Comparative Characterization of Human Meibomian Glands, Free Sebaceous Glands, and Hair-Associated Sebaceous Glands Based on Biomarkers, Analysis of Secretion Composition, and Gland Morphology.

Meibomian gland dysfunction (MGD) is one of the main causes of dry eye disease. To better understand the physiological functions of human meibomian glands (MGs), the present study compared MGs with free sebaceous glands (SGs) and hair-associated SGs of humans using morphological, immunohistochemical, and liquid chromatography-mass spectrometry (LCMS)-based lipidomic approaches. Eyelids with MGs, nostrils, lips, and external auditory canals with free SGs, and scalp with hair-associated SGs of body donors were probed with antibodies against cytokeratins (CK) 1, 8, 10, and 14, stem cell markers keratin 15 and N-cadherin, cell-cell contact markers desmoglein 1 (Dsg1), desmocollin 3 (Dsc3), desmoplakin (Dp), plakoglobin (Pg), and E-cadherin, and the tight junction protein claudin 5. In addition, Oil Red O staining (ORO) was performed in cryosections. Secretions of MGs as well as of SGs of nostrils, external auditory canals, and scalps were collected from healthy volunteers, analyzed by LCMS, and the data were processed using various multivariate statistical analysis approaches. Serial sections of MGs, free SGs, and hair-associated SGs were 3D reconstructed and compared. CK1 was expressed differently in hair-associated SGs than in MGs and other free SGs. The expression levels of CK8, CK10, and CK14 in MGs were different from those in hair-associated SGs and other free SGs. KRT15 was expressed differently in hair-associated SGs, whereas N-cadherin was expressed equally in all types of glands. The cell-cell contact markers Dsg1, Dp, Dsc3, Pg, and E-cadherin revealed no differences. ORO staining showed that lipids in MGs were more highly dispersed and had larger lipid droplets than lipids in other free SGs. Hair-associated SGs had a smaller number of lipid droplets. LCMS revealed that the lipid composition of meibum was distinctively different from that of the sebum of the nostrils, external auditory canals, and scalp. The 3D reconstructions of the different glands revealed different morphologies of the SGs compared with MGs which are by far the largest type of glands. In humans, MGs differ in their morphology and secretory composition and show major differences from free and hair-associated SGs. The composition of meibum differs significantly from that of sebum from free SGs and from hair-associated SGs. Therefore, the MG can be considered as a highly specialized type of holocrine gland that exhibits all the histological characteristics of SGs, but is significantly different from them in terms of morphology and lipid composition.

Ganglioglioma with adverse clinical outcome and atypical histopathological features were defined by alterations in PTPN11/KRAS/NF1 and other RAS-/MAP-Kinase pathway genes.

Exome-wide sequencing studies recently described PTPN11 as a novel brain somatic epilepsy gene. In contrast, germline mutations of PTPN11 are known to cause Noonan syndrome, a multisystem disorder characterized by abnormal facial features, developmental delay, and sporadically, also brain tumors. Herein, we performed a deep phenotype-genotype analysis of a comprehensive series of ganglioglioma (GG) with brain somatic alterations of the PTPN11/KRAS/NF1 genes compared to GG with common MAP-Kinase signaling pathway alterations, i.e., BRAFV600E. Seventy-two GG were submitted to whole exome sequencing and genotyping and 84 low grade epilepsy associated tumors (LEAT) to DNA-methylation analysis. In 28 tumours, both analyses were available from the same sample. Clinical data were retrieved from hospital files including disease onset, age at surgery, brain localization, and seizure outcome. A comprehensive histopathology staining panel was available in all cases. We identified eight GG with PTPN11 alterations, copy number variant (CNV) gains of chromosome 12, and the commonality of additional CNV gains in NF1, KRAS, FGFR4 and RHEB, as well as BRAFV600E alterations. Histopathology revealed an atypical glio-neuronal phenotype with subarachnoidal tumor spread and large, pleomorphic, and multinuclear cellular features. Only three out of eight patients with GG and PTPN11/KRAS/NF1 alterations were free of disabling-seizures 2 years after surgery (38% had Engel I). This was remarkably different from our series of GG with only BRAFV600E mutations (85% had Engel I). Unsupervised cluster analysis of DNA methylation arrays separated these tumours from well-established LEAT categories. Our data point to a subgroup of GG with cellular atypia in glial and neuronal cell components, adverse postsurgical outcome, and genetically characterized by complex alterations in PTPN11 and other RAS-/MAP-Kinase and/or mTOR signaling pathways. These findings need prospective validation in clinical practice as they argue for an adaptation of the WHO grading system in developmental, glio-neuronal tumors associated with early onset focal epilepsy.

DNA methylation-based classification of malformations of cortical development in the human brain.

Malformations of cortical development (MCD) comprise a broad spectrum of structural brain lesions frequently associated with epilepsy. Disease definition and diagnosis remain challenging and are often prone to arbitrary judgment. Molecular classification of histopathological entities may help rationalize the diagnostic process. We present a retrospective, multi-center analysis of genome-wide DNA methylation from human brain specimens obtained from epilepsy surgery using EPIC 850 K BeadChip arrays. A total of 308 samples were included in the study. In the reference cohort, 239 formalin-fixed and paraffin-embedded (FFPE) tissue samples were histopathologically classified as MCD, including 12 major subtype pathologies. They were compared to 15 FFPE samples from surgical non-MCD cortices and 11 FFPE samples from post-mortem non-epilepsy controls. We applied three different statistical approaches to decipher the DNA methylation pattern of histopathological MCD entities, i.e., pairwise comparison, machine learning, and deep learning algorithms. Our deep learning model, which represented a shallow neuronal network, achieved the highest level of accuracy. A test cohort of 43 independent surgical samples from different epilepsy centers was used to test the precision of our DNA methylation-based MCD classifier. All samples from the test cohort were accurately assigned to their disease classes by the algorithm. These data demonstrate DNA methylation-based MCD classification suitability across major histopathological entities amenable to epilepsy surgery and age groups and will help establish an integrated diagnostic classification scheme for epilepsy-associated MCD.

Multilobar unilateral hypoplasia with emphasis on the posterior quadrant and severe epilepsy in children with FCD ILAE Type 1A.

OBJECTIVE: Focal cortical dysplasia (FCD) Type 1 and its three subtypes have yet not been fully characterized at the clinical, anatomopathological, and molecular level (International League Against Epilepsy [ILAE] FCD classification from 2011). We aimed to describe the clinical phenotype of patients with histopathologically confirmed FCD1A obtained from a single epilepsy center between 2002 and 2016. METHODS: Medical records were retrieved from the hospital's archive. Results from electroencephalography (EEG) video recordings, neuroimaging, and histopathology were reevaluated. Magnetic resonance imaging (MRI) post-processing was retrospectively performed in nine patients. DNA methylation studies were carried out from archival surgical brain tissue in 11 patients. RESULTS: Nineteen children with a histopathological diagnosis of FCD1A were included. The average onset of epilepsy was 0.9 years (range 0.2-10 years). All children had severe cognitive impairment and one third had mild motor deficits, yet fine finger movements were preserved in all patients. All patients had daily seizures, being drug resistant from disease onset. Interictal electroencephalography revealed bilateral multi-regional epileptiform discharges. Interictal status epilepticus was observed in 8 and countless subclinical seizures in 11 patients. Regional continuous irregular slow waves were of higher lateralizing and localizing yield than spikes. Posterior background rhythms were normal in 16 of 19 children. Neuroimaging showed unilateral multilobar hypoplasia and increased T2-FLAIR signals of the white matter in 18 of 19 patients. All children underwent tailored multilobar resections, with seizure freedom achieved in 47% (Engel class I). There was no case with frontal involvement without involvement of the posterior quadrant by MRI and histopathology. DNA methylation profiling distinguished FCD1A samples from all other epilepsy specimens and controls. SIGNIFICANCE: We identified a cohort of young children with drug resistance from seizure onset, bad EEG with posterior emphasis, lack of any focal neurological deficits but severe cognitive impairment, subtle hypoplasia of the epileptogenic area on MRI, and histopathologically defined and molecularly confirmed by DNA methylation analysis as FCD ILAE Type 1A.

Modulation of Pancreatic Neuroendocrine Neoplastic Cell Fate by Autophagy-Mediated Death.

INTRODUCTION: Autophagic cell death in cancer cells can be mediated by inhibition of deacetylases. Although extensive studies have focused on the autophagic process in cancer, little is known about the role of autophagy in degrading cytosolic and nuclear components of pancreatic neuroendocrine neoplastic (pNEN) cells leading to cell death, thus improving the therapy of patients affected by pNEN. METHODS: 2D and 3D human pNEN and pancreatic stellate cells were treated with panobinostat and bafilomycin. Autophagy markers were detected by RT-qPCR, immunofluorescence, and Western blot. Autophagosomes were detected by electron microscopy and their maturation by real-time fluorescence of LC3B stable transfected cells. ChIP was performed at the cAMP responsive element. Immunofluorescence was performed in murine pancreatic tissue. RESULTS: We observed that pan-deacetylase inhibitor panobinostat treatment causes autophagic cell death in pNEN cells. We also found that although AMPK-α phosphorylation is counterbalanced by phosphorylated AKT, it is not capable to inhibiting autophagic cell death. However, the binding activity of the cAMP responsive element is prompted by panobinostat. Although autophagy inhibition prevented autophagosome synthesis, maturation, and cell death, panobinostat treatment induced the accumulation of mature autophagosomes in the cytosol and the nucleus, leading to disruption of the organelles, cellular digestion, and decay. Observation of autophagosome membrane proteins Beclin1 and LC3B aggregation in murine pancreatic islets indicates that autophagy restoration may also lead to autophagosome aggregation in murine insulinoma cells. A basal low expression of autophagy markers was detectable in patients affected by pNEN, and, interestingly, the expression of these markers was significantly lower in metastatic pNEN. DISCUSSION/CONCLUSION: Our study highlights that the autophagy functional restoration and prolongation of this catabolic process, mediated by inhibition of deacetylase, is responsible for the reduction of pNEN cells. Prompting of autophagy cell death could be a promising strategy for the therapy of pNEN.

Artificial intelligence facilitates decision-making in the treatment of lumbar disc herniations.

PURPOSE: Apart from patients with severe neurological deficits, it is not clear whether surgical or conservative treatment of lumbar disc herniations is superior for the individual patient. We investigated whether deep learning techniques can predict the outcome of patients with lumbar disc herniation after 6 months of treatment. METHODS: The data of 60 patients were used to train and test a deep learning algorithm with the aim to achieve an accurate prediction of the ODI 6 months after surgery or the start of conservative therapy. We developed an algorithm that predicts the ODI of 6 randomly selected test patients in tenfold cross-validation. RESULTS: A 100% accurate prediction of an ODI range could be achieved by dividing the ODI scale into 12% sections. A maximum absolute difference of only 3.4% between individually predicted and actual ODI after 6 months of a given therapy was achieved with our most powerful model. The application of artificial intelligence as shown in this work also allowed to compare the actual patient values after 6 months with the prediction for the alternative therapy, showing deviations up to 18.8%. CONCLUSION: Deep learning in the supervised form applied here can identify patients at an early stage who would benefit from conservative therapy, and on the contrary avoid painful and unnecessary delays for patients who would profit from surgical therapy. In addition, this approach can be used in many other areas of medicine as an effective tool for decision-making when choosing between opposing treatment options, despite small patient groups.

Mosaic trisomy of chromosome 1q in human brain tissue associates with unilateral polymicrogyria, very early-onset focal epilepsy, and severe developmental delay.

Polymicrogyria (PMG) is a developmental cortical malformation characterized by an excess of small and frustrane gyration and abnormal cortical lamination. PMG frequently associates with seizures. The molecular pathomechanisms underlying PMG development are not yet understood. About 40 genes have been associated with PMG, and small copy number variations have also been described in selected patients. We recently provided evidence that epilepsy-associated structural brain lesions can be classified based on genomic DNA methylation patterns. Here, we analyzed 26 PMG patients employing array-based DNA methylation profiling on formalin-fixed paraffin-embedded material. A series of 62 well-characterized non-PMG cortical malformations (focal cortical dysplasia type 2a/b and hemimegalencephaly), temporal lobe epilepsy, and non-epilepsy autopsy controls was used as reference cohort. Unsupervised dimensionality reduction and hierarchical cluster analysis of DNA methylation profiles showed that PMG formed a distinct DNA methylation class. Copy number profiling from DNA methylation data identified a uniform duplication spanning the entire long arm of chromosome 1 in 7 out of 26 PMG patients, which was verified by additional fluorescence in situ hybridization analysis. In respective cases, about 50% of nuclei in the center of the PMG lesion were 1q triploid. No chromosomal imbalance was seen in adjacent, architecturally normal-appearing tissue indicating mosaicism. Clinically, PMG 1q patients presented with a unilateral frontal or hemispheric PMG without hemimegalencephaly, a severe form of intractable epilepsy with seizure onset in the first months of life, and severe developmental delay. Our results show that PMG can be classified among other structural brain lesions according to their DNA methylation profile. One subset of PMG with distinct clinical features exhibits a duplication of chromosomal arm 1q.

Same same but different: A Web-based deep learning application revealed classifying features for the histopathologic distinction of cortical malformations.

OBJECTIVE: The microscopic review of hematoxylin-eosin-stained images of focal cortical dysplasia type IIb and cortical tuber of tuberous sclerosis complex remains challenging. Both entities are distinct subtypes of human malformations of cortical development that share histopathological features consisting of neuronal dyslamination with dysmorphic neurons and balloon cells. We trained a convolutional neural network (CNN) to classify both entities and visualize the results. Additionally, we propose a new Web-based deep learning application as proof of concept of how deep learning could enter the pathologic routine. METHODS: A digital processing pipeline was developed for a series of 56 cases of focal cortical dysplasia type IIb and cortical tuber of tuberous sclerosis complex to obtain 4000 regions of interest and 200 000 subsamples with different zoom and rotation angles to train a neural network. Guided gradient-weighted class activation maps (Guided Grad-CAMs) were generated to visualize morphological features used by the CNN to distinguish both entities. RESULTS: Our best-performing network achieved 91% accuracy and 0.88 area under the receiver operating characteristic curve at the tile level for an unseen test set. Novel histopathologic patterns were found through the visualized Guided Grad-CAMs. These patterns were assembled into a classification score to augment decision-making in routine histopathology workup. This score was successfully validated by 11 expert neuropathologists and 12 nonexperts, boosting nonexperts to expert level performance. SIGNIFICANCE: Our newly developed Web application combines the visualization of whole slide images with the possibility of deep learning-aided classification between focal cortical dysplasia IIb and tuberous sclerosis complex. This approach will help to introduce deep learning applications and visualization for the histopathologic diagnosis of rare and difficult-to-classify brain lesions.

Contribution of LTi and TH17 cells to B cell aggregate formation in the central nervous system in a mouse model of multiple sclerosis.

BACKGROUND: In a subgroup of patients suffering from progressive multiple sclerosis (MS), which is an inflammation-mediated neurodegenerative disease of the central nervous system (CNS), B cell aggregates were discovered within the meninges. Occurrence of these structures was associated with a more severe disease course and cortical histopathology. We have developed the B cell-dependent MP4-induced experimental autoimmune encephalomyelitis (EAE) as a mouse model to mimic this trait of the human disease. The aim of this study was to determine a potential role of lymphoid tissue inducer (LTi) and TH17 cells in the process of B cell aggregate formation in the MP4 model. METHODS: We performed flow cytometry of cerebellar and splenic tissue of MP4-immunized mice in the acute and chronic stage of the disease to analyze the presence of CD3-CD5-CD4+RORγt+ LTi and CD3+CD5+CD4+RORγt+ TH17 cells. Myelin oligodendrocyte glycoprotein (MOG):35-55-induced EAE was used as B cell-independent control model. We further determined the gene expression profile of B cell aggregates using laser capture microdissection, followed by RNA sequencing. RESULTS: While we were able to detect LTi cells in the embryonic spleen and adult intestine, which served as positive controls, there was no evidence for the existence of such a population in acute or chronic EAE in neither of the two models. Yet, we detected CD3-CD5-CD4-RORγt+ innate lymphoid cells (ILCs) and TH17 cells in the CNS, the latter especially in the chronic stage of MP4-induced EAE. Moreover, we observed a unique gene signature in CNS B cell aggregates compared to draining lymph nodes of MP4-immunized mice and to cerebellum as well as draining lymph nodes of mice with MOG:35-55-induced EAE. CONCLUSION: The absence of LTi cells in the cerebellum suggests that other cells might take over the function as an initiator of lymphoid tissue formation in the CNS. Overall, the development of ectopic lymphoid organs is a complex process based on an interplay between several molecules and signals. Here, we propose some potential candidates, which might be involved in the formation of B cell aggregates in the CNS of MP4-immunized mice.

Alarin in different human intestinal epithelial cell types.

Alarin (AL), a new member of the galanin family, has been localized in various CNS regions, mainly in rodents. Among other effects, it modulates food intake. Therefore, we analyzed the immunohistochemical distribution pattern of AL in human intestinal epithelia. Cryosections of 12 human bowel samples were immunohistochemically double-stained for AL and α-defensin 5 (αD; first set). Two further sets of sections were quadruple-stained either (second set) for AL, chromogranin (CG), synaptophysin (SY), and somatostatin (SO) or (third set) for AL, CG, Peptide Y (PY), and 5-hydroxytryptamine (5-HT). Slides were digitized and quantitative analysis of co-localization rates was undertaken. Small bowel: most of AL-positive cells (56%) were αD-positive Paneth cells located within the base of the crypts (first set). In the second set, about 27% of AL-labeled cells were co-reactive for SY and CG, likely representing entero-endocrine cells. In the third set, the largest subpopulation of AL-positive cells was not co-reactive for other markers applied (89%); most of them were likely Paneth cells. Large bowel: co-localization of AL with αD was not detected (first set). In the second set, AL was frequently co-localized with the other three markers applied (68%). In the third set, AL was frequently co-localized with 5-HT and CG (31%) as well as with PY and 5-HT (22%). Due to its presence in various enteroendocrine as well as Paneth cells, AL may be involved in different physiological and pathological processes.

Digital pathology imaging and computer-aided diagnostics as a novel tool for standardization of evaluation of aganglionic megacolon (Hirschsprung disease) histopathology.

Based on a recently introduced immunohistochemical panel (Bachmann et al. 2015) for aganglionic megacolon (AM), also known as Hirschsprung disease, histopathological diagnosis, we evaluated whether the use of digital pathology and 'machine learning' could help to obtain a reliable diagnosis. Slides were obtained from 31 specimens of 27 patients immunohistochemically stained for MAP2, calretinin, S100ß and GLUT1. Slides were digitized by whole slide scanning. We used a Definiens Developer Tissue Studios as software for analysis. We configured necessary parameters in combination with 'machine learning' to identify pathological aberrations. A significant difference between AM- and non-AM-affected tissues was found for calretinin (AM 0.55% vs. non-AM 1.44%) and MAP2 (AM 0.004% vs. non-AM 0.07%) staining measurements and software-based evaluations. In contrast, S100ß and GLUT1 staining measurements and software-based evaluations showed no significant differences between AM- and non-AM-affected tissues. However, no difference was found in comparison of suction biopsies with resections. Applying machine learning via an ensemble voting classifier, we achieved an accuracy of 87.5% on the test set. Automated diagnosis of AM by applying digital pathology on immunohistochemical panels was successful for calretinin and MAP2, whereas S100ß and GLUT1 were not effective in diagnosis. Our method suggests that software-based approaches are capable of diagnosing AM. Our future challenge will be the improvement of efficiency by reduction of the time-consuming need for large pre-labelled training data. With increasing technical improvement, especially in unsupervised training procedures, this method could be helpful in the future.

5-HT3A serotonin receptor in the gastrointestinal tract: the link between immune system and enteric nervous system in the digestive form of Chagas disease.

Chagas disease is caused by Trypanosoma cruzi and remains one of the most neglected diseases in Latin America. One of its clinical forms is Chagas megacolon. Despite being known for more than half a century, detailed causes are still obscure. Recent evidence indicates a close relationship between the immune system and the enteric nervous system in the etiology of chagasic megacolon pathology. It is believed that low expression of the 5-HT3A serotonin receptor on lymphocytes could be linked to megacolon development. To test this hypothesis, this work investigated the distribution of CD4, CD8, and CD20 lymphocytes and their 5-HT3A receptor expression. The results demonstrated that Chagas patients without megacolon present a higher expression of the 5-HT3A receptor in all analyzed lymphocytes compared with Chagas patients with megacolon. These data suggest that the high expression of this receptor may lead to immunomodulation and prevent the development of Chagas megacolon.

Morphological and Immunohistochemical Characterization of Human Intrinsic Gastric Neurons.

Our knowledge about human gastric enteric neuron types is even more limited than that of human intestinal types. Here, we immunohistochemically stained wholemounts and sections of gastric specimens obtained from 18 tumor-resected patients. Myenteric wholemounts were labeled for choline acetyl transferase (ChAT), neuronal nitric oxide synthase (NOS), and the human neuronal protein HuC/D (as pan-neuronal marker for quantitative analysis) or alternatively for neurofilament (for morphological evaluation). ChAT-positive neurons outnumbered NOS-positive neurons (56 vs. 27%), and neurons negative for both markers accounted for 17%. Two larger groups of neurons (each between 12 and 14%) costained for ChAT and vasoactive intestinal peptide (VIP) or for NOS and VIP, respectively. Clear morphochemical correlation was found for uniaxonal stubby type I neurons (ChAT+; putative excitatory inter- or motor neurons), for uniaxonal spiny type I neurons (NOS+/VIP+; putative inhibitory motor or interneurons), and for multiaxonal type II neurons (ChAT+; putative afferent neurons; immunostaining of additional wholemounts revealed their coreactivity for somatostatin). Whereas these latter neuron types were already known from the human intestine, the morphology of gastric myenteric neurons coreactive for ChAT and VIP was newly described: they had numerous short, extremely thin dendrites and resembled, together with their cell bodies, a "hairy" head. In our sections, nerve fibers coreactive for ChAT and VIP were commonly found only in the mucosa. We suggest these myenteric ChAT+/VIP+/hairy neurons to be mucosal effector neurons. In contrast to myenteric neurons, the much less common submucosal neurons were not embedded in a continuous plexus and did not display any clear morphochemical phenotypes.

Calbindin D28k-Immunoreactivity in Human Enteric Neurons.

Calbindin (CALB) is well established as immunohistochemical marker for intrinsic primary afferent neurons in the guinea pig gut. Its expression by numerous human enteric neurons has been demonstrated but little is known about particular types of neurons immunoreactive for CALB. Here we investigated small and large intestinal wholemount sets of 26 tumor patients in order to evaluate (1) the proportion of CALB⁺ neurons in the total neuron population, (2) the colocalization of CALB with calretinin (CALR), somatostatin (SOM) and vasoactive intestinal peptide (VIP) and (3) the morphology of CALB+ neurons. CALB+ neurons represented a minority of myenteric neurons (small intestine: 31%; large intestine: 25%) and the majority of submucosal neurons (between 72 and 95%). In the submucosa, most CALB⁺ neurons co-stained for CALR and VIP (between 69 and 80%) or for SOM (between 20 and 3%). In the myenteric plexus, 85% of CALB+ neurons did not co-stain with the other markers investigated. An unequivocal correlation between CALB reactivity and neuronal morphology was found for myenteric type III neurons in the small intestine: uniaxonal neurons with long, slender and branched dendrites were generally positive for CALB. Since also other neurons displayed occasional CALB reactivity, this protein is not suited as an exclusive marker for type III neurons.

Epithelial cell types and their proposed roles in maintaining the mucosal barrier in human chagasic-megacolonic mucosa.

Patients suffering from chagasic megacolon must have an intact mucosal barrier as they survive this chronic disease for decades. A key structure of the mucosal barrier are epithelial cells. Vasoactive-intestinal-peptide (VIP)-positive nerve fibres are involved in influencing, e.g., epithelial cell proliferation, mucus secretion (e.g., mucin 2 and trefoil factor 3 of goblet cells) and inflammation or autoimmunity, all putative and/or known factors altered in chagasic megacolon. We analyzed qualitatively and quantitatively goblet cells, their specific markers, such as mucin 2 (MUC2) and trefoil factor 3 (TFF3) and enterocytes, the relation of VIP-immunoreactive nerve fibres to the epithelia, the distribution of gelsolin, a protein involved in chronic inflammation processes in the epithelia, and the proliferation rate of epithelial cells by combined 4',6-diamidino-2-phenylindole (DAPI) and phosphohistone-H3 (PHH3) staining. Goblet cells were the dominating epithelial cell type. They accounted for 38.4% of all epithelial cells in controls and changed to 58.9% in the megacolonic parts. In contrast to the overall expression in goblet cells of control epithelia, TFF3 was confined to goblet cells at the base of the crypts whereas MUC2 was found only in luminal goblet cells. Gelsolin-positive goblet cells were predominantly recognized within the controls. Finally, the mean value of mitosis increased from 1.5% within the controls up to 2.6% in the anal parts of the chagasic sepcimens. Taken together, increased cell proliferation, preponderance of goblet cells, differential MUC 2, and TFF 3 expression might all be factors maintaining an intact mucosal barrier within chagasic megacolon.

The enteric nervous system is a potential autoimmune target in multiple sclerosis.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) in young adults that has serious negative socioeconomic effects. In addition to symptoms caused by CNS pathology, the majority of MS patients frequently exhibit gastrointestinal dysfunction, which was previously either explained by the presence of spinal cord lesions or not directly linked to the autoimmune etiology of the disease. Here, we studied the enteric nervous system (ENS) in a B cell- and antibody-dependent mouse model of MS by immunohistochemistry and electron microscopy at different stages of the disease. ENS degeneration was evident prior to the development of CNS lesions and the onset of neurological deficits in mice. The pathology was antibody mediated and caused a significant decrease in gastrointestinal motility, which was associated with ENS gliosis and neuronal loss. We identified autoantibodies against four potential target antigens derived from enteric glia and/or neurons by immunoprecipitation and mass spectrometry. Antibodies against three of the target antigens were also present in the plasma of MS patients as confirmed by ELISA. The analysis of human colon resectates provided evidence of gliosis and ENS degeneration in MS patients compared to non-MS controls. For the first time, this study establishes a pathomechanistic link between the well-established autoimmune attack on the CNS and ENS pathology in MS, which might provide a paradigm shift in our current understanding of the immunopathogenesis of the disease with broad diagnostic and therapeutic implications.

Neurovascular Interface in Porcine Small Intestine: Specific for Nitrergic rather than Nonnitrergic Neurons.

In the 1970s, by using classic histological methods, close topographical relationships between special areas of enteric ganglia and capillaries were shown in the pig. In this study, by application of double and triple immunohistochemistry, we confirmed this neurovascular interface and demonstrated that these zones are mainly confined to nitrergic neurons in the myenteric and the external submucosal plexus. In the upper small intestine of the pig, the respective neurons display type III morphology, i.e. they have long, slender and branched dendrites and a single axon. In another set of experiments, we prepared specimens for electron-microscopical analysis of these zones. Both ganglia and capillaries display continuous basement membranes, the smallest distances between them being 1,000 nm at the myenteric and 300 nm at the external submucosal level. The capillary endothelium was mostly continuous but, at the external submucosal level, scattered fenestrations were observed. This particular neurovascular relationship suggests that nitrergic neurons may require a greater amount of oxygen and/or nutrients. In guinea pig and mouse, previous ischemia/reperfusion experiments showed that nitrergic neurons are selectively damaged. Thus, a preferential blood supply of enteric nitrergic neurons may indicate that these neurons are more vulnerable in ischemia.

What neurons hide behind calretinin immunoreactivity in the human gut?

Calretinin (CALR) is often used as an immunohistochemical marker for the histopathological diagnosis of human intestinal neuropathies. However, little is known about its distribution pattern with respect to specific human enteric neuron types. Prior studies revealed CALR in both myenteric and submucosal neurons, most of which colabel with choline acetyl transferase (ChAT). Here, we specified the chemical code of CALR-positive neurons in small and large intestinal wholemounts in a series of 28 patients. Besides other markers, we evaluated the labeling pattern of CALR in combination with vasoactive intestinal peptide (VIP). In colonic submucosa, CALR and VIP were almost completely colocalized in about three-quarters of all submucosal neurons. In the small intestinal submucosa, both the colocalization rate of CALR and VIP as well as the proportion of these neurons were lower (about one-third). In the myenteric plexus of both small intestine and colon, CALR amounted to 11 and 10 %, respectively, whereas VIP to 5 and 4 % of the whole neuron population, respectively. Colocalization of both markers was found in only 2 and 3 % of myenteric neurons, respectively. In section specimens, nerve fibers coreactive for CALR and VIP were found in the mucosa but not in the muscle coat. Summarizing the present and earlier results, CALR was found in at least one submucosal and two myenteric neuron populations. Submucosal CALR+/VIP+/ChAT± neurons innervate mucosal structures. Furthermore, CALR immunoreactivity in the myenteric plexus was observed in morphological type II (supposed primary afferent) and spiny type I (supposed inter- or motor-) neurons.