Plasma gelsolin promotes re-epithelialization.

Woundhealing disorders characterized by impaired or delayed re-epithelialization are a serious medical problem that is painful and difficult to treat. Gelsolin (GSN), a known actin modulator, supports epithelial cell regeneration and apoptosis. The aim of this study was to estimate the potential of recombinant gelsolin (rhu-pGSN) for ocular surface regeneration to establish a novel therapy for delayed or complicated wound healing. We analyzed the influence of gelsolin on cell proliferation and wound healing in vitro, in vivo/ex vivo and by gene knockdown. Gelsolin is expressed in all tested tissues of the ocular system as shown by molecular analysis. The concentration of GSN is significantly increased in tear fluid samples of patients with dry eye disease. rhu-pGSN induces cell proliferation and faster wound healing in vitro as well as in vivo/ex vivo. TGF-ß dependent transcription of SMA is significantly decreased after GSN gene knockdown. Gelsolin is an inherent protein of the ocular system and is secreted into the tear fluid. Our results show a positive effect on corneal cell proliferation and wound healing. Furthermore, GSN regulates the synthesis of SMA in myofibroblasts, which establishes GSN as a key protein of TGF-ß dependent cell differentiation.

Efficacy of aflibercept (EYLEA®) on inhibition of human VEGF in vitro.

INTRODUCTION: Pathological formation of blood vessels plays a key role in the growth and metastasis of tumors and also in several serious ophthalmological diseases such as wet age-related macular degeneration (AMD) or diabetic retinopathy. In AMD treatment, aflibercept (tradename EYLEA®) is used to deactivate the underlying pathological neovascularisation. Aflibercept is a recombinant fusion protein which binds to vascular endothelial growth factor (VEGF) receptors, thereby inhibiting VEGF pathway activation. VEGF is one of the most important angiogenesis factors. OBJECTIVE: This analysis investigates lasting efficacy of aflibercept in vitro for later application as therapeutic agent against macular degeneration (AMD). MATERIAL AND METHODS: VEGF-ELISA assays were performed to investigate binding affinities at different aflibercept concentrations. The impact of VEGF on the proliferation of human umbilical vein endothelial cells (HUVEC) was investigated using proliferation assays. Moreover, time-dependent kinetic studies were performed to analyze different aflibercept storage durations with regard to its inhibitory capabilities on human VEGF. RESULTS AND CONCLUSION: Our results reveal that aflibercept significantly lowers the amount of unbound VEGF as well as the proliferation rate of HUVEC. Moreover, in contrast to specifications given by the manufacturer, aflibercept retains its full inhibitory effect up to at least 120h after transference from the original vial into the injection syringe.

Trefoil factor family peptide 3 at the ocular surface. A promising therapeutic candidate for patients with dry eye syndrome?

Dry eye syndrome is a widespread disease accompanied by discomfort and potential visual impairments. Basic causes are tear film instability, hyperosmolarity of the tear film, increased apoptosis as well as chronic inflammatory processes. During the last decades, our understanding of dry eye syndrome has considerably increased. However, the molecular mechanisms of the disease remain largely elusive. In this context, our group focuses on trefoil factor 3 (TFF3). Among other factors, TFF3 performs a broad variety of protective functions on surface epithelium. Its main function seems to be in enhancing wound healing by promoting a process called 'restitution'. Studies evaluating TFF3 properties and effects at the ocular surface using in vivo as well as in vitro models have revealed a pivotal role of TFF3 in corneal wound healing. Subsequent studies in osteoarthritic cartilage seem to draw a different picture of TFF3, which still needs further elucidation. This manuscript summarizes the findings concerning TFF3 in general and its role in the cornea as well as articular cartilage - two tissues which have some things in common. It also discusses the potential of TFF3 as a candidate therapeutic agent for the treatment of, for example, ocular surface disorders.

Expression and regulation of human beta-defensin-2 in osteoarthritic cartilage.

Defensins are antibiotic peptides that are involved in host defence at epithelial and mesenchymal surfaces. Previous studies have shown the induction of human beta-defensin-3 (HBD-3) in osteoarthritic joints, suggesting that these molecules have functions in addition to their ability to kill microbes. The aim of this study was to investigate the production of a further human beta-defensin, named HBD-2, in osteoarthritis (OA) and to determine its regulation by inflammatory cytokines. Healthy and osteoarthritic cartilage was assessed for HBD-2 expression by RT-PCR, immunohistochemistry, and ELISA. C28/I2 chondrocytes, primary chondrocytes, and cartilage explants were cultured for in vitro studies. After 24 h of stimulation with tumour necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) or IL-6, real-time RT-PCR and ELISA experiments were performed to evaluate the effect of these cytokines on the production of HBD-2. In contrast to healthy cartilage, HBD-2 expression was identified in most of the OA samples examined (eight of ten). Cytokines that are potentially involved in the pathogenesis of OA, namely TNF-alpha, IL-1, and IL-6, were transcriptional inducers of HBD-2 in cultured chondrocytes and cartilage explants in vitro, as measured by real-time RT-PCR and ELISA. These results illustrate the induction of HBD-2 in osteoarthritic cartilage and suggest that it is a further factor in the pathogenesis of OA. However, further studies are required to elucidate the role played by HBD-2 in osteoarthritic cartilage.

[Ultrastructural analysis of secretion-transport through the parotid duct]. / Ultrastukturelle Analysen zum Sekrettransport im Ductus parotideus.

BACKGROUND: Mechanisms of secretion transport through the parotid duct (stenon's duct) and their influence on diseases of the parotid gland have not been investigated sufficiently until today. METHODS: According to this background we performed histologic and scanning electron microscopical investigations of 23 parotid ducts in order to investigate the arrangement of fibrillar structures of the duct as well as get deeper insights into the physiology of secretion transport mechanisms. RESULTS: The subepithelial soft tissue of the parotid duct could be divided into two layers. The inner layer measured about 100-200 microm and consisted of collagen and elastic fibres running in a spiral arrangement. The outer layer varied in its thickness and was composed of collageous fibres, which were mostly arranged longitudinally. CONCLUSIONS: The arrangement of collagen fibrils in the wall of the parotid duct seems to influence secretion transport. Due to the spiral organization of collagen fibrils, distension of the duct is likely to be associated with a "wring-out" mechanism leading to unidirectional transport of saliva into the oral cavity. A muscular sphincter at the outlet of the parotid duct did not exist. The duct pierced the buccinatory muscle. Here, it was surrounded by sceletal muscle fibres that lead to a functional closure during contraction. Our results indicate that an active transport of saliva through the parotid duct--as so far assumed--is unlikely.

Antimicrobial defence mechanisms of the human parotid duct.

The parotid duct transports saliva from the gland into the oral cavity. However, its immune response properties, along with the secretion and moistening principles of the duct, have not yet been fully investigated. These properties may play an important role in protecting the parotid gland from infection and also prevent development of sialodocholithiasis, as the parotid duct -- in contrast to the submandibular salivary duct -- is often free of duct concrements. Up to now, only the parotid gland has been investigated, without regard to its duct. The present study analyses the structures of the parotid duct in their relations to antimicrobial defence mechanisms and rheological properties. Investigations were performed on 23 parotid ducts using histological, histochemical and immunohistochemical methods. Epithelial and goblet cells of the parotid duct synthesize a complex mucous layer that covers the epithelium. The viscosity is influenced by secreted mucins and TFF peptides. This layer contains carbohydrates including N-acetyl-glucosamine, N-acetyl-galactosamine, galactose, mannose, fucose and sialic acids. The lamina propria contains granulocytes, T lymphocytes and macrophages. IgA, produced by plasma cells in the subepithelial layer, is frequently integrated in the secretory product. Synthesized mucins, TFF peptides, carbohydrates and immunoglobulins form a complex layer that can be expected to prohibit infection and enables salivary flow. Our study demonstrates that the steady secretion of the parotid gland, together with the ductal cellular and biochemical immune protection system, is likely to thwart ascending infections in the parotid duct and gland.

Functional anatomy of the papilla Vateri: biomechanical aspects and impact of difficult endoscopic intubation.

BACKGROUND: Problems with intubation of the ampulla Vateri during diagnostic and therapeutic endoscopic maneuvers are a well-known feature. The ampulla Vateri was analyzed three-dimensionally to determine whether these difficulties have a structural background. METHODS: Thirty-five human greater duodenal papillae were examined by light and scanning electron microscopy as well as immunohistochemically. RESULTS: Histologically, highly vascularized finger-like mucosal folds project far into the lumen of the ampulla Vateri. The excretory ducts of seromucous glands containing many lysozyme-secreting Paneth cells open close to the base of the mucosal folds. Scanning electron microscopy revealed large mucosal folds inside the ampulla that continued into the pancreatic and bile duct, comparable to valves arranged in a row. CONCLUSIONS: Mucosal folds form pocket-like valves in the lumen of the ampulla Vateri. They allow a unidirectional flow of secretions into the duodenum and prevent reflux from the duodenum into the ampulla Vateri. Subepithelial mucous gland secretions functionally clean the valvular crypts and protect the epithelium. The arrangement of pocket-like mucosal folds may explain endoscopic difficulties experienced when attempting to penetrate the papilla of Vater during endoscopic retrograde cholangiopancreaticographic procedures.

New insights into the pathophysiology of primary acquired dacryostenosis.

OBJECTIVE: To obtain new insights into the pathophysiology of primary acquired dacryostenosis. DESIGN: Comparative autopsy tissue study with histopathologic correlations. MATERIALS: Tissue specimens from the human nasolacrimal ducts of 36 patients undergoing endonasal dacryocystorhinostomy within a framework of primary acquired dacryostenosis were analyzed by histologic studies and electron microscopic examination. Six lacrimal systems of body donors served as controls. TESTING: One group of tissue specimens from each lacrimal system was prepared and processed with paraffin, sectioned, stained by different methods, and finally examined by light microscopy. The other group was processed with araldite after preparation, sectioned semithin and ultrathin, and examined by transmission electron microscopy. MAIN OUTCOME MEASURES: The degree of dacryostenosis was scored in each tissue specimen by grading the histologic sections as mild (active chronic inflammation), moderate (proliferative sclerotic forms of chronic fibrosis), or severe (total subepithelial fibrosis). RESULTS: Of 36 patients with epiphora, 13 had functional obstruction with a patent lacrimal system on syringing; in 23 cases, the lacrimal passage was completely obstructed. Different pathologic stages correlating to duration of symptoms were found ranging from active chronic inflammation to proliferative sclerotic forms and total subepithelial fibrosis. CONCLUSIONS: Descending inflammation from the eye or ascending inflammation from the nose initiates swelling of the mucous membrane, remodeling of the helical arrangement of connective tissue fibers, malfunctions in the subepithelial cavernous body with reactive hyperemia, and temporary occlusion of the lacrimal passage. In the follow-up, repeated isolated occurrence of dacryocystitis leads to structural epithelial and subepithelial changes, which may lead either to a total fibrous closure of the lumen of the efferent tear duct or to a nonfunctional segment in the lacrimal passage that is manifest on syringing.

Detection of natural peptide antibiotics in human nasolacrimal ducts.

PURPOSE: To determine the expression and production of antimicrobial peptides by mucosal cells of the lacrimal passage in healthy and pathologic states. METHODS: Detection of bactericidal-permeability-increasing protein (BPI), heparin-binding protein (CAP37), human cationic antimicrobial protein (LL-37), human alpha-defensin 5 (HD5), human alpha-defensin 6 (HD6), human beta-defensin 1 (HBD-1), and human beta-defensin 2 (HBD-2) was performed by reverse transcription-polymerase chain reaction (RT-PCR). Intracellular deposition of lysozyme, lactoferrin, secretory phospholipase A(2), human neutrophil defensins (HNP-1, -2, and -3), human beta-defensin 1 (HBD-1), and human beta-defensin 2 (HBD-2) was analyzed immunohistochemically. Samples were obtained from 15 patients by surgery and from 10 cadavers. RESULTS: RT-PCR revealed BPI, CAP37, and HBD-1 mRNA in samples of healthy nasolacrimal duct epithelium. Additionally, HBD-2 mRNA was detected in epithelial samples from patients with dacryocystitis. Messenger RNAs for LL-37 and alpha-defensin 5 and 6 were absent in all samples investigated. Immunohistochemistry revealed lysozyme, lactoferrin, secretory phospholipase A(2), and HNP-1, -2, and -3 to be present in all samples, whereas HBD-1 was present only in some of the healthy and inflamed samples. Immunoreactive HBD-2 peptide was visible only in some of the inflamed samples. CONCLUSIONS: The data suggest that the human efferent tear ducts produce a broad spectrum of antimicrobial peptides. Under inflammatory conditions, changes in the expression pattern occurred, revealing induction of the human inducible defensin HBD-2 and in some cases downregulation of HBD-1 and CAP37. Antimicrobial peptides have a therapeutic potential in dacryocystitis, in that they have a broad spectrum of antimicrobial activity and accelerate epithelial healing. However, caution is appropriate, because defensins also promote fibrin formation and cell proliferation, which are key elements in scarring processes, such as dacryostenosis.

Similarities and differences in lectin cytochemistry of laryngeal and tracheal epithelium and subepithelial seromucous glands in cases of sudden infant death and controls.

BACKGROUND: It has been speculated that non-specific defence mechanisms of the epithelium and subepithelial seromucous glands play a role in the larynx and lungs in cases of sudden infant death. METHODS: The larynx and trachea from five children who had died of sudden infant death (SID) syndrome and five control cases of comparable age were compared for the presence of lectin binding sites (12 different lectins tested). RESULTS: The secretory product of mucin producing cells contained carbohydrates including galactose and sialic acids. Binding sites for fucose and N-acetyl-galactosamine were only present in some of the specimens and distribution revealed no correlation between cases of SID and controls. Epithelial cells and serous cells of seromucous glands contained binding sites for sialic acid in cases of SID and controls. Moreover, binding sites for mannose were detected in these cells but were only present in SID cases. The difference between the SID and control groups as to the presence/expression of concanavalin A was highly significant. CONCLUSIONS: It is suggested that mucus hypersecretion in SID occurs in response to bacterial toxins or viral infection and is not specific. The different binding sites for mannose in cases of SID and controls could indicate differences in the production of antimicrobial peptides. A disturbed expression pattern of antimicrobial peptides in children who later succumb to SID could be responsible for an imbalance of the local microflora with a higher density of microorganisms on the mucosa. Further studies are required to elucidate the pattern of expression of antimicrobial peptides in subsequent SID victims.

Mucosa-associated lymphoid tissue in human efferent tear ducts.

The recent description of primary marginal zone lymphomas in human efferent tear ducts with typical features of lymphomas arising in mucosa-associated lymphoid tissue (MALT) infers the presence of MALT in the human efferent tear ducts. To date, studies have not established clearly whether organised MALT occurs in normal human efferent tear ducts. To elucidate this problem, efferent lacrimal pathways from unselected body donors with unknown prior history of efferent tear duct, ocular, or nasal disease were examined for the presence of organised MALT. Organised lymphoid tissue was found with the cytomorphological and immunophenotypic features of MALT in 41% of the cases examined. These findings suggest that MALT is a feature that, although it need not be present in normal efferent tear ducts, is acquired during life in a proportion of apparently asymptomatic individuals.

The cricoarytenoid joint capsule and its relevance to endotracheal intubation.

UNLABELLED: Impaired movement of the cricoarytenoid joint with hoarseness and immobility of the vocal ligament may occur as a consequence of endotracheal intubation. Little is known about the cricoarytenoid joint capsule and its role in intubation. We investigated the joint capsules of 48 cricoarytenoid joints by means of gross anatomy microscopy, histology, and scanning electron microscopy; 30 unfixed cadaver larynges were also subjected to attempts to simulate traumata such as those that may occur during intubation trials. The larynges were intubated with the arytenoid tip entering the lumen of the tracheal tube or extubated with the cuff of the tube only partially deflated. Subsequently, i.e., after dissecting the left and right cricoarytenoid joint from each larynx, the morphologic changes induced experimentally were analyzed by using histologic methods. The cricoarytenoid joint was found to be lined by a wide joint capsule. Unexpectedly large and intensively vascularized synovial folds projected into the joint cavity. After simulation of intubation and extubation, histologic analysis revealed injuries to the synovial folds and joint surface impressions, but no trauma or rupture of the outer joint capsule. We conclude that laxity of the joint capsule and the large synovial folds are predisposing factors for intubation trauma of the cricoarytenoid joint, potentially leading to hemarthros and finally to cricoarytenoid joint dysfunction. IMPLICATIONS: The present study illustrates by morphological investigations and intubation experiments that laxity of the joint capsule and large synovial folds are predisposing factors for intubation trauma of the cricoarytenoid joint, potentially leading to hemarthrosis and finally to cricoarytenoid joint dysfunction.

The cavernous body of the human efferent tear ducts: function in tear outflow mechanism.

PURPOSE: To determine the structure and function of a system of large blood vessels integrated in the bony canal between the orbit and the inferior nasal duct. METHODS: Thirty-one dissected lacrimal systems of adults were analyzed by using gross anatomy, histology, and electron microscopy as well as corrosion vascular casts. RESULTS: More than two thirds of the bony canal between orbit and inferior nasal duct is filled by a plexus of wide-lumened veins and arteries. The vascular system is embedded in the wall of the lacrimal sac and nasolacrimal duct and is connected to the cavernous tissue of the inferior turbinate. Three types of blood vessels can be distinguished inside the vascular tissue that surrounds the lumen of the lacrimal passage: barrier arteries, capacitance veins, and throttle veins. CONCLUSIONS: The surrounding vascular plexus of the lacrimal sac and nasolacrimal duct is comparable to a cavernous body. While regulating the blood flow, the specialized blood vessels permit opening and closing of the lumen of the lacrimal passage, effected by the bulging and subsiding of the cavernous body, and at the same time regulate tear outflow. Other functions such as drainage of absorbed tear fluid components and a role in immunologic response are under discussion as well. Malfunctions in the cavernous body may lead to disturbances in the tear outflow cycle, ocular congestion, or total occlusion of the lacrimal passages. Variations in the conditions for swelling of the cavernous tissue may have led to the (mistaken) description of valves in the lacrimal passage.

Osteoarthritis in cricoarytenoid joint.

OBJECTIVE: Occurrence of osteoarthritis is a frequent event of limb joints in people over 40 years of age. The human cricoarytenoid joint is comparable with the joints of the limbs despite its structure and extracellular matrix composition. To date, little is known about the occurrence of osteoarthritis in the human cricoarytenoid joint. METHODS: Sixty-eight cricoarytenoid joints (42 male and 26 female, age 25-98 years) were analysed by means of histology, lectin histochemistry, immunohistochemistry as well as scanning and transmission electron microscopy. RESULTS: About 50% of the investigated cricoarytenoid joints aged over 40 years show degenerative changes in their joint surface structure at varying levels of intensity. The articular cartilage surface is fibrillated in some places and sometimes shows fissures. A demascing of collagen fibrils next to the joint surface and a loss of proteoglycans in the upper cartilage layers can be observed. Chondrocytes near the joint surface appear as voluminous chondrocyte clusters. The clusters and the superficial cartilage layer show a positive reaction to type VI collagen antibodies. The distribution patterns of lectins are completely changed in fibrillated cartilage areas. CONCLUSION: Degenerative alterations in diarthrodial joints resembling osteoarthritis can occur in the joints of the larynx. These structural changes of the articular cartilage are comparable to osteoarthritis of the limb joints. Osteoarthritis in the cricoarytenoid joint may lead to impaired movements of the arytenoid cartilages. Functionally the structural changes may lead to negative consequences during vocal production, such as impaired vocal quality and reduced vocal intensity.

New insights into the pathomechanism of postintubation arytenoid subluxation.

BACKGROUND: Impaired movement of the cricoarytenoid joint with hoarseness and immobility of the vocal ligament can occur as a consequence of endotracheal intubation. The biomechanics and pathomechanism of cricoarytenoid subluxation have not been demonstrated to date. METHODS: The present study attempts to simulate the trauma that has been associated with arytenoid cartilage subluxation in intubation trials on 37 unfixed larynges in cadavers from persons aged 25 to 89 years. Larynges were intubated or extubated according to former conceptions of arytenoid subluxation, which assume that the arytenoid tip enters the lumen of the tracheal tube, or that a deflection of the arytenoid occurs during withdrawal of the endotracheal tube with the cuff of the tube only partially deflated. Also, manual attempts were carried out to subluxate the arytenoid cartilage. Subsequently after dissecting the left and right cricoarytenoid joint from each larynx, the morphologic changes induced experimentally were analyzed using gross microscopic and histologic methods. RESULTS: Within the scope of the experiment, it proved impossible to produce any subluxation of a cricoarytenoid joint. Histologic analysis revealed injuries of synovial folds, joint-surface impressions of the articular cartilage, and fractures in the area of the subchondral bone in some joints. CONCLUSIONS: Based on the morphologic results, it was concluded that intubation trauma of the cricoarytenoid joint does not cause subluxation per se, but rather that formation of a hemarthros or fractures of the joint bodies lead to fixation of the joint surfaces in an abnormal position. Subsequent ankylosis may occur.

Degenerative changes in the human cricoarytenoid joint.

OBJECTIVE: Changes in the human voice occur during the natural aging process. Occurrence of compromising alterations in the cricoarytenoid joint has been hypothesized as a possible reason for voice changes seen in advanced age and has been discussed controversially until today. METHODS: The present study analyzes degenerative changes in 42 cricoarytenoid joints from 21 body donors (13 men and 8 women; age range, 42-98 years) by means of histological, immunohistochemical, and scanning electron microscopic methods. RESULTS: Many patients older than 40 years show distinctly altered joint surfaces at varying levels of intensity. The articular cartilage surface is fibrillated in some places. Chondrocytes near the joint surface appear as voluminous chondrocyte clusters. The superficial cartilage layer shows a positive reaction to type III and type I collagen antibodies. CONCLUSIONS: Chondrocyte proliferation next to the joint surface, changed collagen synthesis, and fibrillation of the joint surface indicate degenerative alterations. Such changes are well known in cases of limb diarthroses. The changes may impair gross positional or postural movements of the arytenoid cartilages and reduce the degree and extent of vocal ligament closure. The structural changes may also lead to negative functional consequences during vocal production, such as impaired vocal quality and reduced vocal intensity due to air leakage through incompletely or loosely approximated vocal ligaments.