Neurotransmitter and neurotransmitter receptor expression in the saccule of the human vestibular system.

The saccule is one of the vestibular sensory organs of the inner ear. It detects head movements and provides information to maintain balance and orient in space. Despite its critical role, very little is known about its neurotransmission and regulation. Multiple disease entities and medications affect balance, which is why information on neurotransmission in the vestibular end organs including the saccule could have important pharmacological implications. To the best of our knowledge, this is the first paper to describe immunohistochemical expression of a large panel of neurotransmitters and receptors in the human saccule. Saccular tissue was sampled freshly during surgery. Based partly on previous findings in non-humans and partly on potential biological relevance, the neurotransmitters cholecystokinin, dopamine, GABA, glutamate, histamine and serotonin as well as receptors for these were selected for the tested panel. The neuroepithelium expressed glutamate receptor 1 (GluR1), metabotropic glutamate receptor (mGluR), GABA A receptor α (GABAARα), GABA B receptor 2 and cholecystokinin receptor B (CCKBR), whereas l-glutamate, GluR1, CCKBR, GABAARα, dopamine and serotonin receptor 1D were expressed in the subepithelial stroma. The non-sensory epithelium expressed GluR1, mGluR, histamine receptor 3, CCKAR and dopamine transporter. These findings provide a basis for pharmacological research and potential drug development.

Intravitreal bevacizumab upregulates transthyretin in experimental branch retinal vein occlusion.

Purpose: To identify retinal protein changes that mediate beneficial effects of intravitreal bevacizumab in experimental branch retinal vein occlusion (BRVO). Methods: In six Danish Landrace pigs, BRVO was induced with argon laser in both eyes. After BRVO was induced, the right eye of each animal was given an intravitreal injection of bevacizumab while the left eye was treated with saline water. The retinas were collected 15 days after BRVO, and differentially expressed proteins were analyzed with tandem mass tags-based mass spectrometry. Validation of statistically significantly changed proteins was performed with immunohistochemistry and western blotting. Results: Fluorescein angiography showed no recanalization of the occluded vessels. A total of 4,013 proteins were successfully identified and quantified. Nine proteins were statistically significantly changed following bevacizumab intervention. In experimental BRVO, bevacizumab treatment resulted in upregulation of transthyretin (TTR) and pantothenate kinase 3. Bevacizumab downregulated protocadherin 7, protein FAM192A, and ATP synthase protein 8. Immunohistochemistry revealed that TTR was highly abundant in the choroid following bevacizumab intervention. Conclusions: Bevacizumab intervention in experimental BRVO resulted in an increased level of TTR. This is the second study in which we showed an increased retinal level of TTR following anti-vascular endothelial growth factor (VEGF) intervention in experimental BRVO. We hypothesize that there is an interaction between TTR and VEGF and that bevacizumab may exert a beneficial effect on the retina by upregulating TTR.

Modifications of cholera toxin subunit B binding to human large intestinal epithelium. An immunohistochemical study.

Binding of cholera toxin subunit B (CTB) to its receptor and toxin transport into the intestinal epithelial cells are the causative events for the potentially lethal disease cholera. The five sugar mono-sialo ganglioside GM1 is the cell surface receptor for cholera toxin B-subunit. CTB binding was determined by use of immobilized GM1 to microtiter plates and by immunohistochemistry. Sections from the human colon and the human soft palate were incubated with FITC-conjugated CTB and with anti-MUC2. Both the luminal surface of the intestine and the secretory goblet cells exhibited strong binding. Addition of simple carbohydrates and milk to the incubation medium showed that a combination of lactose and non-fat dry milk was potent inhibitors of toxin- and mucin binding. Both CTB and ant-MUC2 stained to the cytoplasm (mucin granules) in the goblet cells from the human soft palate. In the colon CTB stained the entire cytoplasm of the goblet cells while anti-MUC2 detected only the supranuclear region of some cells, suggesting carbohydrate heterogeneity between goblet cell mucin granules in different regions of the human body. Both CTB- and MUC2 binding were inhibited when GM1 was added to the incubation medium. It is proposed that the human colonic goblet cells play a role in the secretory diarrhea in patients with cholera and that milk might have a prophylactic or therapeutic application in the management of cholera.

Neuronal Fibers and Neurotransmitter Receptor Expression in the Human Endolymphatic Sac.

INTRODUCTION: Recent studies suggest that the human endolymphatic sac (ES) may have multiple functions, including an ion-transport capacity comparable to the kidney, an immunological capacity and a possible natriuretic capacity. Further, there have been speculations of a yet undefined role in intracranial pressure homeostasis. The anatomical location towards the sigmoid sinus would suggest a possible endo- and/or paracrine signaling. However, neuronal connections may also apply, but it remains very scarcely explored in the human ES. STUDY DESIGN: DNA micro-arrays and immunohistochemistry were used for analyses of fresh human ES tissue samples. METHODS: A total of 30 tissue samples from the human ES were obtained during translabyrinthine surgery for vestibular schwannoma. Microarray technology was used to investigate tissue sample gene expression, using adjacent dura mater as control. The expression of genes specific for neuronal signaling was determined and results for selected key molecules verified by immunohistochemistry. Transmission electron microscopy was used for ultrastructural analysis. RESULTS: For the transmission electron microscopy analysis, a direct innervation of the ES was observed with unmyelinated fibers imbedded in the ES epithelial lining. The microarrays confirmed, that several molecules involved in neuronal signaling were found expressed significantly in the ES DNA profile, such as the Cholecystokinin peptide and related receptors, Dopamine receptors 2 and 5, vesicular monoamine transporter 2 (VMAT2), plasma monoamine transporter (PMAT), and Serotonin 1D. All peptides were verified by immunohistochemistry. CONCLUSIONS: Based on global gene expression profiling and immuno-histochemical labeling, we conclude that the human ES expresses neuropeptide receptors and monoamine transporters. Combined with the ultrastructural demonstration of unmyelinated axons imbedded within the epithelial lining, the findings suggest that neuro-signaling mechanisms are involved in functions exerted by the ES.

The human endolymphatic sac expresses natriuretic peptides.

OBJECTIVES/HYPOTHESIS: The function of the human endolymphatic sac (ES) has been enigmatic for decades. Hypotheses include controlling endolymphatic fluid homeostasis and inner ear immunological defense. Additionally, several studies indicate a possible endocrine capacity and a yet undefined role in intracranial pressure homeostasis. However, no direct evidence of such capacity exists. This study aims to explore and identify the hypothesized endocrine capacity of the human ES. STUDY DESIGN: DNA microarrays and immunohistochemistry were used for analyses of fresh human ES tissue samples. METHODS: Twelve tissue samples from the human ES were obtained during translabyrinthine surgery for vestibular schwannoma. Microarray technology was used to investigate tissue sample gene expression. Genes specific for an endocrine function were determined, and results were verified by immunohistochemistry. RESULTS: Several natriuretic peptides were found expressed significantly in the ES, including uroguanylin and brain natriuretic peptide, but also peptides regulating vascular tone, including adrenomedullin 2. In addition, both neurophysin and oxytocin (OXT) were found significantly expressed. All peptides were verified by immunohistochemistry. CONCLUSION: The present data support the hypothesis that the human ES may have an endocrine/paracrine capacity through expression of several peptides with potent natriuretic activity. Furthermore, the ES may influence the hypothalamo-pituitary-adrenal axis and may regulate vasopressin receptors and aquaporin-2 channels in the inner ear via OXT expression. We hypothesize that the ES is likely to regulate inner ear endolymphatic homeostasis, possibly through secretion of several peptides, but it may also influence systemic and/or intracranial blood pressure through direct and indirect action on the vascular system and the kidney. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:E201-E208, 2017.

Innate immune defense in the inner ear - mucines are expressed by the human endolymphatic sac.

The human endolymphatic sac has been shown recently to have immunological capacities and has thus been proposed as the main entity protecting the inner ear from pathogen invasion, equivalent to mucosa-associated lymphoid tissue (MALT). Although the sac expresses molecules of the innate immune system, the potential expression of members of the important mucin family has not been detailed. Thus, this paper explores endolymphatic sac expression of a number of mucins and mucin precursors. Twelve fresh tissue samples from the human endolymphatic sac were obtained during translabyrinthine surgery. The expression of Mucin 1, 2, 5B/AC and 16, as well as the core structure elements (mucin precursors) T-antigen, Tn-antigen and Sialyl-Tn-antigen was investigated by immunohistochemistry. The endolymphatic sac epithelium expressed MUC1 (both apically towards the endolymphatic sac (ES) lumen and basally towards the capillary network), MUC 16 and Tn-antigen. There was no labeling after incubation with antibodies against T-antigen, sialyl-Tn-antigen, MUC2 and MUC5B/AC. We conclude that the human endolymphatic sac epithelium expresses a number of mucin molecules, which supports the hypothesis of the sac as the primary immunological tissue structure of the inner ear, equivalent to MALT in other organs. The mucins may also play a role in the formation and continuous homeostasis of the inner ear fluids, as well as the pathogenesis of Meniere's disease.

Ionized calcium-binding adaptor molecule 1 positive macrophages and HO-1 up-regulation in intestinal muscularis resident macrophages.

Small intestinal muscularis externa macrophages have been associated with interstitial cells of Cajal. They have been proposed to play various roles in motility disorders and to take part in a microbiota-driven regulation of gastrointestinal motility. Our objective was to understand the reaction of resident macrophages of the musculature to a pro-inflammatory stimulator, lipopolysaccharide (LPS). Mice were injected with LPS or saline and sacrificed after 6 hr. Whole mounts were stained with antibodies toward CD169, ionized calcium-binding adaptor molecule 1 (iba1) (microglial/macrophage marker) and heme oxygenase-1 (HO-1). Cell densities were measured using unbiased stereology. RESULTS: iba1pos cells showed an overall higher density than CD169pos and HO-1pos cells. Most HO-1pos and iba1pos cells were positive for CD 169 in serosa and at Auerbach's plexus (AP). At the deep muscular plexus, mainly iba1pos cells were present, and were mostly CD169neg ; a few HO-1pos cells were present. CONCLUSIONS: A new subset of resident macrophages in the intestinal muscularis externa was discovered, identified as iba1pos CD169neg . HO-1 is constitutively present in most macrophages in serosa and at AP, suggesting a M2 phenotype. LPS-treatment results in an up-regulation of HO-1pos /CD169neg cells in serosa and at AP. Anat Rec, 300:1114-1122, 2017. © 2016 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Gene expression demonstrates an immunological capacity of the human endolymphatic sac.

OBJECTIVES/HYPOTHESIS: The purpose of the present study is to explore, demonstrate, and describe the expression of genes related to the innate immune system in the human endolymphatic sac. It is hypothesized that the endolymphatic sac has a significant immunological function in the human inner ear. STUDY DESIGN: DNA microarrays and immunohistochemistry were used for analyses of fresh human endolymphatic-sac tissue samples. METHODS: Twelve tissue samples from the human endolymphatic sac were obtained during translabyrinthine surgery for vestibular schwannoma. Microarray technology was used to investigate tissue sample gene expression using adjacent dura mater as control. The expression of genes specific for the innate immune system was determined and results for selected key molecules verified by immunohistochemistry. RESULTS: A comprehensive overview of expressed genes of the innate immune system was obtained. Multiple key elements of both the cellular and humoral innate immune system were expressed, including Toll-like receptors 4 and 7, as well as beta-defensin and lactoferrin. CONCLUSIONS: The present data provides the first direct evidence of an immunological capacity of the human endolymphatic sac. At the molecular level, the endolymphatic sac is capable of antigen recognition and processing for initiation of an immune response. In addition, potent molecules directly toxic to invading pathogens are expressed by the sac epithelium. This evidence strongly supports the endolymphatic sac as a significant immunological entity of the inner ear. LEVEL OF EVIDENCE: N/A.

Gene expression in the human endolymphatic sac: the solute carrier molecules in endolymphatic fluid homeostasis.

OBJECTIVES/HYPOTHESIS: The purpose of the present study is to explore, demonstrate, and describe the expression of genes related to the solute carrier (SLC) molecules of ion transporters in the human endolymphatic sac. STUDY DESIGN: cDNA microarrays and immunohistochemistry were used for analyses of fresh human endolymphatic sac tissue samples. METHODS: Twelve tissue samples of the human endolymphatic sac were obtained during translabyrinthine surgery for vestibular schwannoma. Microarray technology was used to investigate tissue sample expression of solute carrier family genes, using adjacent dura mater as control. Immunohistochemistry was used for verification of translation of selected genes, as well as localization of the specific protein within the sac. RESULTS: An extensive representation of the SLC family genes were upregulated in the human endolymphatic sac, including SLC26a4 Pendrin, SLC4a1 sodium-bicarbonate transporter, SLC9a2 sodium-hydrogen transporter, SLC12a3 thiazide-sensitive Na-Cl transporter, and SLC34a2 sodium-phosphate transporter. CONCLUSIONS: Several important ion transporters of the SLC family are expressed in the human endolymphatic sac, including Pendrin, the thiazide-sensitive Na-Cl transporter, and the Na-phosphate transporter SLC34a2. The data provide a new knowledge base considering the ion-dependent metabolic mechanisms maintaining inner ear homeostasis. More specifically, the results indicate a strong similarity with the ion transportation occurring in the kidney collecting ducts. In addition, the findings prompt a revision of the theories behind contemporary pharmacological treatment of Ménière's disease and may broaden the understanding of the pathogenesis of BPPV.

Carbohydrate determinants in ferret conjunctiva are affected by infection with influenza H1N1 virus.

BACKGROUND: Carbohydrates often accomplish as cell-surface receptors for microorganisms and influenza virus preferentially binds to sialic acid through the viral haemagglutinin. The virus may attach not only to the epithelium in the airways, but also to the surface ocular epithelium. PURPOSE: To decide if ferrets can be used to study virus induced conjunctivitis and to evaluate changes in the conjunctival glycosylation pattern during an influenza attack. METHODS: Ferrets were infected with H1N1 influenza virus via nasal inoculation. The in situ carbohydrate expressions in eyelid sections from ferrets 0 to 10 days after infection was examined using lectin- and immunohistochemistry. RESULTS: The conjunctival cells became hypertrophic with appearance of both PAS positive and PAS + Alcian Blue stained cells 5-6 days after inoculation. The binding of three sialic acid detecting lectins were investigated: WGA, MAA2 and SNA1. While none of them stained conjunctival epithelial cells in the non-infected ferrets to any extent, there was a positive conjunctival reaction in the infected ferret after incubation with all three lectins. Binding of a MUC1 antibody that seems to detect sialylated determinants in the mucin molecule indicates that MUC1 is de novo expressed in most of the squamous conjunctival cells at the start of the influenza infection. MUC5AC positive epithelial cells, probably goblet cells, proliferate in the diseased conjunctiva. CONCLUSION: Nasal inoculation of H1N1 virus to ferrets has an effect on the conjunctival cells and change their expression of glycans. Synthesized glycans are an integral part of the tear film and the present study contributes to reveal the changes that occur in the surface epithelium in the eyelid and thereby to elucidate the pathophysiology of the virus mediated conjunctivitis. Ferrets are suitable animal models to study human conjunctivitis mediated by human influenza virus.

Expression of the carcinoma markers: the sialylated Lewis A and X carbohydrate antigens in normal laryngeal surface epithelium and submucosal glands from old humans.

Aberrant surface expression of the carbohydrate ABH and Lewis antigens are often used as markers for the diagnosis of cancer, but while the distribution of these histo-blood group antigens is relatively well-described in tissues and organs from young and middle-aged humans little is known of their expression in old age. The objective for this study was to estimate if the Lewis A and X antigens together with their sialylated modifications, are expressed in sections of normal laryngeal tissue from old humans. Antibodies directed against the tumor markers Sialyl Lewis A and Sialyl Lewis X showed positive reaction in the surface epithelia from normal larynx autopsies obtained from people aged 77-90 years. The sialylated and non-sialylated Lewis A antigens were more frequently expressed in the pseudostratified epithelium than in squamous surface epithelium. Both the sialylated and the non-sialylated carbohydrates were stained in the submucosal glands in all the autopsies. In conclusion, visualization of Lewis tumor markers in the larynx should be interpreted with great care, as they may be present in normal laryngeal epithelial cells from old humans.

MUC1 and the simple mucin-type antigens: Tn and Sialyl-Tn are differently expressed in salivary gland acini and ducts from the submandibular gland, the vestibular folds, and the soft palate.

OBJECTIVE AND DESIGN: Autopsies of the submandibular gland, the vestibular folds and the soft palate from 65-87 old humans were examined to record the immunohistochemical expression of MUC1 and the simple mucin-type antigens Tn and Sialyl-Tn. RESULTS: (1) The serous acini in the submucosal glands from the larynx and the soft palate expressed MUC1-associated glycans that were not detectable in the serous acini from the submandibular gland. (2) Virtually all the submucosal acini at oral site of the soft palate are mucous, and in contrast to mucous acini in the vestibular folds and submandibular gland, the palatinal acini in the submucosa underneath the oral mucosa showed a well-defined cytoplasmic reaction with anti-MUC1 antibodies as wells as with anti-Tn. (3) Both the mucous acini and the ducts at the oral site of the soft palate showed reaction for Sialyl-Tn while in the vestibular folds and in the submandibular gland expression for this carbohydrate was observed only in the acini. (4) The staining obtained after incubation with the Tn antibodies showed no cross localization with the staining obtained after incubation with an anti-A blood group antibody. (5) All the autopsies showed reaction in the glands after incubation with the MUC1 antibodies while some autopsies reacted with the anti-Tn antibodies and/or with the anti-Sialyl-Tn antibodies and others did not. CONCLUSION: The mucin expression in the acini and ducts from the upper human aerodigestive tract strongly depended on the location of the glandular tissue.

Asthma induction in mice leads to appearance of alpha2-3- and alpha2-6-linked sialic acid residues in respiratory goblet-like cells.

Allergic asthmatic inflammation in mice was induced by sensitization with ovalbumin and lipopolysaccharide from Escherichia coli and visualized in the airways of asthmatic mice by spatial and temporal changes of carbohydrates containing sialic acid residues. Immunohistochemistry was used to demonstrate binding of lectins and antibodies that detect alpha2-3- and alpha2-6-linked sialic acid residues. After sensitization and challenge, the histology of the lung changed markedly, and goblet-like cells appeared, most likely caused by Clara cell metaplasia. Normal Clara cells showed no reaction after incubation with the sialic acid detecting agents, while the goblet-like cells expressed both alpha2-3- and alpha2-6-linked sialic acid residues in the asthmatic animals. The lectins but not the antibodies reacted with intestinal goblet cells. Instead, an antibody recognizing a disialoganglioside, stained large mononuclear cells in the submucosa, indicating a difference in sialylation between goblet cells in the intestine and goblet-like cells developed from Clara cells.

The mink as an animal model for Pseudomonas aeruginosa adhesion: binding of the bacterial lectins (PA-IL and PA-IIL) to neoglycoproteins and to sections of pancreas and lung tissues from healthy mink.

Lung infection with Pseudomonas aeruginosa, leading to chronic lung disease with impaired function, is the major course of morbidity and mortality among cystic fibrosis patients. The bacterium produces two lectins that bind to alpha-D-galactose (PA-IL) and L-fucose (PA-IIL), respectively, and lectin-carbohydrate interactions may be involved in microbial pathogenicity by creating bacterial adherence to epithelial and endothelial cells. An ideal animal model for P. aeruginosa infection has until now not been established, but the mink seems to be the only animal that has been reported to develop spontaneous P. aeruginosa infections in the airways. Since cystic fibrosis also severely may affect pancreatic function, we incubated sections from mink lungs and pancreas with a medium containing Pseudomonas lectins in order to detect in situ binding of the bacterial lectins. In the lungs, both lectins adhered to seromucinous glands located in the submucosa of the larger bronchi. Additionally, PA-IL reacted with the capillaries in the alveolar walls and with the small blood vessels forming the vasa vasorum around the larger vessels, while PA-IIL marked the goblet cells in the bronchial surface epithelium. In the pancreas, both lectins bound to the epithelium in the excretory ducts, and additionally, PA-IL strongly stained the pancreatic capillaries while PA-IIL staining was noticed in the apical part of acinar cells in the exocrine part of the gland while no lectin reaction could be recorded in the endocrine cells. Judging from the results in the present paper the mink should be considered a suitable model to study P. aeruginosa adherence.

Quantitative immunohistochemistry of fluorescence labelled probes using low-cost software.

This paper describes simple procedures to process digital images in quantitative immunofluorescence microscopy. Monoclonal antibodies directed against the sarcoplasmic myosin heavy chain isoforms and against laminin, located on the basement membranes, were applied to sections of human skeletal muscle. The localisation and staining intensity of a fluorescent secondary antibody were recorded using an indirect histochemical method. The digitised images were pre-processed and the luminosities of appropriate structures were determined using existing tools in the widely used image processing software Photoshop from Adobe. Procedures to obtain a quantitative measure for the specific fluorescence signal (the background corrected fluorescence in the object) were developed. In addition, antibody binding to individual cells could be quantified whether these cells are well separated or not. The relation between the specific fluorescence signal and the dilution factor of the primary antibody could be measured to determine a suitable concentration of the antibody for incubation of the sections. The potential fading of the fluorescence signal with time and prolonged exposure to light from the microscope was explored and analysed. With the tools described in the present report it is thus possible also to optimize the topical immunohistochemical protocol in order to quantify the fluorescence signal.

Comparison of the binding properties of the mushroom Marasmius oreades lectin and Griffonia simplicifolia I-B isolectin to alphagalactosyl carbohydrate antigens in the surface phase.

The binding of two alpha-galactophilic lectins, Marasmius oreades agglutinin (MOA), and Griffonia simplicifolia I isolectin B(4) (GS I-B(4)) to neoglycoproteins and natural glycoproteins were compared in a surface phase assay. Neoglycoproteins carrying various alpha-galactosylated glycans and laminin from basement membrane of mouse sarcoma that contains the xenogenic Galalpha1-3Gal1-4GlcNAc epitope were immobilized in microtiter plate wells and lectin binding determined with an enzyme-linked assay. After 24 h of incubation, MOA had higher affinity for the xenogenic pentasaccharide (Galalpha1-3Gal1-4GlcNAcbeta1-3Galbeta1-4Glc) than for the Galalpha-monosaccharide. The binding properties of MOA and GS I-B(4) to the xenogenic disaccharide (Galalpha1-3Galbeta1) were comparable while the binding of MOA to the xenogenic pentasaccharide was much stronger than the binding of GS I-B(4) to the same epitope. Non-xenogenic disaccharide-coupled neoglycoproteins having galactose end groups linked alpha1-2 or alpha1-4 to Gal or linked alpha1-3 to GalNAc bound very weakly to MOA, whereas GS I-B(4) recognized all of these disaccharides with similarly high affinity. MOA also showed high affinity for laminin. The results indicate that the Marasmius oreades lectin has nearly the same affinities as does GS I-B(4) for the simple xenogenic carbohydrate antigens, but MOA has greater affinity for the pentasaccharide and is far more specific in its binding preferences than the Griffonia lectin.