Luminal Surface Research Articles

Modification of low-density lipoproteins by low molecular weight carbonyl products of free-radical oxidation of lipids and carbohydrates plays a key role in atherosclerotic lesion of the vascular wall and in endothelial dysfunction

The review presents evidence of the participation of low-density lipoproteins (LDL) modified by low molecular weight dicarbonyl compounds formed during freeradical oxidation of lipids (malondialdehyde) and carbohydrates in the development of endothelial dysfunction and atherosclerotic vascular lesions. The authors believe that it is they, and not oxidized (hydroperoxide-containing) LDL, that are the main factors of pathogenesis. The role of dicarbonyl-modified LDL in LOX-1 dependent induction of processes leading to the development of endothelial dysfunction is discussed. The results of studies proving that damage to the glycocalyx (a layer of macromolecules that prevent the development of endothelial dysfunction) covering the luminal surface of the endothelium is caused by hyperproduction of reactive oxygen species. Ways of pharmacological correction of free-radical oxidation processes are discussed, due to which inhibition of atherogenesis and diabetogenesis can be achieved.

Fast-track preparation of lung specimens for electron microscope observations of the pulmonary endothelial glycocalyx.

The glycocalyx (GCX) covers the luminal surface of blood vessels and regulates vascular permeability. As GCX degradation predicts various types of vasculopathy, confirming the presence of this structure is useful for diagnosis. Since the GCX layer is very fragile, careful fixation is necessary to preserve its structure. We explored appropriate and feasible methodologies for visualizing the GCX layer using lung tissue specimens excised from anesthetized mice. Each specimen was degassed and immersed in Alcian blue (ALB) fixative solution, and then observed using electron microscopy. Specimens from septic mice were prepared as negative GCX controls. Using these immersion-fixed specimens, the GCX layer was successfully observed using both transmission and scanning electron microscopy; these observations were similar to those obtained using the conventional method of lanthanum perfusion fixation. Spherical aggregates of GCX were observed in the septic mouse specimens, and the GCX density was lower in the septic specimens than in the non-septic specimens. Of note, the presently reported methodology reduced the specimen preparation time from 6 to 2days. We, therefore, concluded that our novel method could be applied to human lung specimens and could potentially contribute to the further elucidation of vasculopathies.

Optical microscopy and cross anatomy for the intestinal wall of sheep infected with Tap worm

Ten intestinal mucosa of sheep intestine were obtained for cross and microscopy examination, the specimens from those intestinal wall were taken at pieces half cm and washed well by running water, then processed by histological technique according to Bancoft & Steven 1987 including fixation, dehydration, clearing and embedding with paraffin wax also sectioning at six micrometer by microtome, finally stained by Hematoxylin and Eosin after that mounted with D. P. X and after drying in dry cabinet for 24 hour examined by light microscope , The intestinal wall was congested and patches of blood hemorrhage were seen. The histological examination revealed to sloughing of the intestinal villi from the luminal surface with presence of hyper secretion of mucus from the goblet cells and intestinal glands in the lamina properia also infiltration of white blood cells in the lamina properia in between the intestinal glands. In conclusion; the intestinal wall was damages after infection with Tape worm due to its effect on the villi of mucosa which are vital structures for absorption of nutritional elements in a animals.

Intraluminal neutrophils limit epithelium damage by reducing pathogen assault on intestinal epithelial cells during Salmonella gut infection.

Recruitment of neutrophils into and across the gut mucosa is a cardinal feature of intestinal inflammation in response to enteric infections. Previous work using the model pathogen Salmonella enterica serovar Typhimurium (S.Tm) established that invasion of intestinal epithelial cells by S.Tm leads to recruitment of neutrophils into the gut lumen, where they can reduce pathogen loads transiently. Notably, a fraction of the pathogen population can survive this defense, re-grow to high density, and continue triggering enteropathy. However, the functions of intraluminal neutrophils in the defense against enteric pathogens and their effects on preventing or aggravating epithelial damage are still not fully understood. Here, we address this question via neutrophil depletion in different mouse models of Salmonella colitis, which differ in their degree of enteropathy. In an antibiotic pretreated mouse model, neutrophil depletion by an anti-Ly6G antibody exacerbated epithelial damage. This could be linked to compromised neutrophil-mediated elimination and reduced physical blocking of the gut-luminal S.Tm population, such that the pathogen density remained high near the epithelial surface throughout the infection. Control infections with a ssaV mutant and gentamycin-mediated elimination of gut-luminal pathogens further supported that neutrophils are protecting the luminal surface of the gut epithelium. Neutrophil depletion in germ-free and gnotobiotic mice hinted that the microbiota can modulate the infection kinetics and ameliorate epithelium-disruptive enteropathy even in the absence of neutrophil-protection. Together, our data indicate that the well-known protective effect of the microbiota is augmented by intraluminal neutrophils. After antibiotic-mediated microbiota disruption, neutrophils are central for maintaining epithelial barrier integrity during acute Salmonella-induced gut inflammation, by limiting the sustained pathogen assault on the epithelium in a critical window of the infection.

The Oral Transglutaminase 2 Inhibitor ZED1227 Accumulates in the Villous Enterocytes in Celiac Disease Patients during Gluten Challenge and Drug Treatment.

The enzyme transglutaminase 2 (TG2) plays a key role in celiac disease (CeD) pathogenesis. Active TG2 is located mainly extracellularly in the lamina propria but also in the villous enterocytes of the duodenum. The TG2 inhibitor ZED1227 is a promising drug candidate for treating CeD and is designed to block the TG2-catalyzed deamidation and crosslinking of gliadin peptides. Our aim was to study the accumulation of ZED1227 after oral administration of the drug. We studied duodenal biopsies derived from a phase 2a clinical drug trial using an antibody that detects ZED1227 when bound to the catalytic center of TG2. Human epithelial organoids were studied in vitro for the effect of ZED1227 on the activity of TG2 using the 5-biotin-pentylamine assay. The ZED1227-TG2 complex was found mainly in the villous enterocytes in post-treatment biopsies. The signal of ZED1227-TG2 was strongest in the luminal epithelial brush border, while the intensity of the signal in the lamina propria was only ~20% of that in the villous enterocytes. No signal specific to ZED1227 could be detected in pretreatment biopsies or in biopsies from patients randomized to the placebo treatment arm. ZED1227-TG2 staining co-localized with total TG2 and native and deamidated gliadin peptides on the enterocyte luminal surface. Inhibition of TG2 activity by ZED1227 was demonstrated in epithelial organoids. Our findings suggest that active TG2 is present at the luminal side of the villous epithelium and that inhibition of TG2 activity by ZED1227 occurs already there before gliadin peptides enter the lamina propria.

Theranostic Nanoparticles: Revolutionizing Cancer and Imaging

Diagnostic agents based on nanoparticles are frequently suggested. However, diagnostic nanoparticles, except for iron oxide nanoparticles, have not yet been extensively utilized in clinical settings. This is because of concerns about toxicity, biodegradation, and elimination as well as difficulties in achieving reproducible particle uniformity and acceptable pharmacokinetic properties. The biologic behavior of nanoparticles should be considered when considering reasonable clinical applications. For instance, numerous nanoparticles are taken up by macrophages and accumulate in tissues with a lot of macrophages. As a result, they can be utilized to provide contrast in lymph nodes, the liver, the spleen, and inflammatory lesions Nanoparticles can also effectively label cells, making it possible to study cell migration in vivo and locate implanted (stem) cells and tissue-engineered grafts. Because it is difficult to control their pharmacokinetic properties, the potential of using nanoparticles for molecular imaging is limited. While targeted nanoparticle delivery to extravascular structures is frequently limited and difficult to separate from an underlying enhanced permeability and retention (EPR) effect, ideal nanoparticle targets are located on the endothelial luminal surface. In conclusion, nanoparticles hold significant clinical potential for other diagnostic and theranostic applications, even though they are not always the best option for molecular imaging because smaller or larger molecules may provide more specific information. This literature review survey endeavored to portray theranostic nanoparticles and their utilization in the therapy and conclusion of malignant growth. This paper also looked at nanoparticles and their potential to lower the dose of radioactive material and improve the quality of computed tomography scans. This review also attempted to demonstrate the benefits and drawbacks of incorporating these nanoparticles into the modern healthcare system. To gather the pertinent data for this review, 18 scholarly sources were chosen and investigated. Every day, new nanotechnology prototypes and developments are made, created, and analyzed. Positive aspects of incorporating nanoparticles into cancer treatment have been documented. On the other hand, there are a lot of people who still do not like the idea of using this technology and think it is just a fantasy or novelty. These technologies have the potential to completely transform the health care industry, according to studies of their effects on the cancer industry. When all these factors are taken into consideration, it appears that this technology is being used more frequently than ever before. This claim was not accurately met by negative perceptions, fear of change, a lack of specific work, cost, toxicity, or synthesis. The study and manipulation of matter in the range of one to one hundred nanometers is known as nanotechnology. Innovative therapeutic and diagnostic methods are at the heart of nanomedicine, or the use of precisely manufactured materials at this length scale for medical purposes. Due to their extremely small size, high surface area to mass ratio, and high reactivity, nanomaterials are physically and chemically distinct from bulk materials of the same composition. Using these features can alleviate some of the drawbacks of conventional medical and diagnostic drugs.

Glycocalyx-Sodium Interaction in Vascular Endothelium.

The glycocalyx generally covers almost all cellular surfaces, where it participates in mediating cell-surface interactions with the extracellular matrix as well as with intracellular signaling molecules. The endothelial glycocalyx that covers the luminal surface mediates the interactions of endothelial cells with materials flowing in the circulating blood, including blood cells. Cardiovascular diseases (CVD) remain a major cause of morbidity and mortality around the world. The cardiovascular risk factors start by causing endothelial cell dysfunction associated with destruction or irregular maintenance of the glycocalyx, which may culminate into a full-blown cardiovascular disease. The endothelial glycocalyx plays a crucial role in shielding the cell from excessive exposure and absorption of excessive salt, which can potentially cause damage to the endothelial cells and underlying tissues of the blood vessels. So, in this mini review/commentary, we delineate and provide a concise summary of the various components of the glycocalyx, their interaction with salt, and subsequent involvement in the cardiovascular disease process. We also highlight the major components of the glycocalyx that could be used as disease biomarkers or as drug targets in the management of cardiovascular diseases.

P-803 3D human endometrium from noninvasively retrieved primary endometrial cells is a reliable model to mimic the implantation window

Abstract Study question Can human endometrial organoids (hEOs) from a noninvasive source (menstrual blood-MB) mimic the physiology of a receptive endometrium Summary answer Endometrial organoids from MB functionally respond to stimuli and express markers of receptivity and can be modulated. What is known already 3D models are considered a new step in promoting precision medicine and an advanced tool for studying endometrial biology, endometrial-associated diseases, and understanding the complex mechanisms that comprise endometrial-embryo crosstalk. So far, both 2D and 3D models are usually obtained from endometrial biopsy. The recent discovery of molecules crucial for successful embryo implantation has offered researchers valuable insights in this field. However, important questions about the molecular mechanisms that drive this process remain to be deciphered. Study design, size, duration hEOs are usually obtained from endometrial biopsy, in this case a noninvasive source(MB) was used to isolate endometrial epithelial (Ep) and stromal (hESC) cells. The hEOs were generated to study the morphological changes that occur in vivo during the menstrual cycle, particularly in the window of implantation (WOI). The hEOs obtained from MB were exposed to hormones treatments to mimic WOI and were also treated with the progesterone blocker mifepristone to reverse the decidualization process. Participants/materials, setting, methods 3D hEOs were prepared from 6 healthy volunteers and cultured for 4 days in expansion medium supplemented with 10-8 M E2 + 10-6 M P4 and 500 uM cAMP. As a control, hEOs were treated with the progesterone blocker mifepristone to reverse the decidualization process. Finally, hEOs were washed with PBS and fixed for immunofluorescent staining or for scanning-electron-microscopy (SEM) or stored at -80 °C for gene expression analysis. Main results and the role of chance In this study, we were able to establish a reliable 3D model using both epithelial and stromal cells with a less invasive approach to mimic the mid-secretory phase. Immunofluorescence staining of vimentin (marker of stromal cells) and cytokeratin 19 (marker of epithelial cells) confirmed that organoids are formed by both Ep and hESCs. Using SEM, we closely observed the luminal surface of hEOs in proliferative phase, where the inner side of Ep cells was characterized by the presence of pinopods, a specific marker of the implantation window. In addition, hEOs express PP14, an implantation marker that has been shown to be significantly up-regulated during WOI. Finally, the expression of FKBP5, regulated by cAMP, and ZBP16, regulated by progesterone, increased significantly during decidualization, while their expression was up-regulated at dawn by mifepristone, a progesterone blocker, thus confirming that hEOs are an effective model of WOI. Limitations, reasons for caution All results need to be validated in a larger cohort and further implemented with other cell types (e.g., endothelial and immune cells) to implement this model for drug testing and therapy Wider implications of the findings Effective and reliable modeling of embryonic implantation is needed to mimic the cascade of molecular events that occurs in vivo. This model can be used for personalized medicine without the invasiveness of biopsy. Trial registration number Not applicable

433-P: Syndecan-1, a Marker of Endothelial Glycocalyx Degradation, Is Associated with Albuminuria in Type 2 Diabetes

The endothelial glycocalyx (EG) covers the luminal surface of vascular endothelium and is involved in various functions of endothelium including the microvascular permeability. Syndecan (SDC)-1 is a transmembrane heparan sulfate proteoglycan and is a major component of EG. Since the extracellular domain of SDC-1 is shed by inflammatory mediators, increased circulating levels of SDC-1 is considered as a marker of EG degradation. No study has thus far investigated the relationship between EG degradation and albuminuria in type 2 diabetes. This study aimed to investigate the association between EG degradation assessed by serum SDC-1 levels and urinary albumin to creatinine ratio (ACR) in type 2 diabetes. This study enrolled 370 Japanese patients with type 2 diabetes (T2D) and 219 nondiabetic controls (ND). Patients with severe kidney dysfunction whose estimate glomerular filtration rate (eGFR) was less than 30 mL/min/1.73m2 were excluded. T2D patients were divided into normoalbuminuria (A1), microalbuminuria (A2) and macroalbuminuria (A3) by ACR. Median value of serum SDC-1 levels in T2D with A1 stage was 24.5 ng/mL which was higher than that in ND (16.0 ng/mL) (p <0.001). Median values of SDC-1 in T2D with A2 and A3 stages were 29.0 and 38.7 ng/mL, respectively, which were significantly different among albuminuria stages (p <0.001). First, multivariate analysis including T2D patients with A1 stage and ND revealed that the presence of T2D was independently associated with serum SDC-1 level (β = 0.356, p <0.001). Next, multivariate analysis in T2D patients with all albuminuria stages revealed that serum SDC-1 level was independently associated with ACR (β = 0.121, p = 0.012) after adjustment for known other risk factors for albuminuria. In conclusion, serum SDC-1 level is increased in T2D compared to ND and is an independent determinant of ACR in T2D patients. This study suggests a potential involvement of the EG degradation in albuminuria in T2D. Disclosure Y. Kakutani: None. T. Morioka: None. Y. Yamazaki: None. A. Ochi: None. S. Fukumoto: None. T. Shoji: Research Support; Ono Pharmaceutical Co., Ltd., Bayer Inc. Other Relationship; Kowa Company, Ltd. M. Emoto: None.

Aldosterone: Essential for Life but Damaging to the Vascular Endothelium.

The renin angiotensin aldosterone system is a key regulator of blood pressure. Aldosterone is the final effector of this pathway, acting predominantly via mineralocorticoid receptors. Aldosterone facilitates the conservation of sodium and, with it, water and acts as a powerful stimulus for potassium excretion. However, evidence for the pathological impact of excess mineralocorticoid receptor stimulation is increasing. Here, we discussed how in the heart, hyperaldosteronism is associated with fibrosis, cardiac dysfunction, and maladaptive hypertrophy. In the kidney, aldosterone was shown to cause proteinuria and fibrosis and may contribute to the progression of kidney disease. More recently, studies suggested that aldosterone excess damaged endothelial cells. Here, we reviewed how damage to the endothelial glycocalyx may contribute to this process. The endothelial glycocalyx is a heterogenous, negatively charged layer on the luminal surface of cells. Aldosterone exposure alters this layer. The resulting structural changes reduced endothelial reactivity in response to protective shear stress, altered permeability, and increased immune cell trafficking. Finally, we reviewed current therapeutic strategies for limiting endothelial damage and suggested that preventing glycocalyx remodelling in response to aldosterone exposure may provide a novel strategy, free from the serious adverse effect of hyperkalaemia seen in response to mineralocorticoid blockade.

On the major role played by the lumen curvature of intracranial aneurysms walls in determining their mechanical response, local hemodynamics, and rupture likelihood

The properties of intracranial aneurysms (IAs) walls are known to be driven by the underlying hemodynamics adjacent to the IA sac. Different pathways exist explaining the connections between hemodynamics and local tissue properties. The emergence of such theories is essential if one wishes to compute the mechanical response of a patient-specific IA wall and predict its rupture. Apart from the hemodynamics and tissue properties, one could assume that the mechanical response also depends on the local morphology, more specifically, the curvature of the luminal surface, with larger values at highly-curved wall portions. Nonetheless, this contradicts observations of IA rupture sites more often found at the dome, where the curvature is lower. This seeming contradiction indicates a complex interaction between the hemodynamics adjacent to the aneurysm wall, its morphology, and mechanical response, which warrants further investigation. This was the main goal of this work. We accomplished this by analyzing the stress and stretch fields in different regions of the wall for a sample of IAs, which have been classified based on particular hemodynamics conditions and lumen curvature. Pulsatile numerical simulations were performed using the one-way fluid-solid interaction strategy implemented in OpenFOAM (solids4foam toolbox). We found that the variable best correlated with regions of high stress and stretch was the lumen curvature. Additionally, our data suggest a connection between the local curvature and particular hemodynamics conditions adjacent to the wall, indicating that the lumen curvature is a property that could be used to assess both mechanical response and hemodynamic conditions, and, moreover, suggest new rupture indicators based on the curvature.

Development of a Silk Fibroin-Small Intestinal Submucosa Small-Diameter Vascular Graft with Sequential VEGF and TGF-β1 Inhibitor Delivery for In Situ Tissue Engineering.

Proper endothelialization and limited collagen deposition on the luminal surface after graft implantation plays a crucial role to prevent the occurrence of stenosis. To achieve these conditions, a biodegradable graft with adequate mechanical properties and the ability to sequentially deliver therapeutic agents isfabricated. In this study, a dual-release system is constructed through coaxial electrospinning by incorporating recombinant human vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGF-β1) inhibitor into silk fibroin (SF) nanofibers to form a bioactive membrane. The functionalized SF membrane as the inner layer of the graft is characterized by the release profile, cell proliferation and protein expression. It presents excellent biocompatibility and biodegradation, facilitating cell attachment, proliferation, and infiltration. The core-shell structure enables rapid VEGF release within 10 days and sustained plasmid delivery for 21 days. A 2.0-mm-diameter vascular graft is fabricated by integrating the SF membrane with decellularized porcine small intestinal submucosa (SIS), aiming to facilitate the integration process under a stable extracellular matrix structure. The bioengineered graft is functionalized with the sequential administration of VEGF and TGF-β1, and with the reinforced and compatible mechanical properties, thereby offers an orchestrated solution for stenosis with potential for in situ vascular tissue engineering applications.

Development of a universal, oriented antibody immobilization method to functionalize vascular prostheses for enhanced endothelialization for potential clinical application

BackgroundThrombosis is a common cause of vascular prosthesis failure. Antibody coating of prostheses to capture circulating endothelial progenitor cells to aid endothelialization on the device surface appears a promising solution to prevent thrombus formation. Compared with random antibody immobilization, oriented antibody coating (OAC) increases antibody-antigen binding capacity and reduces antibody immunogenicity in vivo. Currently, few OAC methods have been documented, with none possessing clinical application potential.ResultsDopamine and the linker amino-PEG8-hydrazide-t-boc were successfully deposited on the surface of cobalt chromium (CC) discs, CC stents and expanded polytetrafluoroethylene (ePTFE) grafts under a slightly basic condition. CD34 antibodies were immobilized through the reaction between aldehydes in the Fc region created by oxidation and hydrazides in the linker after t-boc removal. CD34 antibody-coated surfaces were integral and smooth as shown by scanning electron microscopy (SEM), had significantly reduced or no substrate-specific signals as revealed by X-ray photoelectron spectroscopy, were hospitable for HUVEC growth as demonstrated by cell proliferation assay, and specifically bound CD34 + cells as shown by cell binding testing. CD34 antibody coating turned hydrophobic property of ePTFE grafts to hydrophilic. In a porcine carotid artery interposition model, a confluent monolayer of cobblestone-shaped CD31 + endothelial cells on the luminal surface of the CD34 antibody coated ePTFE graft were observed. In contrast, thrombi and fibrin fibers on the bare graft, and sporadic cells on the graft coated by chemicals without antibodies were seen.ConclusionA universal, OAC method was developed. Our in vitro and in vivo data suggest that the method can be potentially translated into clinical application, e.g., modifying ePTFE grafts to mitigate their thrombotic propensity and possibly provide for improved long-term patency for small-diameter grafts.

Evaluating protective effects of botanicals under inflammation and oxidative stress in chicken apical-out enteroids

Botanicals (BOTs) are well known for their anti-inflammatory and antioxidant activities. They have been widely used as feed additives to reduce inflammation and improve intestinal functions in agricultural animals. However, the effects of BOTs on chicken intestinal epithelial functions are not fully understood. The 3D apical-out chicken enteroids recapitulate the intestinal tissue, and allow convenient access to the luminal surface, thus serving as a suitable model for investigating gut functions. The aim of this study was to identify the roles of BOTs in protecting the intestinal epithelium in chicken enteroids under challenging conditions. Apical-out enteroids were isolated from the small intestines of 18 days-old chicken embryos. Lipopolysaccharide (LPS, 10 µg/mL) and menadione (400 µM) challenges were performed in the media with or without BOTs. Paracellular Fluorescein isothiocyanate-dextran 4kD (FD4) permeability, inflammatory cytokine gene expression, and reactive oxygen species (ROS) generation were analyzed post-BOTs and challenges treatments. Statistical analysis was performed using one-way ANOVA and post hoc multiple comparisons among treatments. The results showed that the LPS challenge for 24 h induced a 50% increase in FD4 permeability compared with nontreated control; thymol, thyme essential oil, and phenol-rich extract significantly (P < 0.02) reduced FD4 permeability by 25%, 41%, and 48% respectively, in comparison with LPS treatment. Moreover, the gene expression of inflammatory cytokines was upregulated, tight junction proteins and defensins were downregulated (P < 0.05) after 6 h of LPS treatment, while these BOTs treatments significantly restored the LPS-induced gene expression alterations (P < 0.05). Menadione oxidative challenge for 1 h significantly increased the ROS level compared with unchallenged control. Enteroids treated with thymol and thyme essential oils showed 30% reduced ROS levels, while the phenol-rich extract reduced them by 60%, in comparison with the challenged group (P < 0.0001). These data confirmed the role of BOTs in supporting the barrier function and reducing the disruptive effects of inflammation and oxidation in the chicken intestine.

Empiric anti-Giardia therapy in non-diarrheal protein-losing enteropathy: A focus on children with monogenic humoral immunodeficiency.

Background and Aim: Chronic giardia infection can lead to non-erosive gastrointestinal disorders, including protein-losing enteropathy (PLE). This report describes non-diarrheal PLE in chronic giardiasis in children with defective humoral immunity. Methods: The retrospective report is related to 2 children known to have a monogenic inborn error of immunity. The first patient is a 13-year-old boy with X-linked agammaglobulinemia (Patient-1), and the second is a 5-year-old boy with NF-kB inducing kinase (NIK) deficiency infection. Frequency, growth status, and serum immunoglobulin-G (IgG) trough and albumin levels were monitored (Patient-2). Results: Patient-1 had more frequency of pneumonia but reported no symptoms of gastrointestinal disease, including alteration of bowel habits, change in stool consistency, nausea, vomiting, fatigue, bloating, and abdominal pain. No clinical oedema on examination. His weight remains static at 19-20 kg for about 1.5 years. Simultaneously, hypoproteinaemia was noted (Figure-1). A trial of increasing IVIG (0.7 - 1 g/kg) and the use of subcutaneous immunoglobulin (0.2 g/kg) did not reverse the biochemical and clinical situation. Hypoproteinaemia workup revealed normal liver and kidney functions, normal cardiac function, and no proteinuria. Interestingly, his upper endoscopy showed mild duodenitis and the presence of giardia lamblia at the luminal surface (Figure-2). Following this, a 2-week course of oral metronidazole (7.5 mg/kg/dose BID) resulted in a restoration of therapeutic serum IgG trough and albumin levels. Additionally, the child's nutritional status improved, and the frequency of respiratory infections dropped. In Patient-2, progressive hypoproteinaemia was noted over nine months. Similarly, no gastrointestinal complaints; however, the family reported foul-smelling semisolid stools with regular consistency. His IgG trough level was 2 g/l, and his albumin level was 20 g/l. Despite maximizing IVIG, he developed two episodes of pneumonia and once otitis media. An empiric oral metronidazole course resulted in the amelioration of symptoms and restoration of proteinemia. Meanwhile, faeces microscopy confirmed the presence of Giardia lamblia. Conclusions: Progressive hypoproteinaemia in children with humoral immunodeficiency is a clinical concern. Routine stool microscopy can identify giardia infection despite the ambiguity of gastrointestinal symptoms in such patients. However, a trial of empiric metronidazole therapy should be considered.

Comprehensive hemocompatibility analysis on the application of diamond-like carbon to ePTFE artificial vascular prosthesis

The aim of this study was to obtain comprehensive data regarding the hemocompatibility of diamond-like carbon (DLC)-coated expanded polytetrafluoroethylene (ePTFE). DLC increased the hydrophilicity and smoothened the surface and fibrillar structure, respectively, of the ePTFE. DLC-coated ePTFE had more albumin and fibrinogen adsorption and less platelet adhesion than uncoated ePTFE. There were scarce red cell attachments in in vitro human and in vivo animal (rat and swine) whole blood contact tests in both DLC-coated and uncoated ePTFE. DLC-coated ePTFE had a similar but marginally thicker band movement than uncoated-ePTFE with SDS-PAGE after human whole blood contact test. In addition, survival studies of aortic graft replacement in rats (1.5 mm graft) and arteriovenous shunt in goats (4 mm graft) were performed to compare the patency and clot formation between DLC-coated and uncoated ePTFE grafts. Comparable patency was observed in both animal models. However, clots were observed in the luminal surface of the patent 1.5 mm DLC-coated ePTFE grafts, but not in that of uncoated ePTFE grafts. In conclusions, hemocompatibility of DLC-coated ePTFE was high and comparable to that of uncoated ePTFE. However, it failed to improve the hemocompatibility of 1.5 mm ePTFE graft probably because increased fibrinogen adsorption canceled the other beneficial effects of DLC.

Valvular system of the lacrimal drainage pathway and the valve of Rosenmüller

The valves of the lacrimal drainage system are an enigma with a rich eponymous history. The unidirectional flow of tears along with the ultrastructural demonstration of distinctive mucosal folds on the luminal surface has rekindled an interest in them. The first in vivo direct demonstration of the valve of Rosenmüller and its functions has laid to rest some of the controversies of its existence and that of the valve of Huschke. The dynamic assessment of the valve of Rosenmüller has shown a well-defined functional role in the facilitation of unidirectional tear flow. The present mini review describes the embryological aspects, brief overview of the eponymous valves, techniques for their identification along with recent structural and functional aspects of the valve of Rosenmüller.

Intestinal origins of Parkinson's Disease pathologies

Accumulating evidence suggests that Parkinson’s disease (PD) pathology may arise in the gut. This likely occurs through the enteric nervous system (ENS), which facilitates bidirectional communication between the brain and intestines. A hallmark of PD is neuronal accumulation of misfolded α-synuclein (aSyn) proteins which have been shown to travel from the ENS to the brain via the vagus nerve. The goal of this study was to connect related neural and immune phenotypes in ex vivo and in vivo mouse models of PD.Recent data suggests that enteric neuronal fibers containing calcitonin gene related peptide (CGRP) act as chemo-sensors that can be stimulated to release CGRP in response to bacterial and chemical signals. CGRP can activate receptors that influence goblet cells (GCs) and immune cells. GCs are responsible for secreting mucus to maintain mucosal barrier integrity. Deterioration of gut barrier integrity is hallmark of PD patients. We hypothesize that alterations in luminal contents modifies and disturbs GCs disrupting the gut barrier in PD. This may result in increased release of CGRP leading to alterations in mucus production, immune responses, and aSyn aggregation in enteric neurons. In this study, the pesticide rotenone was given to C57BL/6 mice to facilitate aggregation of misfolded aSyn. Lectin and immuno-histochemistry were performed on sections of ileum and colon. GC mucopolysaccharides were fluorescently labeled with the lectin Ulex Europaeus Agglutinin I (UEA) conjugated to rhodamine. UEA-1+ cells in crypts were 25% more prevalent (68.5 +/- 0.5 vs. 52.5 +/-2.5) with rotenone treatment but 53% less prevalent in the luminal portion of crypts (22.5 +/- 2.5 vs. 47.5 +/-2.5). Fewer GCs in luminal positions is consistent with decreased mucus seen in PD patients. T cells were identified by immunoreactive (ir) CD3. Approximately 50% fewer CD3+ cells were in rotenone treated lamina propria (17 +/- 1.4) compared to control (36.2 +/- 3.5) consistent with previous reports. The area of CGRP-ir fibers in the lamina propria was 114% higher in control (6.4%+/-1.3) compared to rotenone treated tissue (5.6%+/-0.6). Rotenone treatment resulted in a 217% increase in the area average of CGRP-ir fibers in the region just below the epithelial monolayer (12.8%+/-1.3) compared to control (5.8%+/-0.7). While we did not observe CGRP fibers beyond the epithelial cell layer, GC cell disruption that could allow luminal contents to access CGRP fibers was suggested by the potential failure of GCs to migrate toward luminal surfaces from crypts. Future studies are needed to test the hypothesis that rotenone disturbance of mitochondrial function in GCs causes intestinal wall failure that leads to altered neuroimmune function. Aggregation of aSyn in enteric neurons may thereby be mediated by altered immune to neural actions as exacerbated by alterations in CGRP containing fibers and T cell populations in the gut. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Coexistence of multiple clear cell papillary renal cell carcinoma with renal oncocytoma: a case report.

The authors report a case of a female 65-year-old suffering from end-stage kidney failure for 10 years, who presented with a double left renal tumor, composed by an oncocytoma associated to multiple CCPRCC, a very rare entity. A radical left nephrectomy was realized by lumbotomy, with an uneventful postoperative course. Histological examination was challenging. Immunohistological examination showed diffuse positivity of cytokertain 7. No local recurrence nor metastatic progression were found during the 12 months of follow-up. CCPRCC, is a new entity, previously known as the unclassified rena cell carcinoma, is a malignant renal tumor, initially reported in patients at end-stage kidney failure. Oncocytoma is a well-known rare benign renal tumor. The association of both is rare, and should be kept in mind, especially when scanoguided diagnosis biopsy is realized. Histopathological confirmation may be challenging, given the recent identification of CCPRCC. The nuclei disposal toward the luminal surface is a characteristic pathological landmark of CCPRCC. Immunohistopathological examination is of great help, showing a distinctive profile: diffuse staining for cytokertain 7 and carbonic anhydrase IX. CCPRCC is a new malignant pathological entity in renal tumors. It can be associated with other benign renal lesions. This should be taken into consideration while histopathological examination, mainly of scanoguided biopsy cores.

Abstract 336: Endothelial Cells Communicate with Surrounding Vascular Cells Via Bidirectional and Polarized Secretion of Extracellular Vesicular Cargo: Implications for Atherosclerotic Plaque Development

Introduction: Endothelial cells (EC) can release extracellular vesicle (EV)-encapsulated miRNAs and proteins to mediate cell-cell communication. We hypothesized that EC-EV release is altered by activation state and drives functional changes in surrounding cells via bidirectional release. Methods: EVs were isolated from supernatants of human aortic endothelial cells (HAECs; ± IL-1β, 100pg/ml) via serial ultracentrifugation and quantified as per MISEV2018 guidelines. Cargo was assessed (miRNA sequencing, proteomics). HAEC EVs were added to primary monocytes and vascular smooth muscle cells (VSMCs) at a physiological concentration and RNA-seq was performed. Bioinformatic analysis of EV contents and RNA-seq was performed. To assess polarized EV secretion, EVs were quantified from apical and basolateral compartments, and visualized with total internal reflection fluorescence (TIRF) and cryogenic transmission electron microscopy (cryo-TEM). Results: Activated HAECs have increased EV release (6.73±1.81X10 10 vs. 2.98±1.44 x10 11 particles/ml; p=0.028, N=3). In comparison to quiescent ECs, activated EC-EVs carry miRNA and protein cargo that play shared and distinct roles in several pro-atherogenic pathways. EC-EVs participate in cell-cell communication with circulating monocytes and resident VSMCs, and EVs from activated ECs lead to changes in pathways that are pro-inflammatory and atherogenic. Importantly, ECs are capable of bidirectional communication with luminal and abluminal cells via their unique ability to release EVs to the apical and basolateral compartments as determined by EV-quantification using nanoparticle tracking and western blot analysis, EV-visualization via cryo-TEM, and dynamic basolateral HAEC-EV release via TIRF microscopy. Intriguingly, ECs polarize and secrete compartment-specific cargo to the luminal and abluminal surfaces with in silico analysis implicating unique communication with circulating and resident vascular cells. Conclusions: ECs utilize EV contents to mediate cell-cell communication in quiescent and activated states. Importantly, they can polarize vesicle release bidirectionally, which may specifically govern functional changes in circulating and resident vascular cells.