the iron label in mesenchyme cells within the connective tissue compartment of the respiratory trees exposed to ferritin further suggests directional transfer of materials. Holothurians with respiratory trees are nutritionally bipolar; the anus serves as a second mouth.

The worsening of tibialis anterior muscle atrophy during recovery post-immobilization correlates with enhanced connective tissue area, proteolysis, and apoptosis

Sustained muscle wasting due to immobilization leads to weakening and severe metabolic consequences. The mechanisms responsible for muscle recovery after immobilization are poorly defined. Muscle atrophy induced by immobilization worsened in the lengthened tibialis anterior (TA) muscle but not in the shortened gastrocnemius muscle. Here, we investigated some mechanisms responsible for this differential response. Adult rats were subjected to unilateral hindlimb casting for 8 days (I8). Casts were removed at I8, and animals were allowed to recover for 10 days (R1 to R10). The worsening of TA atrophy following immobilization occurred immediately after cast removal at R1 and was sustained until R10. This atrophy correlated with a decrease in type IIb myosin heavy chain (MyHC) isoform and an increase in type IIx, IIa, and I isoforms, with muscle connective tissue thickening, and with increased collagen (Col) I mRNA levels. Increased Col XII, Col IV, and Col XVIII mRNA levels during TA immobilization normalized at R6. Sustained enhanced peptidase activities of the proteasome and apoptosome activity contributed to the catabolic response during the studied recovery period. Finally, increased nuclear apoptosis prevailed only in the connective tissue compartment of the TA. Altogether, the worsening of the TA atrophy pending immediate reloading reflects a major remodeling of its fiber type properties and alterations in the structure/composition of the extracellular compartment that may influence its elasticity/stiffness. The data suggest that sustained enhanced ubiquitin-proteasome-dependent proteolysis and apoptosis are important for these adaptations and provide some rationale for explaining the atrophy of reloaded muscles pending immobilization in a lengthened position.

Decorin is a part of the ovarian extracellular matrix in primates and may act as a signaling molecule

Is decorin (DCN), a putative modulator of growth factor (GF) signaling, expressed in the primate ovary and does it play a role in ovarian biology? DCN expression in the theca, the corpus luteum (CL), its presence in the follicular fluid (FF) and its actions revealed in human IVF-derived granulosa cells (GCs), suggest that it plays multiple roles in the ovary including folliculogenesis, ovulation and survival of the CL. DCN is a secreted proteoglycan, which has a structural role in the extracellular matrix (ECM) and also interferes with the signaling of multiple GF/GF receptors (GFRs). However, DCN expression and action in the primate ovary has yet to be determined. Archival human and monkey ovarian samples were analyzed. Studies were conducted using FF and GC samples collected from IVF patients. Immunohistochemistry, western blotting, RT-PCR, quantitative RT-PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA) studies were complemented by cellular studies, including the measurements of intracellular Ca²⁺, reactive oxygen species (ROS), epidermal GF receptor (EGFR) phosphorylation by DCN and caspase activity. Immunohistochemistry revealed strong DCN staining in the connective tissue and follicular thecal compartments, but not in GCs of pre-antral and antral follicles. Pre-ovulatory follicles could not be studied, but DCN was associated with connective tissue of CL samples and the cytoplasm of luteal cells. DCN expression in monkey CL doubled (P < 0.05) towards the end of the luteal lifespan. DCN was found in human FF obtained from IVF patients (mean: 12.9 ng/ml; n = 20) as determined by ELISA. DCN mRNA and/or protein were detected in freshly isolated and cultured, luteinized human GCs. In the latter, exogenous human recombinant DCN increased intracellular Ca²⁺ levels and induced the production of ROS in a concentration-dependent manner. DCN, like epidermal GF, phosphorylated EGFR significantly (P < 0.05) and reduced the activity of caspase 3/7 in cultured GCs. The data indicate the expression of DCN in the theca of growing follicles, in FF of ovulatory follicles and in the CL. Therefore, DCN may exert paracrine actions via GF/GFR systems in multiple ovarian compartments. Functional studies were performed in cultures of human luteinized GCs, which are an apt model but may not fully mirror the pre-ovulatory GC compartment or the CL. Other human ovarian cells, including the thecal cells, were not available. In accordance with its evolving roles in other organs, ovarian DCN is an ECM-associated component, which acts as a multifunctional regulator of GF signaling in the primate ovary. DCN may thus be involved in folliculogenesis, ovulation and the regulation of the CL survival in primates. This study was supported by Deutsche Forschungsgemeinschaft (DFG) MA1080/17-3 and in part DFG MA1080/21-1 (to AM), NIH grants HD24870 (S.R.O. and R.L.S.), the Eunice Kennedy Shriver NICHD/NIH through cooperative agreement HD18185 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research (S.R.O.) and 8P51OD011092-53 for the operation of the Oregon National Primate Research Center (G.A.D., J.D.H., S.R.O. and R.L.S).