Development and validation of a stability-indicating method, structural elucidation of new degradation products from misoprostol by LC-MS time-of-flight, and an ex vivo study of vaginal permeation.

Misoprostol (MSP) is commonly prescribed in obstetrics and gynecology clinical practice for labor induction, cervical ripening, first-trimester pregnancy termination, and the treatment of postpartum hemorrhage. Furthermore, there is a lack of comprehensive discussion evaluating how different commercially available formulations influence the overall efficacy of MSP, even though reports indicate issues with the quality of these formulations, particularly regarding stability and vaginal absorption processes. This study investigates the stability of MSP under acidic conditions and its in vitro permeation using swine vaginal mucosa. A forced degradation study was conducted using 0.2 M HCl, and a high-efficiency LC method was developed. Three degradation products were identified and characterized using electrospray ionization-high-resolution quadrupole-time-of-flight-MS, with respective m/z values of 391.2508, 405.2705, and 387.2259, respectively. These results suggest that the degradation mechanism involves dehydration of the ß-hydroxy ketone moiety, followed by isomerization to its most resonance-stable form and de-esterification. Finally, the in vitro permeation study revealed that the esterified form of MSP was unable to permeate the mucosa and required prior degradation for any component to be detected in the receptor fluid.

Campylobacter majalis sp. nov. and Campylobacter suis sp. nov., novel Campylobacter species isolated from porcine gastrointestinal mucosa.

Strains LMG 7974T and LMG 8286T represent single, novel Campylobacter lineages with Campylobacter pinnipediorum and Campylobacter mucosalis as nearest phylogenomic neighbours, respectively. The results of average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) analyses of LMG 7974T, LMG 8286T and type strains of species of the genus Campylobacter confirmed that these strains represent novel species of the genus Campylobacter. The 16S rRNA gene sequences of both strains showed highest identity towards C. mucosalis (97.84 and 98.74 %, respectively). Strains LMG 7974T and LMG 8286T shared 72.5 and 73.7% ANI, respectively, with their nearest phylogenomic neighbours and less than 21 % dDDH. The draft genome sizes of LMG 7974T and LMG 8286T are 1 945429 bp and 1 708214 bp in length with percentage DNA G+C contents of 33.8 and 37.2 %, respectively. Anomalous biochemical characteristics and low MALDI-TOF mass spectrometry log scores supported their designation as representing novel species of the genus Campylobacte. We therefore propose to classify strain LMG 7974T (=CCUG 20705T) as the type strain of the novel species Campylobacter majalis sp. nov. and strain LMG 8286T (=CCUG 24193T, NCTC 11879T) as the type strain of the novel species Campylobacter suis sp. nov.

[Expression of galectin-13 in allergic diseases involving airway, skin and mucous membranes].

To detect the expression of galectin-13 in allergic diseases and provide a new way for the diagnosis and treatment of allergic diseases. A retrospective analysis method was used to screen 216 patients with allergic diseases with house dust mites or aspergillus as allergens who visited the Department of Allergy and Department of Respiratory of Tongji Hospital attached Tongji Medical College, Huazhong University of Science and Technology from March 2018 to May 2021. These allergic diseases included allergic asthma, allergic bronchopulmonary aspergillosis, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, allergic urticaria. 25 subjects without underlying diseases were selected as healthy controls. The galectin-13 content in serum in each group were detected, and the Pearson correlation was used to determine the correlation between the galectin-13 content in serum in each group and blood eosinophil count, blood specific IgE, the score scale of allergic disease. The expression of Galectin-13 was increased in allergic asthma group (71.44±39.44) pg/ml, allergic bronchopulmonary aspergillosis group (100.10±47.62) pg/ml, allergic rhinitis group (54.11±24.81) pg/ml and dermatitis group (44.12±19.51) pg/ml. The expression of galectin-13 was not significantly increased in allergic urticaria group (32.75±10.29) pg/ml and the allergic conjunctivitis group (30.55±9.87) pg/ml. The galectin-13 content in serum, was positively correlated with blood eosinophil count(rs=0.54, P<0.001) and house dust mite specific IgE (rs=0.51, P<0.001) in allergic asthma group, and was positively correlated with blood eosinophil count(rs=0.63, P=0.025) and aspergillus fumigatus specific IgE (rs=0.58, P=0.046) in allergic bronchopulmonary aspergillosis group. It was positively correlated with blood eosinophil count (rs=0.52, P=0.000 2) and house dust mite specific IgE (rs=0.41, P=0.005) in allergic rhinitis group. In allergic conjunctivitis group, the expression of galectin-13 was positively correlated with conjunctivitis symptom score (rs=0.47, P=0.048). In atopic dermatitis group, the expression of galectin-13 was positively correlated with blood eosinophil count (rs=0.58, P<0.001) and house dust mite specificity IgE (rs=0.47, P=0.002). In allergic urticaria group, the expression of galectin-13 was not significantly correlated with blood eosinophil count or house dust mite specific IgE. Galectin-13 may be related to the occurrence and progress of allergic diseases and may be involved in the occurrence of eosinophilic inflammation.

Pseudocarcinomatous Hyperplasia, Squamous Cell Carcinoma, and Keratoacanthoma Associated to Lymphomas of the Skin and External Mucous Membranes: A Case Report and Literature Review.

Pseudocarcinomatous hyperplasia (PCH) is a benign reactive epithelial proliferation that may be associated to lymphomas of the skin or external mucous membranes. We present a case of single lesion mycosis fungoides (Woringer-Kollop's reticulosis pagetoid) associated with PCH that was initially misdiagnosed as squamous cell carcinoma (SCC) and review all PubMed-indexed previously reported cases on lymphomas of the skin or external mucous membranes associated to PCH, SCC, and keratoacanthomas. Including our own case, we collected data of 114 cases of cutaneous or mucosal lymphoproliferative disorders associated to PCH, 3 cases associated to SCC, and other 3 cases associated to keratoacanthomas. All cases were tabulated to the following parameters whenever data was available: sex, age, previous medical conditions, number of lesions (single × multiple), site of involvement (mucosa, skin or both), clinical impression, initial equivocal histopathologic diagnosis, final diagnosis, keratinocytic atypia (presence × absence), lymphocytic atypia (presence × absence), CD30-status, and treatment.

Evaluation of the mucoadhesive strengths of Abelmoschus esculentus and Irvingia gabonensis gums for possible application in veterinary vaccine delivery: the effect of extraction methods.

This study evaluates the effects of different gum extraction methods on the mucoadhesive strengths of Abelmoschus esculentus (AE) and Irvingia gabonensis (IG) gums and the release of vaccine antigen in vaccine-gum formulations. AE and IG gums were extracted employing previously documented methods with acetone or sodium chloride (NaCl) and either oven-dried or freeze-dried. Gum extracts were analyzed for mucoadhesive strengths using a modified rotational cylinder method on animal mucosa. The time taken to detach from the mucosa was taken as the Peak Adhesion Time (PAT). The gum extracts were charged with Peste des petits ruminant vaccine and the antigen release was evaluated using agar gel immunodiffusion technique. The means of the PATS were analyzed using Mann-whitney t-test at p < .05. The NaCl extracted and freeze-dried IG gum showed sustained mean PATs of 1766 ± 73 s; 2116 ± 101 s; 7044 ± 117 s, while the oven-dried IG gum and both AE gums showed short-lived average PATs. Vaccine-gum formulations of IG at ratios 2:1, 1:1 & 1:2 had strong positive reactions while only that of AE at 2:1 showed a strong positive reaction. This study shows that NaCl extracted and freeze-dried IG gum has immunomodulatory potential for mucoadhesive vaccine delivery in ruminants.

Parkinson's disease biomarkers based on α-synuclein.

Parkinson's disease is the second most common neurodegenerative disorder after Alzheimer's disease and is estimated to affect approximately 1-4% of individuals aged over 60 years old. Although considerable efforts have been invested into developing disease-modifying therapies for Parkinson's disease, such efforts have been confounded by the difficulty in accurately diagnosing Parkinson's disease during life to enable accurate patient stratification for clinical trialling of candidate therapeutics. Therefore, the search for effective biomarkers that can be accurately evaluated during life with non-invasive means is a pressing issue in the field. Since the discovery of α-synuclein (α-syn) as a protein linked to a familial form of Parkinson's disease, later identified as the major protein component of the neuropathological hallmark of idiopathic Parkinson's disease, considerable interest has focused on this protein and its distinct conformers. We describe here the progress that has been made in the area of Parkinson's disease biomarker discovery with a focus on α-synuclein. In particular, we highlight the novel assays that have been employed and the increasing complexity in evaluating α-synuclein with regard to the considerable diversity of conformers that exist in the biofluids and peripheral tissues under disease conditions. "This article is part of the Special Issue Synuclein."

Molecular Characterization of Mucus Binding.

Binding of small molecules to mucus membranes in the body has an important role in human health, as it can affect the diffusivity and activity of any molecule that acts in a mucosal environment. The binding of drugs and of toxins and signaling molecules from mucosal pathogens is of particular clinical interest. Despite the importance of mucus-small molecule binding, there is a lack of data revealing the precise chemical features of small molecules that lead to mucus binding. We developed a novel equilibrium dialysis assay to measure the binding of libraries of small molecules to mucin and other mucus components, substantially increasing the throughput of small molecule binding measurements. We validated the biological relevance of our approach by quantifying binding of the antibiotic colistin to mucin, and showing that this binding was associated with inhibition of colistin's bioactivity. We next used a small molecule microarray to identify 2,4-diaminopyrimidine as a mucin binding motif and confirmed the importance of this motif for mucin binding using equilibrium dialysis. Furthermore, we showed that, for molecules with this motif, binding to mucins and the mucus-associated biopolymers DNA and alginate is modulated by differences in hydrophobicity and charge. Finally, we showed that molecules lacking the motif exhibited different binding trends from those containing the motif. These results open up the prospect of routine testing of small molecule binding to mucus and optimization of drugs for clinically relevant mucus binding properties.

Mucous Membrane Color Assessment Variability of Veterinary Students Using Either Colorimetric or Word-Based Scales.

A colorimetric scale has the potential to be very useful as a training tool for students in veterinary training programs. The authors of this report hypothesized that clinically active, graduate level veterinary students would assess mucous membrane color with greater consistency using an image-based system than with traditional word-based techniques. Third- and fourth-year veterinary students were asked to evaluate 10 canine gingival mucosa images and rate them with either an image-based scale designed by the authors or a word-based system. Although the mean absolute deviations from the median values were greater for the word scale (0.22) than for the image scale (0.20) indicating increased variation, mixed model analysis did not demonstrate these differences were significant ( p = .120). Based on this data it is possible that prior image and word-based instruction made it easier for the students to differentiate mucous membrane colors, or that the majority of the students were multimodal learners rather than single-type learners, making previous instruction techniques effective regardless of whether they were visual or read/write based. The authors recommend that future investigators both evaluate previous student instruction and consider administering visual, auditory, read/write, kinesthetic (VARK) tests when investigating potential learning aids in veterinary medicine.

Higher Levels of a Cytotoxic Protein, Vaginolysin, in Lactobacillus-Deficient Community State Types at the Vaginal Mucosa.

Vaginolysin (VLY), a cytotoxic protein produced by Gardnerella vaginalis, may contribute to bacterial vaginosis. We observed that women with G. vaginalis, low levels of lactobacilli, history of vaginal douching, higher Nugent scores, and higher vaginal pH had increased VLY. Inflammatory markers were not highly expressed with increasing VLY. Vaginolysin's role in bacterial vaginosis warrants further evaluation.

Nanoparticle mucoadhesive system as a new tool for fish immune system modulation.

Recently, chitosan-based nanoparticles with mucoadhesive properties emerged as a strategy for mucosal drug release. This study aimed to characterize the interaction of mucoadhesive system chitosancoated PLGA nanoparticles (NPMA) with fish external mucus. NP suspensions with fluorescent probe were prepared and characterized by size, polydispersity, zeta potential and pH measures. In post-exposure fish were observed an increase in fluorescence imaging over time and it was significantly influenced by NPMA concentration. We also observed the main predominance the fluorescence in the spleen, followed by liver, gill and other tissues. The use of mucoadhesive nanocarriers becomes an alternative for administration of drugs and immunomodulators in immersion systems since the nanosystem can adhere to the mucosal surface of the fish with little residual effect in the water.

Evaluating the electrical impedance and mucus-related gene expression of uterine endometrial tissues in mares.

We investigated the electrical impedance of the reproductive tracts (vagina and uterine endometrial tissues) and the expression of mucus-related genes to identify the stage of the estrous cycle in mares. We first examined vaginal impedance in native Hokkaido mares during their estrous cycle and found no significant differences. However, impedance levels tended to decrease towards ovulation. Furthermore, we investigated the estrous cycle by measuring the electrical impedance of the uterine endometrial tissues obtained from carcasses of mares. We found that impedance levels in the endometrial tissues decreased in the regressed phase of the corpus luteum (CL). Expression of mucus-related genes (ATP1A1, CFTR, AQP3, and AQP5) varied at different stages of the estrous cycle. Among them, AQP3 expression was consistent with previous reports. We concluded that electrical impedance in the uterine endometrial tissues of mares could be potentially used to verify the presence of active CL in horses for experimental purposes. However, further studies are needed to determine the reference value and to identify the day of the estrous cycle in mares.

Effects of the Chemical Structures of Oligoarginines Conjugated to Biocompatible Polymers as a Mucosal Adjuvant on Antibody Induction in Nasal Cavities.

We have been investigating the potential of oligoarginine-linked polymers as an adjuvant for mucosal vaccination that induces immunoglobulin G (IgG) in systemic circulation and immunoglobulin A (IgA) secreted on the mucosa. Our latest infection experiments demonstrated that mice immunized nasally with a mixture of inactivated influenza viruses and poly(N-vinylacetamide-co-acrylic acid) (PNVA-co-AA) modified with D-octaarginine were perfectly protected from homologous virus infection. On the contrary, virus infection was observed in mice immunized with the antigen alone. This difference was presumably due to insignificant induction of secreted IgA on the nasal mucosa in the latter mice. Since it was unclear whether the current induction level was sufficient for heterologous virus infection, we evaluated the effects of the chemical structures of oligoarginines conjugated to PNVA-co-AA on induction of intranasal IgA. The number and optical activity of the arginine residues and the degree of modification with oligoarginines in the polymer backbone were listed as a factor that would influence IgA induction. Mouse experiments revealed that maximization of the modification resulted in an increase in adjuvant activities of oligoarginine-linked polymers most effectively. Glycine segments inserted between oligoarginines and the polymer backbone were a prerequisite for the maximization. The highest IgA level was observed when antigens were coadministered with diglycine-D-octaarginine-linked PNVA-co-AA.

Antibody Drug-Target Engagement Measurement in Tissue Using Quantitative Affinity Extraction Liquid Chromatography-Mass Spectrometry: Method Development and Qualification.

We demonstrate a novel strategy using affinity extraction (AE) LC-MS to directly measure drug exposure and target engagement, two critical pharmacological questions, with a single assay. The assay measures total drug and target concentration at the site of therapeutic action, as well as the amount of target bound to drug. The case study presented applies the strategy to measure drug engagement of a membrane bound receptor (CD40) that is critical to immune regulation in colon biopsies collected from monkey dosed with an anti-CD40 antibody. Unlike other techniques that measure receptor occupancy, such as flow cytometry, this technique does not rely on viable cells allowing measurement of frozen samples in a remote setting from the clinic.

Single-dose pharmacokinetics of tenofovir alafenamide and its active metabolite in the mucosal tissues.

Objectives: Tenofovir alafenamide, a prodrug of tenofovir, produces higher PBMC concentrations of tenofovir diphosphate (tenofovir-dp) than tenofovir disoproxil fumarate. To understand tenofovir alafenamide's mucosal tissue distribution and its implications for pre-exposure prophylaxis, we characterized tenofovir-dp in female genital tract (FGT) and lower gastrointestinal (GI) tissues. Methods: Healthy seronegative women were given 5, 10 or 25 mg of tenofovir alafenamide ( n = 8/group). Each participant provided plasma, PBMC and cervical, vaginal and rectal tissue samples over 14 days. Plasma, cell lysate and tissue homogenate concentrations were analysed by LC-MS/MS. Dose proportionality was declared in plasma and PBMCs if the natural log AUC versus natural log dose regression line 90% CI was within 0.57-1.43. In vitro tenofovir-dp formation was assessed in PBMCs and ectocervical (Ect1/E6E7) and vaginal (VK2/E6E7) cells incubated in 0.5 and 10 µM tenofovir alafenamide or tenofovir. clinicaltrials.gov: NCT02357602. Results: Following single doses of 5, 10 and 25 mg, median (IQR) tenofovir plasma AUC 0-14 days was 52.8 (49.5-59.6), 78.1 (68.2-86.9) and 169.7 (131.2-211.4) ng·h/mL and tenofovir-dp PBMC AUC 0-14 days was 2268 (1519-4090), 4584 (3113-5734) and 9306 (6891-10785) fmol·h/10 6 cells, respectively. Tenofovir was quantifiable in 52% and 92% of FGT and GI tissues, whereas tenofovir-dp was quantifiable in only 5% and 19% of FGT and GI tissues, respectively. Plasma tenofovir and PBMC tenofovir-dp were dose proportional (90% CI = 0.87-1.15 and 0.62-1.02, respectively). In vitro tenofovir-dp was 1.7-17-fold higher in epithelial cells than PBMCs. Conclusions: After tenofovir alafenamide dosing in vivo , tenofovir-dp was unquantifiable in most tissues (91%) although cervical and vaginal epithelial cells efficiently formed tenofovir-dp from tenofovir alafenamide in vitro . These findings warrant further investigation of tenofovir alafenamide's pharmacology.

Defensins at the Mucosal Surface: Latest Insights into Defensin-Virus Interactions.

Defensins are innate immune effector peptides expressed at mucosal surfaces throughout the human body and are potently antiviral in vitro The role of defensins in viral pathogenesis in vivo is poorly understood; however, recent studies have revealed that defensin-virus interactions in vivo are complicated and distinct from their proposed antiviral mechanisms in vitro These findings highlight the need for additional research that connects defensin neutralization of viruses in cell culture to in vivo antiviral mechanisms.

A critical review about methodologies for the analysis of mucoadhesive properties of drug delivery systems.

Mucoadhesion is a useful strategy for drug delivery systems, such as tablets, patches, gels, liposomes, micro/nanoparticles, nanosuspensions, microemulsions and colloidal dispersions. Moreover, it has contributed to many benefits like increased residence time at application sites, drug protection, increased drug permeation and improved drug availability. In this context, investigation into the mucoadhesive properties of pharmaceutical dosage forms is fundamental, in order to characterize, understand and simulate the in vivo interaction between the formulation and the biological substrate, contributing to the development of new mucoadhesive systems with effectiveness, safety and quality. There are a lot of in vivo, in vitro and ex vivo methods for the evaluation of the mucoadhesive properties of drug delivery systems. However, there also is a lack of standardization of these techniques, which makes comparison between the results difficult. Therefore, this work aims to show an overview of the most commonly employed methods for mucoadhesion evaluation, relating them to different proposed systems and using artificial or natural mucosa from humans and animals.

A Fluorescent Probe for Glycosaminoglycans Applied to the Detection of Dermatan Sulfate by a Mix-and-Read Assay.

Glycosaminoglycans are complex biomolecules of great biological and medical importance. The quantification of glycosaminoglycans, in particular in complex matrices, is challenging due to their inherent structural heterogeneity. Heparin Red, a polycationic, fluorescent perylene diimide derivative, has recently emerged as a commercial probe for the convenient detection of heparins by a mix-and-read fluorescence assay. The probe also detects glycosaminoglycans with a lower negative charge density than heparin, although with lower sensitivity. We describe here the synthesis and characterization of a structurally related molecular probe with a higher positive charge of +10 (vs. +8 of Heparin Red). The superior performance of this probe is exemplified by the quantification of low dermatan sulfate concentrations in an aqueous matrix (quantification limit 1 ng/mL) and the detection of dermatan sulfate in blood plasma in a clinically relevant concentration range. The potential applications of this probe include monitoring the blood levels of dermatan sulfate after administration as an antithrombotic drug in the absence of heparin and other glycosaminoglycans.

The Synaptojanins in the murine small and large intestine.

The expression of the phosphoinositides phosphatases Synaptojanins (Synjs) 1 and 2 has been shown in brain and in some peripheral tissues, but their expression in the intestine has not been reported. Herein we show that the small and large intestine express Synj1 and Synj2. Their mRNA levels, measured by RT-PCR, are not affected by development in the small intestine but in the colon they increase with age. Immunostaining assays reveal that both Synjs localize at the apical domain of the epithelial cells and at the lamina propria at sites also expressing the neuron marker calretinin. Synj2 staining at the lamina propria is fainter than that of Synj1. In colonocytes Synjs are at the apical membrane and cytosolic membrane vesicles. Synj2 is also at the mitochondria. Western blots reveal that the intestinal mucosa expresses at least two Synj1 (170- and 139-kDa) and two Synj2 (160- and 148-kDa) isoforms. The observations suggest that Synj1-170, Synj2-160, and Synj2-148 in colonocytes, might participate in processes that take place mainly at the apical domain of the epithelial cells whereas Synj1-139 in those at the enteric nervous system. Experimental colitis augments the mRNA abundance of both Synjs in colon but only Synj2 mRNA levels are increased in colon tumors. In conclusion, as far as we know, this is the first report showing expression, location and isoforms of Synj1 and Synj2 in the small and large intestine and that they might participate in intestinal pathology.

Ex vivo construction of a novel model of bioengineered bladder mucosa: A preliminary study.

OBJECTIVE: To generate and to evaluate ex vivo a novel model of bioengineered human bladder mucosa based on fibrin-agarose biomaterials. METHODS: We first established primary cultures of stromal and epithelial cells from small biopsies of the human bladder using enzymatic digestion and selective cell culture media. Then, a bioengineered substitute of the bladder lamina propria was generated using cultured stromal cells and fibrin-agarose scaffolds, and the epithelial cells were then subcultured on top to generate a complete bladder mucosa substitute. Evaluation of this substitute was carried out by cell viability and histological analyses, immunohistochemistry for key epithelial markers and transmission electron microscopy. RESULTS: The results show a well-configured stroma substitute with a single-layer epithelium on top. This substitute was equivalent to the control bladder mucosa. After 7 days of ex vivo development, the epithelial layer expressed pancytokeratin, and cytokeratins CK7, CK8 and CK13, as well as filaggrin and ZO-2, with negative expression of CK4 and uroplakin III. A reduction of the expression of CK8, filaggrin and ZO-2 was found at day 14 of development. An immature basement membrane was detected at the transition between the epithelium and the lamina propria, with the presence of epithelial hemidesmosomes, interdigitations and immature desmosomes. CONCLUSIONS: The present results suggest that this model of bioengineered human bladder mucosa shared structural and functional similarities with the native bladder mucosa, although the epithelial cells were not fully differentiated ex vivo. We hypothesize that this bladder mucosa substitute could have potential clinical usefulness after in vivo implantation.

A unique gene cluster for the utilization of the mucosal and human milk-associated glycans galacto-N-biose and lacto-N-biose in Lactobacillus casei.

The probiotic Lactobacillus casei catabolizes galacto-N-biose (GNB) and lacto-N-biose (LNB) by using a transport system and metabolic routes different from those of Bifidobacterium. L. casei contains a gene cluster, gnbREFGBCDA, involved in the metabolism of GNB, LNB and also N-acetylgalactosamine. Inactivation of gnbC (EIIC) or ptsI (Enzyme I) of the phosphoenolpyruvate : sugar phosphotransferase system (PTS) prevented the growth on those three carbohydrates, indicating that they are transported and phosphorylated by the same PTS(Gnb) . Enzyme activities and growth analysis with knockout mutants showed that GnbG (phospho-ß-galactosidase) hydrolyses both disaccharides. However, GnbF (N-acetylgalactosamine-6P deacetylase) and GnbE (galactosamine-6P isomerase/deaminase) are involved in GNB but not in LNB fermentation. The utilization of LNB depends on nagA (N-acetylglucosamine-6P deacetylase), showing that the N-acetylhexosamine moieties of GNB and LNB follow different catabolic routes. A lacAB mutant (galactose-6P isomerase) was impaired in GNB and LNB utilization, indicating that their galactose moiety is channelled through the tagatose-6P pathway. Transcriptional analysis showed that the gnb operon is regulated by substrate-specific induction mediated by the transcriptional repressor GnbR, which binds to a 26 bp DNA region containing inverted repeats exhibiting a 2T/2A conserved core. The data represent the first characterization of novel metabolic pathways for human milk oligosaccharides and glycoconjugate structures in Firmicutes.