Staining In Order Research Articles

Artificial Intelligence-Based Sentinel Lymph Node Metastasis Detection in Cervical Cancer

Background/objectives: Pathological ultrastaging, an essential part of sentinel lymph node (SLN) mapping, involves serial sectioning and immunohistochemical (IHC) staining in order to reliably detect clinically relevant metastases. However, ultrastaging is labor-intensive, time-consuming, and costly. Deep learning algorithms offer a potential solution by assisting pathologists in efficiently assessing serial sections for metastases, reducing workload and costs while enhancing accuracy. This proof-of-principle study evaluated the effectiveness of a deep learning algorithm for SLN metastasis detection in early-stage cervical cancer. Methods: We retrospectively analyzed whole slide images (WSIs) of hematoxylin and eosin (H&E)-stained SLNs from early-stage cervical cancer patients diagnosed with an SLN metastasis with either H&E or IHC. A CE-IVD certified commercially available deep learning algorithm, initially developed for detection of breast and colon cancer lymph node metastases, was employed off-label to assess its sensitivity in cervical cancer. Results: This study included 21 patients with early-stage cervical cancer, comprising 15 with squamous cell carcinoma, five with adenocarcinoma, and one with clear cell carcinoma. Among these patients, 10 had macrometastases and 11 had micrometastases in at least one SLN. The algorithm was applied to evaluate H&E WSIs of 47 SLN specimens, including 22 that were negative for metastasis, 13 with macrometastases, and 12 with micrometastases in the H&E slides. The algorithm detected all H&E macro- and micrometastases with 100% sensitivity. Conclusions: This proof-of-principle study demonstrated high sensitivity of a deep learning algorithm for detection of clinically relevant SLN metastasis in early-stage cervical cancer, despite being originally developed for adenocarcinomas of the breast and colon. Our findings highlight the potential of leveraging an existing algorithm for use in cervical cancer, warranting further prospective validation in a larger population.

#1918 L-NAME accelerates the progression of diabetic kidney disease in mice

Abstract Background and Aims Diabetic kidney disease (DKD) represents one of the major causes of end stage kidney disease worldwide. Despite DN having such a high incidence, mouse models of DN rarely mimic all aspects of the human disease, besides showing very slow progression and sometimes maintenance issues. For this reason, in this project we aimed to develop an accelerated, reproducible and accessible mouse model of DN. Method For our experiments we chose 9-week-old female and male of the leptin receptor knock-out (BKS.Cg-Dock7m +/+ Leprdb/J) mice as type 2 diabetes model. As these mice are still not prone to develop severe kidney injury despite their diabetic condition, we administered the eNOS inhibitor L-NAME for 6 weeks, in order to suppress nitric oxide production and therefore to aggravate kidney damage. Mice were divided in three groups: one vehicle control group, one receiving 40/mg/kg/day of L-NAME and one receiving 80/mg/kg/day of L-NAME dissolved in drinking water. Blood pressure and glomerular filtration rate (GFR) were measured at different time points, together with glycemia and albuminuria. Weight and water intake were also monitored throughout the study. At the end of the experiment, animals were killed and blood and organs collected for further analysis. Results Mice treated with 80 mg/kg/day of L-NAME showed an average increase of 10 mmHg in systolic blood pressure, with a peak of 30 mmHg increase at week 4 of treatment. On the other hand, 40 mg/kg/day of L-NAME did show any effect on blood pressure. Despite the different effect on blood pressure, three weeks of L-NAME administration caused a significant reduction in GFR at both doses, with 5% reduction for the 40 mg-group and 23% for the 80 mg-group. For the 80 mg-group we observed an even further GFR decline after 5 weeks of treatment, with a 28% reduction. No change in GFR was observed in the controls. This decrease in GFR was accompanied by a 10-fold increase in urinary albumin for both experimental groups already after 3 weeks of treatment, which after 4 weeks slightly declined but remained significantly higher than the controls (p < 0.0001). At a histological level, PAS staining of kidney sections revealed that in particular mice treated with 80 mg of L-NAME have enlarged Bowman capsule and glomerular tuft area, besides a reduced glomerular capillary density, coinciding with mesangial expansion. We also stained for overall collagen deposition with picrosirius red staining in order to detect possible fibrosis formation. We then observed that 80 mg of L-NAME accelerates interstitial fibrosis in the cortical area of the kidney, which also overlapped with an increased deposition of collagen I. This increase in collage I was also detected in kidney cortex of mice treated with 40 mg of L-NAME, although their overall collagen deposition and therefore fibrotic tissue did not differ from the control group. The medullar region of kidneys was also analyzed, but none of the two experimental groups revealed significant differences in terms overall collagen or collagen I deposition. Unlike mesangial expansion and fibrosis, L-NAME did not influence microphages infiltration, as revealed by CD68 staining in kidney sections. This suggests that L-NAME treatment does not accelerate kidney damage by triggering or accelerating inflammatory processes. This was also confirmed by qPCR analysis of kidney tissue, where the expression pro-inflammatory markers, such as IL1β, TNFα and MCP-1, did not change in the experimental groups. Conclusion Our results show that L-NAME considerably accelerates the progression of DKD in diabetic mice, with GFR declining and progressive albuminuria already after 3 weeks. Such disfunction is confirmed at 6 weeks by histological analysis, where mesangial expansion and fibrosis were detected. This model can be used to assess the therapeutic potential of novel interventions aimed to slow down DKD.

HELICOBACTER PYLORI – BIOLOGICAL FEATURES AND METHODS OF LABORATORY DIAGNOSIS

The spring period - in that time the relevance of diagnostics related to Helicobacter pylori infection forces us to pay more attention to the cohort of patients with gastrointestinal tract pathology. Most often, these are patients with exacerbation of chronic gastritis (CH) and peptic ulcer disease (UD) of the stomach and duodenum, the typical course of which involves the seasonality of exacerbations: spring and autumn. Among other criteria of a "typical" course, infection with H. pylori, which is the cause of these diseases and without its destruction in the body, it is impossible to achieve clinical remission of H. pylori-associated diseases and prevent their recurrence. Before prescribing therapy, the causative agent must be identified, and after treatment, its eradication must be confirmed. The severity of chronic diseases of helicobacterial etiology depends on the degree of pathogenicity of the strains, the presence of certain cytotoxic genes. The review analyzes modern information on the biological properties of the causative agent of helicobacteriosis and methods of its diagnosis. They can be divided into invasive (requires taking a biopsy during endoscopic examination) and non-invasive. Bacteriological and morphological research methods are distinguished among the invasive ones. The histological method is recognized as the "gold standard" for the diagnosis of helicobacteriosis. The essence of the method consists in the preparation of preparations of the gastric mucosa and their Giemsa staining in order to detect bacterial cells in the preparation. The method allows you to determine the characteristics of the causative agent and assess the condition of the gastric mucosa. The bacteriological method is considered indispensable for checking strains for resistance to certain antibacterial drugs, which allows predicting the results of treatment. Currently, non-invasive diagnostic methods have become the most widespread. Along with the respiratory urease test, serological methods are used (immunoenzyme analysis, immunoblotting), as well as the immunochromatographic method. The molecular diagnostic method, namely PCR, is used to study the genotypic and phenotypic characteristics of H. pylori strains in gastric biopsy samples, saliva, stools, gastric juice, and dental plaque. PCR provides excellent sensitivity and specificity of over 95% compared to other tests.

Unveiling the Therapeutic Potential of Vitamin-E in Preventing Inflammation and Stromal Congestion in Alcoholic Liver Injury.

The objective of the research was to investigate and assess how effective Vitamin E is in preventing or reducing liver inflammation and stromal congestion associated with alcoholic liver injury. This is a laboratory-based experimental study. A total of 18 domestic rabbits were divided into groups A, B, and C. Group A was the control group and treated with normal saline as a placebo. Groups B and C were given 30% ethanol in a daily dose of 30 ml/kg/day. Additionally, group C was treated with vitamin E at 50 mg/kg/day. All three groups were sub-divided into two sub-groups I and II on the basis of experimental duration of eight weeks and four weeks respectively. The subgroups with eight weeks of experimental time duration were categorized as "category E8" and subgroups with an experimental duration of four weeks were categorized as "category E4". Liver tissue samples from each animalwere subjected to staining using hematoxylin and eosin (H&E) stain for histological staining in order to assess portal inflammation and to measure the sizes of hepatic sinusoids and central veins to evaluate hepatic congestion. A statistically significant variance was observed in the size of central veins, hepatic sinusoids, and invasion of inflammatory cells in portal areas across and between the groups within categories E4 and E8. Animals treated with vitamin E exhibited lower invasion of inflammatory cells and larger central veins and sinusoids compared to those not treated with vitamin E. Vitamin E may have a significant role in reducing or limiting the infiltration of inflammatory cells and could help prevent hepatic congestion in cases of alcoholic liver injury.

Evaluation of the TLR3 involvement during Schistosoma japonicum-induced pathology

BackgroundDespite the functions of TLRs in the parasitic infections have been extensively reported, few studies have addressed the role of TLR3 in the immune response to Schistosoma japonicum infections. The aim of this study was to investigate the properties of TLR3 in the liver of C57BL/6 mice infected by S. japonicum.MethodsThe production of TLR3+ cells in CD4+T cells (CD4+CD3+), CD8+T cells (CD8+CD3+), γδT cells (γδTCR+CD3+), NKT cells (NK1.1+CD3+), B cells (CD19+CD3−), NK (NK1.1−CD3+) cells, MDSC (CD11b+Gr1+), macrophages (CD11b+F4/80+), DCs (CD11c+CD11b+) and neutrophils (CD11b+ Ly6g+) were assessed by flow cytometry. Sections of the liver were examined by haematoxylin and eosin staining in order to measure the area of granulomas. Hematological parameters including white blood cell (WBC), red blood cell (RBC), platelet (PLT) and hemoglobin (HGB) were analyzed. The levels of ALT and AST in the serum were measured using biochemical kits. The relative titers of anti-SEA IgG and anti-SEA IgM in the serum were measured by enzyme-linked immunosorbent assay (ELISA). CD25, CD69, CD314 and CD94 molecules were detected by flow cytometry.ResultsFlow cytometry results showed that the expression of TLR3 increased significantly after S. japonicum infection (P < 0.05). Hepatic myeloid and lymphoid cells could express TLR3, and the percentages of TLR3-expressing MDSC, macrophages and neutrophils were increased after infection. Knocking out TLR3 ameliorated the damage and decreased infiltration of inflammatory cells in infected C57BL/6 mouse livers.,The number of WBC was significantly reduced in TLR3 KO-infected mice compared to WT-infected mice (P < 0.01), but the levels of RBC, platelet and HGB were significantly increased in KO infected mice. Moreover, the relative titers of anti-SEA IgG and anti-SEA IgM in the serum of infected KO mice were statistically decreased compared with the infected WT mice. We also compared the activation-associated molecules expression between S.japonicum-infected WT and TLR3 KO mice.ConclusionsTaken together, our data indicated that TLR3 played potential roles in the context of S. japonicum infection and it may accelerate the progression of S. japonicum-associated liver pathology.

Abstract 5332: Characterization and comparison of acridine orange/propidium iodide and acridine/DAPI viability detection methods for cell and gene therapy development

Abstract Cell viability is one of the critical quality attributes (CQAs) for cell and gene therapy products. Characterization of cell viability, in addition to cell identity, cell count and other CQAs, are essential to ensure the quality, safety and efficacy throughout various developmental stages of cell and gene therapies. To determine cell viability, a variety of viability detection methods have been used, such as trypan blue (TB) exclusion assay, and fluorescence-based dyes including acridine orange/propidium iodide (AO/PI), acridine orange/4′,6-diamidino-2-phenylindole (AO/DAPI), all of which have been employed on flow or image-based cytometry systems. In this work, we employed the Cellaca MX high-throughput cell counter to characterize and compare AO/PI and AO/DAPI viability detection methods. First, we measured the viabilities of different ratios of viable and non-viable Jurkat cells created via two cell dying processes. Next, viabilities of the stained cells were measured multiple times within a 30 min staining time for each method in order to monitor the trend of viability changes. The viability results showed that the AO/DAPI method were generally higher than AO/PI method with differences ranging from 0% to 24%. We also observed the discrepancy of the measured viability differences between naturally-dying and heat-killed Jurkat cells, suggesting that the viability difference might be dependent on the cell dying process. In the time-dependent viability characterization, results from AO/DAPI method showed increasing dead cell counts leading to decreasing viability, revealing an on-going staining process. In contrast, results from AO/PI method showed more consistent cell count and viability measurements, indicating a more instantaneous staining process. Lastly, we conducted a cell proliferation assay as an orthogonal method to confirm replicability of the low viability cell samples. The proliferation assay results showed no significant increase in cell concentrations within the 4-day time frame. The viability results before the culture were approximately 0% for both naturally-dying and heat-killed Jurkat cells, using the AO/PI staining method, and 12% and 9% using the AO/DAPI respectively. Furthermore, spiked samples of natural-dying and heat-killed cells with healthy cells (&amp;gt;90% viability) were generated and measured, which showed exponential growth. The viability results of these spiked samples before the culture were 11% and 9% by AO/PI and 17% and 18% by AO/DAPI, respectively. Taken together, these results suggest that cell viability methods need to be optimized for different fluorescent stains in order to generate reliable, robust, and fit-for-purpose results for the downstream assays for cell and gene therapy products.. Citation Format: Yongyang Huang, Samir Patel, Mackenzie Pierce, Carolina Franco Nitta, Bo Lin, Leo Chan. Characterization and comparison of acridine orange/propidium iodide and acridine/DAPI viability detection methods for cell and gene therapy development. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5332.

Biological Activity of Combretum erythrophyllum: Antioxidant, Apoptotic, and Cytotoxic Activity of the Leaf and Stembark Extract

Species of Combretum are highly valued in Africa due to the plethora of traditional medicinal uses they may offer and the medicinally important phytometabolites they are known to contain. Traditionally, C. erythrophyllum is used to treat bacterial infections, venereal diseases, abdominal pain, sores, infertility, and labour pains, while displaying, anti-viral, anti-parasitic, cytotoxic, and mutagenic activities. There are numerous published works available on the bioactivity of phytometabolites of the leaf extracts of C. erythrophyllum; however there have been limited or no studies published on the bioactivity of the stembark. Hence, this study aimed to provide a comparative analysis of the biological activity of the leaf and stembark extracts of C. erythrophyllum. The following characters were evaluated through the emanating study: total flavonoid and phenolic content, as well as the antioxidant, cytotoxic, and apoptosis activities of the leaf and stembark extract. Methanolic extracts appeared to have the highest possible antioxidant potential among all of the tested extracts and displayed the lowest IC50 values (leaf 5.29 and stembark 4.29 µg/mL) when evaluated using the DPPH assay, the methanolic extracts appeared to quantify the largest amount of compositional phenolic content (1341.05 ± 4.4 mg/GAE/g). Methanolic extracts were the best performing, with the overall lowest IC50 values when tested against HeLa and HEK293 cells (leaf 54.53 µg/mL and stembark 18.30 µg/mL). A positive correlation between % inhibition and extract concentrations was noted for all of the assays. The extent/level of antioxidant activity was seen to be directly proportional to the flavonoid and phenolic content. Extracts with the highest total phenolic content appeared to display the strongest cytotoxic activity. This study integrated the use of fluorescence microscopy with acridine orange staining in order to accurately determine the viability of cells. A direct correlation was observed between the results obtained from the cytotoxicity and apoptosis assay. It may be concluded that the antioxidant properties, total phenolic, and total flavonoid content were directly proportional to the apoptotic and cytotoxic activity expressed by the tested extracts. Focus should now be placed on isolating phytocompounds of importance from the best performing extracts. The transformation of an isolate into a drug of pharmacological importance has yet to be appraised on a large scale. Therefore, further evaluation of this species and particularly the transformation of the isolates needs to be explored as this species has shown immense medicinal potential.

PCR Based Detection of Cutaneous Leishmaniasis

Background: The second most common illness after malaria that is transmitted by vectors and that may be lethal if untreated is leishmaniasis. Leishmaniasis is caused by parasites of the genus Leishmania, which are classified into two subgenera, Viannia and Leishmania. Methodology: A total of 100 sample collected from presumed clinically as CL patients by dermatologist, with 50 positive cases and 50 negative cases on Geimsa stain microscopy for Leishman Donovan (LD) bodies. After permission from Ethical board in Khyber medical university KPK and inform consent from patient or their guardian in case of children was taken. The lesion and its surroundings were cleaned with 70% ethanol, and the fluid oozing out of the first prick was obtained and disperse over a glass slide, left to dry and fixed with methanol, and stained with Giemsa stain. Two pricks were administered with a sterile BD syringe at the active margin of the lesion. The second prick's fluid was collected and kept in an EDTA tube for PCR usage at -20°C at the IBMS laboratory of KMU in Peshawar. Results: Out of 100 CL patients, 53 were female and 47 were male. The patients age was ranged from two year to 89 year. PCR was positive in 64 cases and negative in 36 cases out 100 for CL. Out of 50 positive microscopic cases 37cases were positive on PCR. Out of 50 microscopic negative cases 27 (54%) were positive by PCR. A statistically significant difference was found between PCR and microscopic method in the detection of CL. Conclusion: The finding in our study specifies that PCR base analysis is superior to microscopy for diagnosis of CL. Therefore, we recommend use of PCR for the detection of CL along with Giemsa staining in order to improve the quality and sensitivity of routine diagnosis of the disease. Keywords: Polymerase Chain Reaction, Giemsa staining, Cutaneous leishmaniasis

Effect of Sodium Fluorescein Use on Surgical Outcomes and Survival in Cases with High Graded Glial Tumor : A retrospective study

Objective: The most effective factor on malignant glioma (MGs) treatment affecting overall survival is the extent of resection. The use of sodium fluorescein (FL) staining in order to increase the amount of resection, is applied more effectively and safely at lower doses with the aid of 560nm filtered surgical microscopes. Our aim was to investigate the effects of the use of FL in MGs surgery on the gross total resection rate (GTR), duration of surgery, length of hospital stays, and survival time.Methods: A retrospective study was conducted on 17 patients whose histopathological evaluations were reported as MGs and operated under surgical white light (Group 1), and 23 patients who were operated under FL560 module surgical microscope (Group 2) with a low dose of (3mg/kg body weight) FL dye. The blood loss in the course of surgery, GTR, surgical time, and hospital stay were compared for both groups with the student-t-test. Kaplan-Meier method was used for the survival time analysis.Results: GTR rates were found to be 82.3% for patients operated under surgical white light, and the percentage for FL-utilized patients was 91.3%. There was no significant difference in blood loss or hospital stay between the two groups; however, the surgical time for FL-utilized patients was found to be significantly low in comparison to the other group. The overall survival time for patients who were operated under surgical white light was found to be 64 weeks (448 ± 64 days) while it was determined as 84.7 weeks (593±55 days) for patients operated with the use of FL, however, the difference between them was not found out to be statistically significant. The use of FL enabled the surgeon to determine the cortical incision area in 13 cases, where the location of the tumor was close to the cortex.Conclusions: While the use of FL shortens the surgical time in contrast-enhancing MGs, it increases the GTR rate. FL also functions well with the determination of the location of the surgical site for tumors close to the cortex.

Metronomic doses and drug schematic combination response tested within chambered coverslips for the treatment of breast cancer cells (JIMT-1).

Low-dose metronomic (LDM) chemotherapy is an alternative to conventional chemotherapy and is the most frequently used approach in low dose chemotherapy regimens. The selection of patients, drug dosages, and dosing intervals in LDM is empirical. In this study, we systematically examined the schedule-dependent interaction of drugs on a breast cancer cell line (BCC) cultured in chambered coverslips. The LDM studies were combined with cell staining in order to better characterize different cell states and cell death modes, including caspase-dependent apoptosis, caspase-independent cell death and autophagy-dependent cell death. Microscope images were examined using the Fiji Trainable Weka Segmentation plugin to analyse cell area in 7500 images showing different modes of cell death. Paclitaxel combined with LDM chemotherapy demonstrated a reduction in the area covered by live cells. In contrast, there was an induction of high levels of cell death due to caspase-dependent apoptosis.

Comparison of engineered cartilage based on BMSCs and chondrocytes seeded on PVA-PPU scaffold in a sheep model.

In this study, polyvinyl alcohol hydrogel chains were crosslinked by polyurethane in order to synthesize a suitable substrate for cartilage lesions. The substrate was fully characterized, and in vitro and in vivo investigations were conducted based on a sheep model. In vitro tests were performed based on the chondrocyte cells with the Alcian Blue and safranin O staining in order to prove the presence of proteoglycan on the surface of the synthesized substrate, which has been secreted by cultures of chondrocytes. Furthermore, the expression of collagen type I, collagen type II, aggrecan, and Sox9 was presented in the chondrocyte cultures on the synthesized substrate through RT-PCR. In addition, the H&amp;E analysis and other related tests demonstrated the formation of neocartilage tissue in a sheep model. The results were found to be promising for cartilage tissue engineering and verified that the isolated chondrocyte cultures on the synthesized substrate retain their original composition.

Evaluation of clinical profile in patients from Western and Southern Algeria indicates prevalence of karyotypic, autosomal and gonosomal abnormalites

This work presents objectives that are based on the identification of the clinical profile of patients with polymalformative syndromes. It consists on a determination of the prevalence and the type of chromosomal abnormality for each group of patients, and constitutes the first retrospective study on a sample of 196 cases referred for postnatal cytogenetic study in the service of cytogenetic and molecular biology laboratory within the University Hospital of Oran November 1, 1954. Through this study which spanned a year and a half from January 2020 until July 2021, the practical interest and the important role of genetic counseling were highlighting. The results show that 30.6% of the referred patients have an abnormal karyotype, 69.4% have a normal karyotype, 78.3% have an autosomal abnormality, and 21.7% have a gonosome abnormality. Down's syndrome represented the most frequent 78.4% reference pattern among the category of abnormal numbers and autosomes. The karyotypes were carried out for these patients in standard Giemsa staining in order to highlight a possible anomaly in number or structure and to confirm the diagnosis made, to provide them with better therapeutic management by the following doctors. FISH also used to confirm some results. These results are mostly in agreement with the data in the literature. It’s important to note that the care is not only medical; it is also social and rehabilitative. In order to improve the vital prognosis, as well as the quality and life expectancy of patients.

The function of IL-33/ST2 signaling axis in treg cells activating fibrosis in IgG4-related disease

ObjectsTo explore whether IL-33/ST2 signaling axis can activate Treg cells in promoting tissue fibrosis in IgG4-related disease. MethodsPeripheral blood from patients diagnosed as IgG4-related disease and healthy volunteers matched with age and sex from September 2019 to December 2020 in Sun Yat-sen Memorial Hospital was collected and disposed to separate the peripheral blood mononuclear cells and serum. The concentration of serum IL-33, IL-13 and ST2 was measured using ELISA kits. And the ratio of Treg cells was measured with flow cytometry. Patients diagnosed as type1 autoimmune pancreatitis from September 2019 to December 2020 in Sun Yat-sen Memorial Hospital were enrolled in this study. Patients diagnosed as pancreatic cancer and chronic pancreatitis were enrolled as controls. Pathological sections of these patients were collected and treated with Immunohistochemical staining and Masson trichrome staining in order to observe the expression of FoxP3, IL-33, IL-13 and ST2 as well as the grade of tissue fibrosis. ResultsWe found that no matter in blood circulation or at the affected sites in IgG4-related disease, the expression of IL-33, IL-13 and ST2 was up regulated and Treg cells expanded. In type1 autoimmune pancreatitis, the degree of fibrosis was positively correlated to IL-33, IL-13, ST2 and FoxP3. Moreover, IL-33, IL-13, ST2 and Treg cells affected each other both in blood and in pancreas. ConclusionsIL-33/ST2 may affect the expanding of Treg and the secretion of IL-13 in the fibrosis process in IgG4-RD.

Особенности накопления липидов в створках эпоксиобработанных ксеногенных биопротезов клапанов сердца

Introduction.Xenograft heart valves are prone to structural valve degeneration accompanied by lipid accumulation. Research data suggest that the lipid insudation of bioprosthetic valve is secondary to cellular infiltration, since glycosaminoglycans produced by the recipient's cells are responsible for the lipid storage in the xenograft tissue. The objective of this research was to study the mechanisms of lipid accumulation in the heart valve xenograft, and to test the hypothesis that cells and glycosaminoglycans play a role in this process. Materials and methods.The study included 10 explanted bioprosthetic mitral valves (5 xenoaortic and 5 xenopericardial). We separated the leaflets of these valves from the stent, then the material was fro-zen and transferred to the cryotome for section preparation. The sections were stained with Gill's H&amp;E staining in order to study the cellular infiltration and detect any degenerative changes. To analyze the component composition of the leaflets the sections were stained with Movat's pentachrome, whereas Oil Red O was used to visualize the lipids. We used light microscopy to analyze the stained samples. Results. Lipids and clusters of foam cells were present in all xenoaortic heart valves, but they were not found in the xenopericardial heart valves. Most lipid deposits and cellular infiltrates were not colocalized. Movat’s pentachrome staining revealed an absence of glycosaminoglycans in the leaflets of both types of bioprostheses. Conclusion. The intensity of lipid insudation in the leaflets of bioprosthetic heart valves depends on the xenograft biomaterial. Xenoaortic bioprosthetic heart valves are revealed to be prone to lipid accumulation, unlike xenopericardial bioprostheses. The presence of cell infiltration and glycosaminoglycans in the leaflets is not a determining factor contributing to the accumulation of lipids. Keywords: bioprosthetic heart valves, structural valve degeneration, cell infiltration, blood lipids, glycos-aminoglycans

Causes and Management of Tooth Discoloration: A Review

Tooth discoloration is a common dental occurrence that is linked to both clinical and aesthetic issues. The treatment of tooth discoloration has grown into highly complex, scientific, and therapeutic field by the twenty-first century. Dental surgeons must understand the etiology of tooth staining in order to provide an accurate diagnosis and thus management. Extrinsic causes produce tooth darkening, whereas intrinsic congenital or systemic influences cause tooth discoloration. Tooth bleaching is a safe and effective way to lighten the color of the teeth. Today, the physician has a number of options, including at-home tray-based bleaching treatments with low hydrogen peroxide or carbamide peroxide concentrations. Surgical options include dental restorations which is may be indicated for some cases. This review aims to summarize current evidence regarding causes, classification and management of tooth discoloration. Medline and PubMed public database searches were carried out for papers written all over the world on tooth discoloration. No predictive analytics technology was used.

Immunohistochemistry: A Method to Analyze M. ulcerans Infected Skin Tissue.

Immunohistochemistry (IHC) is a combination of immunological, biochemical, and pathological methods to visualize the presence and distribution of specific epitopes in tissue sections. Selected antigens are stained by differently labeled antibodies binding to their target antigens in situ. Here we describe sample preparation and sample staining in order to diagnose and analyze tissue samples infected with M. ulcerans from human as well as animal source.

Single Dose of N-Acetylcysteine in Local Anesthesia Increases Expression of HIF1α, MAPK1, TGFβ1 and Growth Factors in Rat Wound Healing.

In this study, we aimed to investigate the influence of N-acetylcysteine (NAC) on the gene expression profile, neoangiogenesis, neutrophils and macrophages in a rat model of incisional wounds. Before creating wounds on the backs of 24 Sprague–Dawley rats, intradermal injections were made. Lidocaine–epinephrin solutions were supplemented with 0.015%, 0.03% or 0.045% solutions of NAC, or nothing (control group). Scars were harvested on the 3rd, 7th, 14th and 60th day post-surgery. We performed immunohistochemical staining in order to visualize macrophages (anti-CD68), neutrophils (anti-MPO) and newly formed blood vessels (anti-CD31). Additionally, RT-qPCR was used to measure the relative expression of 88 genes involved in the wound healing process. On the 14th day, the number of cells stained with anti-CD68 and anti-CD31 antibodies was significantly larger in the tissues treated with 0.03% NAC compared with the control. Among the selected genes, 52 were upregulated and six were downregulated at different time points. Interestingly, NAC exerted a significant effect on the expression of 45 genes 60 days after its administration. In summation, a 0.03% NAC addition to the pre-incisional anesthetic solution improves neovasculature and increases the macrophages’ concentration at the wound site on the 14th day, as well as altering the expression of numerous genes that are responsible for the regenerative processes.

Features of establishing the presence of blood in old stains by the method of thin-layer vertical chromatography

Due to the fact that the bloodstain pattern analysis takes the first place in the structure of the study of biological objects, the question of the study of blood patterns is relevant today. The main task (that will allow to solve the following, which puts the investigation before the experts), is to prove that these patterns, which are examined, contain blood, (regardless of their remoteness, attempts to destroy patterns of blood), find out its types or refute traces of blood. If the expert fails to prove the presence of blood, he must refuse to address the following issues. Otherwise, the expert may obtain results that will ultimately lead him to an erroneous conclusion.&#x0D; The article considers options for extraction of old blood stains in order to establish the presence of blood by the method of thin-layer vertical chromatography. This question is still relevant, as biological material can change under the influence of environmental factors and time and this can be an obstacle to establishing the fact of its presence on physical evidence, and also it can do harm to the quality of the study and the correctness of the conclusions, made as a result of the study. In order to select substances, that could provide rapid extraction of old blood stains, a study was performed using weak solutions of alkalis and acids and subsequent determination of the presence of blood by thin layer vertical chromatography. In immunological studies, there have been cases where blood crusts on non-hygroscopic surfaces under the influence of natural factors (such as high temperatures and direct sun rays) have not been extracted in saline and distilled water for several days. In addition, when using known blood samples in the reactions, the dependence of the saturation of the extracts on the age of the samples and the time of their extraction. These observations prompted researches in the sector of biological research and accounting of the Cherkasy NDEKTs of the Ministry of Internal Affairs on methods for extracting old blood stains, which would allow to detect blood in the shortest possible time by the method of thin-layer vertical chromatography.

Immunolocalization of decorin, a small leucin-rich proteoglycan, in the normal and injured horse tendon

The aim of this study was to evaluate the intensity of decorin in normal and the injured equine cadaver limbs. The study was carried out using superficial digital flexor tendon (SDFT) samples harvested from equine cadaver limbs. Randomly assigned left or right equine distal forelimbs were collected in normal (7 - 14 years old ± 2.86 SD) and injured (6 - 16 years old ± 3.64 SD) SDFT. Small pieces of approximately 5 x 5 x 3 mm were collected from normal and the injured areas at the mid-most metacarpal regions and fixed in 4% paraformaldehyde at room temperature. They were routinely prepared for H&E staining in order to confirm whether they were normal or injured. Then they were prepared for immunological assay, the primary and the secondary antibodies were prepared in a standard ratio. The results showed that the distribution of decorin was varied between fascicles and the interfascicular matrix (IFM), and the latter showed greater intensity than the fascicles. The injured tendons showed up-regulation of decorin, it was intensively expressed in all the cases of injured tendon, particularly fascicles showed significant up-regulation when compared to the normal tendon. It was concluded that decorin was varied regionally, and the injured tendons presented a significant up-regulation.

Forensic Application of Monoclonal Anti-Human Glycophorin A Antibody in Samples from Decomposed Bodies to Establish Vitality of the Injuries. A Preliminary Experimental Study.

Glycophorins are an important group of red blood cell (RBC) transmembrane proteins. Monoclonal antibodies against GPA are employed in immunohistochemical staining during post-mortem examination: Through this method, it is possible to point out the RBC presence in tissues. This experimental study aims to investigate anti-GPA immunohistochemical staining in order to evaluate the vitality of the lesion from corpses in different decomposition state. Six cases were selected, analyzing autopsies’ documentation performed by the Institute of Legal Medicine of Rome in 2010–2018: four samples of fractured bones and three samples of soft tissues. For the control case, the fracture region of the femur was sampled. The results of the present study confirm the preliminary results of other studies, remarking the importance of the GPA immunohistochemical staining to highlight signs of survival. Moreover, this study suggests that the use of this technique should be routinely applied in cases of corpses with advanced putrefaction phenomena, even when the radiological investigation is performed, the macroscopic investigation is inconclusive, the H&E staining is not reliable. This experimental application demonstrated that the use of monoclonal antibody anti-human GPA on bone fractures and soft tissues could be important to verify whether the lesion is vital or not.