Light Microscopy Research Articles

Effect of a phthalate derivative purified from Bacillus zhangzhouensis SK4 on quorum sensing regulated virulence factors of Pseudomonas aeruginosa

Pseudomonas aeruginosa causes life-threatening diseases and is resistant to almost all conventional antibiotics. The quorum sensing (QS) system of P. aeruginosa contributes to many pathogenic factors some of which are pigment production, motility, and biofilm. The disruption of quorum sensing system may be an impactful strategy to deal with infections. The present study investigates the anti-quorum sensing property of a bioactive molecule extracted from marine epibiotic bacteria present on the surface of seaweeds. Among all the isolates tested against monitor strain Chromobacterium violaceum (MTCC 2656), the one with the highest activity was identified as Bacillus zhangzhouensis SK4. The culture supernatant was extracted with chloroform which was then partially purified by TLC and column chromatography. The probable anti-QS compound was identified as 1,2-benzenedicarboxylic acid, bis (2-methylpropyl ester) by GC-MS and NMR analysis. The treatment of P. aeruginosa MCC 3457 with the lead compound resulted in the reduced production of pyocyanin, rhamnolipids, exopolysaccharide, biofilm, and motility. The observations of light and scanning electron microscopy also supported the biofilm inhibition. The lead compound showed synergism with the meropenem antibiotic and significantly reduced MIC. The molecular docking and pharmacokinetics study predicted 1, 2-benzenedicarboxylic acid, bis (2-methylpropyl ester), a phthalate derivative as a good drug candidate. The molecular dynamics study was also performed to check the stability of the lead compound and LasR complex. Further, lead compounds did not exhibit any cytotoxicity when tested on human embryonic kidney cells. As per our knowledge, this is the first report on the anti-QS activity of B. zhangzhouensis SK4, indicating that epibiotic bacteria can be a possible source of novel compounds to deal with the multidrug resistance phenomenon.

EVALUATION OF TRICHODERMA ASPERELLUM BIOFORTIFIED WITH VERMI COMPOST AGAINST FUSARIUM OXYSPORUM F.SP. LYCOPERSICI

Morphological features are not adequate to accurately categorizedifferent species of the genus Trichoderma. Molecular characters, incombination with morphological characters, are used to identify Trichodermaat the species or subspecies level. The present study was focused oncharacterising Trichoderma asperellum based on morphology and molecularanalysis using genes such as ITS. Light microscopy results showed that Trichodermaasperellum regularly branched and typically paired conidiophores withstraight phialides and globose to subglobose shaped conidia Sequence similarityanalysis with reference T. asperellum isolates available in the NCBIdatabase showed 100 percent nucleotide similarity for ITS4 and ITS5. A dualculture test clearly showed that Trichoderma asperllum inhibited thetested fungal plant pathogen, Fusarium oxysporum f. sp. lycopersici(87.38 percent). Population dynamics of Trichoderma asperellum incompost were found to be stable until 21 days after mixing.

Macroscopic, microscopic, and immunofluorescent characterization of the Greek tortoise (Testudo graeca graeca) oropharyngeal floor with concern to its feed adaptation as a herbivorous land reptile.

The current investigation focuses on gross anatomy, light, and scanning electron microscopy (SEM) of the Testudo graeca oropharyngeal floor, with particular reference to the immunofluorescence technique to examine its tongue. The T. graeca oropharyngeal floor showed many anatomical structures: the lower rhamphotheca, paralingual ridge, lower alveolar ridge, tongue, laryngeal mound, and glottis. The lower rhamphotheca appeared as a V-shaped jaw line with a highly serrated edge and a median tomium (beak). SEM observations of the lingual apex and the lingual body showed rectangular and conical filiform papillae with porous surfaces and taste pores. Meanwhile, the lingual root had two wings that carried papillae with different shapes: dagger-shaped, conical, bifurcated, and leaf-like papillae, and these papillae lacked taste pores. The laryngeal mound had openings for the laryngeal mucus gland and its secretions. Light microscopy findings showed mucous glands in the propria submucosa and near the mucosal surface of the lingual apex. The lingual root had lingual papillae and two hyaline cartilaginous skeletons between skeletal muscles, and the lingual papillae were elongated filiform, rectangular filiform papillae, and fungiform papillae. The lamina propria constituted the core of the lingual papillae and the mucous gland, they had a positive reaction with the periodic acid schiff (PAS) reagent. The apical surface of the fungiform papillae had taste pores. Under immunofluorescence, the vimentin was detected in taste bud cells, and synaptophysin reacted to the taste buds and nerve bundles. The current study of the Greek tortoise oropharyngeal floor investigated its herbivorous eating habits using its serrated lower rhamphotheca, a large tongue with differently shaped papillae, and numerous mucous glands. RESEARCH HIGHLIGHTS: The Greek tortoise (T. graeca graeca) oropharyngeal floor showed many anatomical structures: lower rhamphotheca, paralingual ridge, lower alveolar ridge, tongue, laryngeal mound, and glottis. SEM and light microscopy observations of the tongue revealed varied types and shapes of lingual papillae with a porous surface on the tongue apex (rectangular or conical filiform papillae), on the tongue body (filiform and fungiform papillae), and on the tongue root (dagger-shaped, conical, bifurcated, and leaf-like papillae). Light microscopy findings: the lamina propria constituted the core of the lingual papillae and had numerous mucous glands that had a slightly magenta-red color with PAS reagent. The apical surface of the fungiform papillae had taste pores. Vimentin and synaptophysin gave a reaction to the taste buds.

Distribution of prostatic markers in glands of the female urethra and anterior vaginal wall-a rapid autopsy study.

There are varying reports of immunohistochemically detected prostatic marker protein distribution in glands associated with the female urethra that may be related to tissue integrity at the time of fixation. In this study we used tissue derived from rapid autopsies of female patients to determine the distribution of glandular structures expressing prostate-specific antigen (PSA) and prostate-specific acid phosphatase (PSAP) along the female urethra and in surrounding tissues, including the anterior vaginal wall (AVW). Tissue blocks from 7 donors that contained the entire urethra and adjacent AVW were analyzed. These tissue samples were fixed within 4-12hours of death and divided into 5-mm transverse slices that were paraffin embedded. Sections cut from each slice were immunolabeled for PSA or PSAP and a neighboring section was stained with hematoxylin and eosin. The sections were reviewed by light microscopy and analyzed using QuPath software. In tissue from all donors, glandular structures expressing PSA and/or PSAP were located within the wall of the urethra and were present along its whole length. In the proximal half of the urethra from all donors, small glands expressing PSAP, but not PSA, were observed adjacent to the and emptying into the lumen. In the distal half of the urethra from 5 of the 7 donors, tubuloacinar structures lined by a glandular epithelium expressed both PSA and PSAP. In addition, columnar cells at the surface of structures with a multilayered transitional epithelium in the distal half of the urethra from all donors expressed PSAP. No glands expressing PSA or PSAP were found in tissues surrounding the urethra, including the AVW. Greater understanding of the distribution of urethral glands expressing prostatic proteins in female patients is important because these glands are reported to contribute to the female sexual response and to urethral pathology, including urethral cysts, diverticula, and adenocarcinoma. Strengths of the present study include the use of rapid autopsy to minimize protein degradation and autolysis, and the preparation of large tissue sections to demonstrate precise anatomical relations within all the tissues surrounding the urethral lumen. Limitations include the sample size and that all donors had advanced malignancy and had undergone previous therapy which may have had unknown tissue effects. Proximal and distal glands expressing prostate-specific proteins were observed in tissue from all donors, and these glands were located only within the wall of the urethra.

Effect of glazing and thermocycling on the fracture toughness and hardness of a New fully crystallized aluminosilicate CAD/CAM ceramic material

BackgroundThe mechanical properties of fully crystallized lithium aluminosilicate ceramics may be influenced by intraoral temperature variations and postmilling surface treatment. The purpose of this study is to explore the interplay among glazing, thermocycling, and the mechanical characteristics (namely, fracture toughness and hardness) of fully crystallized lithium aluminosilicate ceramics.MethodsBending bars (n = 40) cut from LisiCAD blocks (GC, Japan) were randomly assigned to glazed or unglazed groups (n = 20) and subjected to the single edge v-notch beam method to create notches. A glazing firing cycle was applied to the glazed group, while the unglazed group was not subjected to glazing. Half of the specimens (n = 10) from both groups underwent thermocycling before fracture toughness testing. The fracture toughness (KIC) was evaluated at 23 ± 1 °C using a universal testing machine configured for three-point bending, and the crack length was measured via light microscopy. Seven specimens per group were selected for the hardness test. Hardness was assessed using a Vickers microhardness tester with a 1 kg load for 20 s, and each specimen underwent five indentations following ISO 14705:2016. The Shapiro–Wilk and Kolmogorov-Smirnov tests were used to evaluate the normality of the data and a two-way ANOVA was utilized for statistical analysis. The significance level was set at (α = 0.05).ResultsRegardless of the thermocycling conditions, the glazed specimens exhibited significantly greater fracture toughness than did their unglazed counterparts (P < 0.001). Thermocycling had no significant impact on the fracture toughness of either the glazed or unglazed specimens. Furthermore, statistical analysis revealed no significant effects on hardness with thermocycling in either group, and glazing alone did not substantially affect hardness.ConclusionsThe impact of glazing on the fracture toughness of LiSiCAD restorations is noteworthy, but it has no significant influence on their hardness. Furthermore, within the parameters of this study, thermocycling was found to exert negligible effects on both fracture toughness and hardness.

Functional Morphology of Leg Mechanosensory Organs in Early Postembryonic Development in the Stick Insect (Sipyloidea chlorotica)

The subgenual organ complex of stick insects has a unique neuroanatomical organisation with two elaborate chordotonal organs, the subgenual organ and the distal organ. These organs are present in all leg pairs and are already developed in newly hatched stick insects. The present study analyses for the first time the morphology of sensory organs in the subgenual organ complex for a membrane connecting the two sensory organs in newly hatched insects (Sipyloidea chlorotica (Audinet-Serville 1838)). The stick insect legs were analysed following hatching by axonal tracing and light microscopy. The subgenual organ complex in first juvenile instars shows the sensory organs and a thin membrane connecting the sensory organs resembling the morphology of adult animals. Rarely was this membrane not detected, where it is assumed as not developed during embryogenesis. The connection appears to influence the shape of the subgenual organ, with one end extending towards the distal organ as under tension. These findings are discussed for the following functional implications: (1) the physiological responses of the subgenual organ complex to mechanical stimuli after hatching, (2) the influence of the membrane on the displacement of the sensory organs, and (3) the connection between the subgenual organ and distal organ as a possible functional coupling.

Lysophosphatidic Acid Stimulates Mitogenic Activity and Signaling in Human Neuroblastoma Cells through a Crosstalk with Anaplastic Lymphoma Kinase

Lysophosphatidic acid (LPA) is a well-documented pro-oncogenic factor in different cancers, but relatively little is known on its biological activity in neuroblastoma. The LPA effects and the participation of the tyrosine kinase receptor anaplastic lymphoma kinase (ALK) in LPA mitogenic signaling were studied in human neuroblastoma cell lines. We used light microscopy and [3H]-thymidine incorporation to determine cell proliferation, Western blot to study intracellular signaling, and pharmacological and molecular tools to examine the role of ALK. We found that LPA stimulated the growth of human neuroblastoma cells, as indicated by the enhanced cell number, clonogenic activity, and DNA synthesis. These effects were curtailed by the selective ALK inhibitors NPV-TAE684 and alectinib. In a panel of human neuroblastoma cell lines harboring different ALK genomic status, the ALK inhibitors suppressed LPA-induced phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), which are major regulators of cell proliferation. ALK depletion by siRNA treatment attenuated LPA-induced ERK1/2 activation. LPA enhanced ALK phosphorylation and potentiated ALK activation by the ALK ligand FAM150B. LPA enhanced the inhibitory phosphorylation of the tumor suppressor FoxO3a, and this response was impaired by the ALK inhibitors. These results indicate that LPA stimulates mitogenesis of human neuroblastoma cells through a crosstalk with ALK.

Systematic Implications of Palynomorph Diversity Using Microscopic Trends Among Asteraceous Flora From the Drylands of Baluchistan, Pakistan.

Pollen micromorphological traits with taxonomic implications are first reported from the study area for 50 Asteraceous taxa belonging to nine tribes. Cichorieae (21 taxa), Cardueae (11 taxa), Inuleae (six taxa), and Anthemideae (four taxa) are the leading tribes. The research included Cousinia haeckeliae, Himalaiella afghana, Pterachaenia stewartii (endemic to Afghanistan and Pakistan), and Xylanthemum macropodum (endemic to Baluchistan). Light and scanning electron microscopy were employed for the visualization of pollen photomicrographs. The data was analyzed statistically via SPSS, PAST, and Origin. Significant diagnostic qualitative and quantitative palynological traits were explored for discrimination down to the species level within the tribes. All the investigated taxa possessed radial symmetry, isopolarity, and monad form (characters for distinction at the subdivision level). The aperture types were trizonocolporate, tetrazonocolporate, and tricolporate with number position and character (NPC) formulas N3P4C5, N4P4C5, and N3P4C3. Goniotreme, peritreme, and ptychotreme types of amb were recognized. Echinate, echinate lophate, scabrate, and gemmate sculpturing were present with and without perforated surface patterns. Variations in the shapes in polar and equatorial views and lacuna shapes further assisted the separation of taxa. The observed shape classes were perprolate, prolate spheroidal, prolate, subprolate, oblate spheroidal, suboblate, and oblate. Principal component analysis, correlation, standard probability plots, and ridge line paired features plot for quantitative variables determined the positive correlation between the length and width of colpi in equatorial and polar view with polar axis and equatorial diameter and number of spines between colpi with the number of spines per pollen. The number of spines per pollen was negatively correlated with the width and length of colpi in the polar view. Multiple sample analysis of variance (ANOVA) concluded that a high statistically significant difference exists among the means of analyzed traits. The examined qualitative and quantitative palynological traits revealed noticeable variations, thus providing the source for species discrimination in Asteraceous tribes.

The physical and cellular mechanism of structural color change in zebrafish

Many animals exhibit remarkable colors that are produced by the constructive interference of light reflected from arrays of intracellular guanine crystals. These animals can fine-tune their crystal-based structural colors to communicate with each other, regulate body temperature, and create camouflage. While it is known that these changes in color are caused by changes in the angle of the crystal arrays relative to incident light, the cellular machinery that drives color change is not understood. Here, using a combination of 3D focused ion beam scanning electron microscopy (FIB-SEM), micro-focused X-ray diffraction, superresolution fluorescence light microscopy, and pharmacological perturbations, we characterized the dynamics and 3D cellular reorganization of crystal arrays within zebrafish iridophores during norepinephrine (NE)-induced color change. We found that color change results from a coordinated 20° tilting of the intracellular crystals, which alters both crystal packing and the angle at which impinging light hits the crystals. Importantly, addition of the dynein inhibitor dynapyrazole-a completely blocked this NE-induced red shift by hindering crystal dynamics upon NE addition. FIB-SEM and microtubule organizing center (MTOC) mapping showed that microtubules arise from two MTOCs located near the poles of the iridophore and run parallel to, and in between, individual crystals. This suggests that dynein drives crystal angle change in response to NE by binding to the limiting membrane surrounding individual crystals and walking toward microtubule minus ends. Finally, we found that intracellular cAMP regulates the color change process. Together, our results provide mechanistic insight into the cellular machinery that drives structural color change.

Patient-derived parotid gland pleomorphic adenoma organoid model and its preliminary histological and functional characterization

Objective: To establish patient-derived organoid models of pleomorphic adenomas (PAs) of the parotid gland and preliminarily characterize their histology, related biomarkers and functions. Methods: Fresh tumor tissue specimens were collected from surgical procedures of Oral and Maxillofacial Department. The harvested tissues were processed and cultured in a head and neck tumor organoid culture system, resulting in the successful establishment of organoid models from four cases of parotid gland pleomorphic adenomas. The in vitro growth of PA organoids was recorded by light microscopy. The successfully established organoids were passaged and cryopreserved, and the cryopreserved PA organoids were revived and re-cultured to observe their viability and organoid regeneration ability. Histological characterization, as well as characterization and detection of related markers and functional proteins, were performed on the organoids, comparing them with the patient-derived tissues. Results: The constructed organoid model of pleomorphic adenoma exhibited a dense and compact three-dimensional spherical structure. Hematoxylin and eosin staining indicated morphological similarities between the organoid and its tissue of origin. Immunohistochemistry showed positive cytoplasmic staining for Calponin, CK7, and EMA in both the organoid and the source tumor tissue, suggesting consistent histopathological characteristics between the organoid and its tissue of origin. Periodic acid-Schiff staining of the organoid showed positive staining for glycogen, with positive staining located in the interior and periphery of the organoid, indicating that the organoid possessed secretory functions like the salivary gland. Conclusion: We successfully constructed organoids of pleomorphic adenoma derived from patient samples. This model faithfully replicates the tissue morphology and biomarkers of the source tissue and exhibits biological functions associated with mucus secretion. It serves as a valuable in vitro model for studying the development and progression of salivary gland tumors.

Immunohistochemical properties of embryonic telocytes in a myogenic microenvironment.

Telocytes are a unique interstitial cell type that functions in adulthood and embryogenesis. They have characteristic immunohistochemical phenotypes while acquiring different immunohistochemical properties related to the organ microenvironment. The present study aims to investigate the immunohistochemical features of embryonic telocytes during myogenesis and describe their morphology using light microscopy and TEM. Telocytes represent a major cellular constituent in the interstitial elements. They had distinguished telopodes and podoms and formed a 3D interstitial network in the developing muscles. They formed heterocellular contact with myoblasts and nascent myotubes. Telocytes also had distinctive secretory activity. Telocytes identified by CD34. They also express CD68 and MMP-9 to facilitate the development of new tissues. Expression of CD21 by telocytes may reveal their function in immune defense. They also express VEGF, which regulates angiogenesis. In conclusion, the distribution and immunological properties of telocytes in the myogenic tissue indicate that telocytes provide biological and structural support in the development of the myogenic tissue architecture and organization.

Desmodesmus pseudoperforatus sp. nov., a giant in Desmodesmus, series Maximi

ABSTRACT Series Maximi is a group within Desmodesmus (Chlorophyta) which contains the three species D. maximus, D. perforatus and D. tropicus. Besides their large cell sizes, the most striking morphological trait unique to the members of this genus is the presence of large perforations between the coenobial cells. The perforations are either present (D. perforatus and D. tropicus) or the space between the cells is completely sealed (D. maximus). We report here a fourth species of the series based on two strains (AICB 1155 and AICB 1572) from freshwater environments of the Danube Delta, Romania. The ultrastructure of the cell walls revealed a partial closure of the space between the cells which creates slightly visible perforations in light microscopy. Analyses of DNA sequences and secondary structure of the ITS2 fragment by coalescent (PTP, GMYC), distance (ASAP), and evolutionary modelbased methods (ML, BI) indicated a fourth candidate species which included both the AICB strains investigated and other strains with similar morphological traits from reference literature. Despite its poor representation in public databases, analysis of the SSU rDNA gene for the series Maximi strengthened the ITS2 results. Therefore, based on its larger cell sizes relative to the other members of the series, cell morphology, the presence of partial perforations and other peculiarities of the cell wall ultrastructure, the independent phylogenetic position, CBCs analysis, and a different ‘DNA signature’ within the ITS2 fragment we suggest that this clade should be described as a new taxon of the series Maximi, Desmodesmus pseudoperforatus sp. nov.

Automated quantification of photoreceptor outer segments in developing and degenerating retinas on microscopy images across scales

The functionality of photoreceptors, rods, and cones is highly dependent on their outer segments (POS), a cellular compartment containing highly organized membranous structures that generate biochemical signals from incident light. While POS formation and degeneration are qualitatively assessed on microscopy images, reliable methodology for quantitative analyses is still limited. Here, we developed methods to quantify POS (QuaPOS) maturation and quality on retinal sections using automated image analyses. POS formation was examined during the development and in adulthood of wild-type mice via light microscopy (LM) and transmission electron microscopy (TEM). To quantify the number, size, shape, and fluorescence intensity of POS, retinal cryosections were immunostained for the cone POS marker S-opsin. Fluorescence images were used to train the robust classifier QuaPOS-LM based on supervised machine learning for automated image segmentation. Characteristic features of segmentation results were extracted to quantify the maturation of cone POS. Subsequently, this quantification method was applied to characterize POS degeneration in “cone photoreceptor function loss 1” mice. TEM images were used to establish the ultrastructural quantification method QuaPOS-TEM for the alignment of POS membranes. Images were analyzed using a custom-written MATLAB code to extract the orientation of membranes from the image gradient and their alignment (coherency). This analysis was used to quantify the POS morphology of wild-type and two inherited retinal degeneration (“retinal degeneration 19” and “rhodopsin knock-out”) mouse lines. Both automated analysis technologies provided robust characterization and quantification of POS based on LM or TEM images. Automated image segmentation by the classifier QuaPOS-LM and analysis of the orientation of membrane stacks by QuaPOS-TEM using fluorescent or TEM images allowed quantitative evaluation of POS formation and quality. The assessments showed an increase in POS number, volume, and membrane coherency during wild-type postnatal development, while a decrease in all three observables was detected in different retinal degeneration mouse models. All the code used for the presented analysis is open source, including example datasets to reproduce the findings. Hence, the QuaPOS quantification methods are useful for in-depth characterization of POS on retinal sections in developmental studies, for disease modeling, or after therapeutic interventions affecting photoreceptors.

Extending culture time to improve Mitotic Index for cytogenetic dosimetry

Purpose To analyze the effects of extending lymphocyte cultivation time on the Mitotic Index, frequency of first-division cells, and dose estimation after irradiating blood samples with different doses of radiation. Materials and methods Blood samples from two healthy male volunteers were separately irradiated with three doses (3, 5, and 6 Gy) using a 60Co gamma source (average dose rate: 1.48 kGy.h−1) and cultivated in vitro for conventional (48 h) and extended (56, 68, and 72 h) amounts of time. Colcemid (0.01 µg.mL−1) was added at the beginning of the culture period. Cells were fixed, stained with fluorescence plus Giemsa (FPG), and analyzed under a light microscope. The effects of prolonged culture duration on the Mitotic Index (MI), frequency of first-division cells (M1 cells), and the First-Division Mitotic Index (FDMI) were investigated. The estimation of delivered doses was conducted using a conventional 48h-culture calibration curve. Results Overall, cells presented higher MI (up to 12-fold) with the extension of culture, while higher radiation doses led to lower MI values (up to 80% reduction at 48 h). Cells irradiated with higher doses (5 and 6 Gy) had the most significant increase (5- to 12-fold) of MI as the cultivation was prolonged. The frequency of M1 cells decreased with the prolongation of culture for all doses (up to 75% reduction), while irradiated cells presented higher frequencies of M1 cells than non-irradiated ones. FDMI increased for all irradiated cultures but most markedly in those irradiated with higher doses (up to 10-fold). The conventional 48h-culture calibration curve proved adequate for assessing the delivered dose based on dicentric frequency following a 72-hour culture. Conclusion Compared to the conventional 48-hour protocol, extending the culture length to 72 hours significantly increased the Mitotic Index and the number of first-division metaphases of irradiated lymphocytes, providing slides with a better scorable metaphase density. Extending the culture time to 72 hours, combined with FPG staining to score exclusively first-division metaphases, improved the counting of dicentric chromosomes. The methodology presented and discussed in this study can be a powerful tool for dicentric-based biodosimetry, especially when exposure to high radiation doses is involved.

Collagen hydrogel protects intestinal epithelial cells from indomethacin-induced damage: results of an in vitro experiment

INTRODUCTION. Indomethacin is a derivative of indoleacetic acid and has anti-inflammatory, analgesic and antipyretic effects. However, the results of numerous studies show that indomethacin, like many other nonsteroidal anti-inflammatory drugs (NSAIDs), have an inhibitory effect on the viability and functional activity of enterocytes. In this regard, the search for new ways to reduce the severity of side effects from the use of NSAIDs remains relevant. One of these approaches may be to enrich patients’ diets with non-drug biologically active compounds, including proteins. However, the effect of dietary proteins and biologically active peptides on NSAID-induced damage to the wall of the small intestine and stomach has not been sufficiently studied. AIM. To evaluate the ability of a collagen-containing dietary supplement to protect human duodenal epithelial cells (HuTu-80 line) from indomethacin-induced damage. MATERIALS AND METHODS. The composite collagen-containing hydrogel was provided by «FIRST ALIVE COLLAGEN» LLC (Russia) and is a registered dietary supplement. The work used a commercial culture of human skin fibroblast cells and human duodenal epithelial cells (line HuTu-80). The viability of intestinal cells and fibroblasts was assessed using light and fluorescence microscopy and flow cytometry methods. RESULTS AND DISCUSSION. It has been established that indomethacin inhibits cell growth, causes apoptosis and death of enterocytes, and also leads to the accumulation of cells in the S-phase, which indicates a disruption in the regulation of the cell cycle. It was revealed that collagen hydrogel prevents cell death caused by indomethacin and reduces the number of apoptotic cells in the population. The protective effect of collagen hydrogel is characterized by normalization of the cell cycle of enterocytes and restoration of their growth and proliferative activity. CONCLUSION. Thus, collagen hydrogel, in vitro, is able to reduce the pathogenic effect of indomethacin on human intestinal epithelial cells. The protective effect of collagen hydrogel is characterized by maintaining viability, inhibiting apoptotic processes, and maintaining cell cycle stability. The results obtained indicate the prospects of using a dietary supplement based on a composite collagen hydrogel as a prophylactic agent to reduce the risk of NSAID-associated gastrointestinal diseases. However, to confirm the therapeutic effectiveness of the dietary supplement, further research is necessary, both using experimental animal modeling of NSAID-associated diseases of the human gastrointestinal tract, and clinical studies.

#2862 Genetics and electron microscopy: complementary tools for management of focal segmental glomerulosclerosis?

Abstract Background and Aims Focal segmental glomerular sclerosis (FSGS) defines a histologic lesion that represents a pattern of injury associated to different pathologic conditions affecting both children and adults. It is classified into primary forms, presumably immune-mediated, secondary forms, determined by a maladaptive response to different conditions, genetic and undetermined forms. According to Mayo Clinic classification, primary and secondary forms can de differentiated using electron microscopy (EM), with a podocytes foot process effacement (FPE) ≥80% identifying primary forms. Better understanding the role of Genetics in FSGS and response to treatment in different forms is of vital importance in this rare and multifaceted condition. We analyzed a discrete cohort of patients with FSGS examined both with EM and genetic testing (GT). Main aims of our study were: 1) assessment of GT in primary and secondary forms; 2) analysis of response to therapy according to histologic and genetic classification. Method This is a retrospective observational study on patients that received a histological diagnosis of FSGS, including EM, and underwent NGS genetic testing from January 2020 to June 2023. Primary and secondary forms of FSGS were distinguished by degree of pedicle fusion at EM, according to Mayo Clinic classification. Clinical, laboratory, genetic data and used therapies were recorded. Complete response (CR) to therapy was defined as a reduction of proteinuria to 0.3 g/day, with normalization of albuminemia. Partial response (PR) was defined as halving proteinuria into a sub-nephrotic range and increasing albuminemia up to 3.5 g/dL. Absent response was defined as a persisting nephrotic range proteinuria. Results In the study period, 33 patients receiving a histologic diagnosis of FSGS with EM examination underwent NGS testing. Of these, 25 had available genetic results and were included. Ten patients (40%) were females, 15 (60%) were males; mean age was 40.8 years (SD ±14.5); 20 (80%) were Caucasian, 4 (16%) Black, 1 (4%) Asian. Based on EM, 11 (44%) were classified as primary forms, 14 (56%) as secondary forms. Twelve (48%) patients presented with nephrotic syndrome. Of these, 10 had a primary form. Seven (28%) had nephrotic-range proteinuria without hypoalbuminemia, 6 (24%) had subnephrotic range proteinuria. Six (54.5%) of the primary forms and 8 (57.1%) of the secondary forms had a positive genetic testing (p = 0.78). Among secondary forms, through GT, one diagnosis of Fabry disease and one of Tuberous Sclerosis were made in patients without clear systemic symptoms. Fabry disease was confirmed by presence of zebra bodies at EM. All primary forms received immunosuppressive therapies, all being rituximab-based regimens; all secondary forms received support therapy. Nine (81.1%) primary forms and 9 (64.3%) secondary forms had at least a PR to treatment (4 CR in primary group and 1 CR in secondary group) (p = 0.33). Among primary forms, 5 of 6 patients (83%) with a positive GT had at least a PR with a strong immunosuppressive therapy. In secondary forms group 4 of 8 patients (50%) had at least PR with offered support therapy. Fabry and Tuberous Sclerosis were offered specific treatments with benefit. Conclusion Percentage of positive GT did not differ in primary and secondary forms of FSGS as defined by Mayo Clinic classification. Thus a positive GT should not be considered a unique feature of secondary forms. GT was critical for an unexpected diagnosis of systemic hereditary diseases, thus enabling adequate target therapies. We believe GT should be offered to all patients with FSGS. In primary FSGS, defined according to EM, more than 80% of patients had at least a PR following immunosuppressive therapy, even in patients with positive GT. In conclusion, as clinical presentation and light microscopy are not sufficient for an accurate diagnosis, combining EM and GT is of utmost importance in proper classification and management of FSGS.

#2168 Lupus nephritis histopathological classification system: a questionnaire-based survey of the Renal Pathology Society (RPS)

Abstract Background and Aims Kidney biopsy is the gold standard for lupus nephritis diagnosis, defining histologic class, disease activity and chronicity in detail and providing guidance for treatment. The updated 2018 ISN/RPS histopathological classification in lupus nephritis is widely used for prognosis and treatment decisions. Given the evolving phenotypes of lupus nephritis and the success of new targeted agents in clinical trials, there is ongoing discussion about the need for updating existing classification systems. A survey to assess the current use of the lupus nephritis histopathological classification systems in everyday practice was conducted on behalf of the RPS. Method An online survey was sent between September 27th and October 24th, 2023 to active members of the RPS. The survey used an internet platform and contained both multiple-choice and open-ended questions; results were analyzed anonymously. Results 185 of 562 RPS members replied to the questionnaire. 179 (97%) were pathologists and the level of expertise in lupus nephritis was good: 120 (65%) participants indicated they encounter more than 20 biopsies with lupus nephritis per year, while only 13% encounter fewer than 10. Almost 90% of responders discuss kidney biopsy results in a multidisciplinary meeting involving both clinicians and pathologists, on a weekly or monthly basis. The 2018 ISN/RPS lupus nephritis classification is used in most cases (92%), while 7% of participants prefer the earlier 2004 version. The average grade on the utility of the 2018 ISN/RPS classification was 8 (IQR 7-9) on a scale from 0 (not useful) to 10 (extremely useful). Almost 90% of the pathologist responders indicated they include the NIH activity/chronicity indices in biopsy reports. Reasons for not adding the activity/chronicity indices were given as responses to open-ended questions: (1) clinicians usually do not ask for activity/chronicity indices—(2) the extra workload outweighs the perceived clinical benefit—(3) a quantitative description of the degree of activity/chronicity is less time-consuming and more understandable than the indices—(4) reproducibility is not guaranteed. The average grade on the utility of the activity/chronicity indices was 7 (IQR 7-9) on a scale from 0 (not useful) to 10 (extremely useful). Pathologists rated treating clinicians' understanding of kidney biopsy reports and lupus classification at light microscopy with an average score of 8 (IQR 7-9) on a scale from 0 (no understanding) to 10 (complete understanding). Suggested improvements of the 2018 ISN/RPS lupus nephritis classification were further standardization and simplicity. Clearer definitions were requested for: class III vs class IV; segmental sclerosis vs fibrotic crescents; the role of globally sclerosed glomeruli; lupus-like entities; and lupus podocytopathy. Furthermore, there was a request on guidelines for new parameters: e.g., focus on extraglomerular involvement, laboratory and clinical features and biomarkers; CD68+ staining to detect endocapillary hypercellularity; and EXT1/2 immunostaining. Conclusion Our survey shows the 2018 ISN/RPS lupus nephritis classification is widely used in everyday practice by pathologists, and well known by clinicians. The results obtained by the RPS survey serve as a starting point for the new Working Group of lupus nephritis classification. We will consider the ideas and suggestions of the international community to evaluate biopsies from extensively phenotyped cohorts, to modify the classification and to facilitate improved targeted treatment decisions—a current unmet need.

Dental stem cells improve memory and reduce cell death in rat seizure model.

Epilepsy is a common neurological disorder that significantly affects the quality of life of patients. In this study, we aim to evaluate the effectiveness of dental pulp stem cell (DPSC) transplantation in decreasing inflammation and cell death in brain cells, thus reducing seizure damage. We induced seizures in rats using intraperitoneal injections of pentylenetetrazole (PTZ). In the PTZ + DPSC group, we conducted bilateral hippocampal transplantation of DPSCs in PTZ-lesioned rat models. After 1 month, we performed post-graft analysis and measured some behavioral factors, such as working memory and long-term memory, using a T-maze test and passive avoidance test, respectively. We investigated the immunohistopathology and distribution of astrocyte cells through light microscopy and Sholl analysis. Additionally, we employed the Voronoi tessellation method to estimate the spatial distribution of the cells in the hippocampus. Compared to the control group, we observed a reduction in astrogliosis, astrocyte process length, the number of branches, and intersections distal to the soma in the hippocampus of the PTZ + DPSC group. Further analysis indicated that the grafted DPSCs decreased the expression of caspase-3 in the hippocampus of rats with induced seizures. Moreover, the DPSCs transplant protected hippocampal pyramidal neurons against PTZ toxicity and improved the spatial distribution of the hippocampal neurons. Our findings suggest that DPSCs transplant can be an effective modifier of astrocyte reactivation and inflammatory responses.

#710 Rare manifestation of minimal change disease associated with prostate cancer

Abstract Background and Aims Prostate cancer is the most common malignancy in men. Secondary nephrotic syndrome as part of the paraneoplastic syndrome occurs in 11% of cases and is most often associated with membranous glomerulopathy. The association of minimal change disease (MCD) and prostate cancer is extremely rare, where only one case has been described in the available literature. Method We present the case of a 77-year-old patient who was admitted to the Transplantation-Nephrology Department of the University Hospital Martin with 3rd degree acute kidney injury and anasarca with fully developed nephrotic syndrome. Results During hospitalization, there was a gradual elevation of renal parameters and oliguria with the need for hemodialysis treatment. However, as part of the oncological screening, the level of prostate specific antigen was elevated (10.69 ng/l) with subsequent biopsy confirmed adenocarcinoma of the prostate in stage IIB (pT1cN0M0). Renal biopsy was performed with the finding of MCD, in light microscopy without pathological findings, with fully vital, normocellular glomeruli, without interstitial fibrosis and tubular damage, immunofluorescence without significant findings, in the electron microscope, pedicel podocyte fusions were present. Despite the generally valid recommendations for the treatment of prostate cancer of the given stage and age of the patient (follow-up), antiandrogen therapy was started in cooperation with the urologist along with the treatment of MCD with corticosteroids at a dose of 1 mg/kg. The patient experienced a significant improvement in renal parameters with the onset of diuresis without the need for dialysis treatment. Conclusion In the reported case, we describe a rare form of MCD associated with prostate cancer and a rare manifestation in the form of renal failure and the need for dialysis treatment with subsequent complete restitution of renal functions after KS treatment and antiandrogen treatment of the underlying oncological disease.

#1941 About a case: ABBA disease in a patient with thymoma

Abstract Background and Aims ABBA (antibrush border antibody) disease is a rare autoimmune tubulointerstitial kidney disease with few descriptions in the literature. Diagnosis is based on light microscopy, immunofluorescence, electron microscopy and immunostaining for LRP2 (LDL receptor related protein 2), also known as megalin. Method We report the case of a 47-year-old Caucasian man admitted in January 2023 for oedema and acute kidney injury (creatinine 3 mg/dl). Biology revealed nephrotic syndrome with hypoalbuminemia (12 g/L) and albuminuria (13 g/g). Haematuria was also noted (75/mm3). His medical history was notable for myasthenia gravis and epithelial Gougerot. Serological tests were negative for HIV, hepatitis B and hepatitis C. Anti-GBM and anti-PLA2R were negative, antinuclear antibodies and antineutrophil cytoplasmic antibodies were both positive at 1/80 without specificity. Serum complement (C3 and C4) and protein electrophoresis were normal. Renal biopsy showed membranous nephropathy, anti-PLA2R, anti-THSD7A, NELL1 and EXT1/2 negative. There was also IgG staining along Bowman's capsule and along part of the tubular basement membrane (TBM). The acute tubular injury was severe. Morphological studies with thoracic tomodensitometry, thoracic MRI and PET scan revealed a thymoma. The patient underwent surgery in May. It was a stage 3 thymoma with pulmonary and pericardial invasion. In June 2023, the patient started haemodialysis due to progression of chronic renal failure. We performed a second renal biopsy, which confirmed the atypical membranous nephropathy with glomerular and tubular deposits and 10% interstitial sclerosis. The patient received two injections of RITUXIMAB (30 June and 14 July 2023). Results In October 2023, we performed a third renal biopsy due to dialysis dependency three months after RITUXIMAB. The glomerular lesions were similar to the previous biopsy, with minimal extra-membranous deposits. However, tubulointerstitial lesions were more pronounced than before. There was 25% tubular atrophy and 25% interstitial sclerosis associated with interstitial inflammation. We also identified anti-LRP2 immune deposits along the TBM. All these histopathological features lead to the diagnosis of ABBA. Conclusion Anti-LRP2 nephropathy is an orphan disease. It's caused by autoantibodies directed against the proximal tubular brush border, leading to a progressive decline in kidney function. The largest recently published cohort includes 67 patients. Many patients may be misdiagnosed due to the insidious onset and atypical presentation with concomitant glomerular or tubulointerstitial disease. In the cohort, 30% of patients had cancer, including solid organ malignancies (including 1 thymoma). It seems that combined treatment with corticosteroids and another immunosuppressive therapy (cyclophosphamide or rituximab) has better results. In our case, despite his thymoma surgery and rituximab perfusions, the patient is still on dialysis. The immunosuppressive treatment will be discussed with the oncologist soon.