Number Of Cells In Tissue Research Articles

Abstract P3-07-09: Single cell transcriptomic analysis reveals the effects of BRCA1 and BRCA2 mutations on distinct signaling networks and cancer susceptibility

Abstract Background: Inheritance of BRCA1 and BRCA2 mutations is associated with increased risk of breast and ovarian cancers. Previous studies with low-throughput flow cytometry-based assays suggested elevated number of luminal progenitor cells in the breast tissues of BRCA1 mutation carriers compared to breast tissues of non-carriers. However, breast epithelial cell-specific transcriptome differences between BRCA1, BRCA2 mutation carriers, and non-carriers and how these differences alter susceptibility to transformation are yet to be elucidated. Methods: We generated a single cell transcriptome atlas of breast tissues from BRCA1 (six samples, 17,220 cells), BRCA2 (four samples, 25,046 cells) mutation carriers and non-carriers (11 samples, >50,000 cells). Using previously described markers, epithelial cells were sub-clustered into basal, luminal progenitor, and mature luminal cells. Genes differentially expressed in epithelial cells of BRCA1 and BRCA2 mutation carriers compared to those in non-carrier donors were subjected to Ingenuity Pathway Analysis to determine signaling pathways uniquely active in BRCA1 and BRCA2 mutant epithelial cells. Breast epithelial cells derived from three donor types were immortalized using hTERT and then transformed with PIK3CAH1047R mutant or H-RasG12V ± SV40-T/t antigens, and tumorigenicity was determined in vivo. Results: BRCA1 but not BRCA2 mutations altered the ratio between basal, luminal progenitor and mature luminal cells in breast tissues compared to breast tissues in non-carriers. A unique cluster of cells within luminal progenitors was underrepresented in case of BRCA2 mutation carriers compared to non-carriers or BRCA1 mutation carriers. BRCA1 or BRCA2 mutations specifically altered transcriptomes which are an integral part of mTOR and MYCN signaling, and the translational machinery. Signaling pathway alterations in epithelial cells unique to BRCA1 mutations included YAP1, BRD4, SMARCA4, and TGFβ1 signaling. BRCA2 mutations were associated with upregulation of IL-6, FOXO3, and TNFSF11 signaling. Breast epithelial cells from BRCA2 mutation carriers but not BRCA1 mutation carriers or non-carriers modified to overexpress hTERT + PIK3CAH1047R generated tumors in NSG mice. These tumors displayed large cystic structures with basilar epithelial cells lining the rim of cysts with focal areas of cellular hyperplasia and neoplastic cells that extended into the lumen. However, BRCA1/2 mutation status did not influence tumorigenicity by hTERT+ H-RASG12V +SV40-T/t antigens. Conclusions: Our studies provide a high resolution transcriptome atlas of breast epithelial cells of BRCA1 and BRCA2 mutation carriers, which also reveal potentially targetable signaling networks uniquely deregulated in these cells. BRCA2 mutations are associated with distinct susceptibility to PIK3CA mutation-driven transformation. Since PIK3CA mutations are observed in clinically normal breast tissues, screening for such mutations in BRCA2 mutation carriers may help to detect pre-neoplastic or early stage breast cancer. Citation Format: Harikrishna Nakshatri, Poornima Bhat-Nakshatri, Duojiao Chen, Katie Chen, Henry Mang, Christopher A Herodotou, Aditi S Khatpe, Patrick C McGuire, Xiaoling Xuei, Yunlong Liu, George Sandusky, Anna Maria Storniolo. Single cell transcriptomic analysis reveals the effects of BRCA1 and BRCA2 mutations on distinct signaling networks and cancer susceptibility [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-07-09.

Escherichia coli Nissle 1917 Enhances Efficacy of Oral Attenuated Human Rotavirus Vaccine in a Gnotobiotic Piglet Model.

Human rotavirus (HRV) infection is a major cause of viral gastroenteritis in young children worldwide. Current oral vaccines perform poorly in developing countries where efficacious vaccines are needed the most. Therefore, an alternative affordable strategy to enhance efficacy of the current RV vaccines is necessary. This study evaluated the effects of colonization of neonatal gnotobiotic (Gn) pigs with Escherichia coli Nissle (EcN) 1917 and Lacticaseibacillus rhamnosus GG (LGG) probiotics on immunogenicity and protective efficacy of oral attenuated (Att) HRV vaccine. EcN-colonized pigs had reduced virulent HRV (VirHRV) shedding and decreased diarrhea severity compared with the LGG-colonized group. They also had enhanced HRV-specific IgA antibody titers in serum and antibody secreting cell numbers in tissues pre/post VirHRV challenge, HRV-specific IgA antibody titers in intestinal contents, and B-cell subpopulations in tissues post VirHRV challenge. EcN colonization also enhanced T-cell immune response, promoted dendritic cells and NK cell function, reduced production of proinflammatory cytokines/Toll like receptor (TLR), and increased production of immunoregulatory cytokines/TLR expression in various tissues pre/post VirHRV challenge. Thus, EcN probiotic adjuvant with AttHRV vaccine enhances the immunogenicity and protective efficacy of AttHRV to a greater extent than LGG and it can be used as a safe and economical oral vaccine adjuvant.

MicroRNA-101a-3p mimic ameliorates spinal cord ischemia/reperfusion injury.

miR-101a-3p is expressed in a variety of organs and tissues and plays a regulatory role in many diseases, but its role in spinal cord ischemia/reperfusion injury remains unclear. In this study, we established a rat model of spinal cord ischemia/reperfusion injury by clamping the aortic arch for 14 minutes followed by reperfusion for 24 hours. Results showed that miR-101a-3p expression in L4-L6 spinal cord was greatly decreased, whereas MYCN expression was greatly increased. Dual-luciferase reporter assay results showed that miR-101a-3p targeted MYCN. MYCN immunoreactivity, which was primarily colocalized with neurons in L4-L6 spinal tissue, greatly increased after spinal cord ischemia/reperfusion injury. However, intrathecal injection of an miR-101a-3p mimic within 24 hours before injury decreased MYCN, p53, caspase-9 and interleukin-1β expression, reduced p53 immunoreactivity, reduced the number of MYCN/NeuN-positive cells and the number of necrotic cells in L4-L6 spinal tissue, and increased Tarlov scores. These findings suggest that the miR-101a-3p mimic improved spinal ischemia/reperfusion injury-induced nerve cell apoptosis and inflammation by inhibiting MYCN and the p53 signaling pathway. Therefore, miR-101a-3p mimic therapy may be a potential treatment option for spinal ischemia/reperfusion injury.

Dynamics of quantitative changes of diffuse lymphoid tissue cells of bronchi and lungs of guinea pigs sensitized with ovalbumin

Background. There is a progressive increase of respiratory allergic diseases nowadays in the world, made it necessary to study the participation of the components of the immune system in these processes. From the point of view of modern aspects about the organs of the immune system, interesting are the patterns of morphogenesis and function of bronchus associated lymphoid tissue, occupied a special place in the immunological protection of the body due to the large area of contact with various antigens. Morphogenesis and reactive changes in the local immunity in respiratory system in conditions of allergic inflammation remain urgent issue of modern morphology. Objective. To study the changes in diffuse lymphoid tissue of bronchi and lungs of guinea pigs sensitized with ovalbumin. Methods. We have studied the lung of 48 guinea pigs, using histological, immunohistochemical, morphometric, statistical methods, under conditions of experimental ovalbumin-induced allergic inflammation, assessed the average number of lymphocytes, macrophages and plasma cells in the diffuse lymphoid tissue. Results. The average number of lymphocytes in diffuse lymphoid tissue of bronchi and lungs increased from the 23rd day of observation and remained at a high level until the end of the experiment, the maximum was during the early period of the development of allergic inflammation, the increasing coefficient was 4.7. The average number of plasma cells also acquired maximum elevation in the early period of allergic process, the increasing coefficient was 2.0. The most significant average number of macrophages was on the 23rd day of observation with same increasing coefficient. Among all types of immunocompetent cells of diffuse lymphoid tissue in bronchi and lungs, T-lymphocytes prevailed during the experiment elevated almost by 5 times. Conclusions. In the early period of development of experimental ovalbumin-induced allergic inflammation, the specific resistance of the respiratory system manifests itself in the form of activation of local links of cellular and humoral adaptive immunity, as evidenced by the dynamics of changes in the average number of lymphocytes (the maximum increasing coefficient 4.7 in the 1st experimental group), macrophages and plasma cells (maximum increasing coefficient 2.0 in the 1st experimental group) of diffuse lymphoid tissue of bronchi and lungs of guinea pigs.

Number of cells in parathyroid tissue in primary hyperparathyroidism cases and its relationship with serum calcium value.

Background:The relationship between serum calcium (Ca) level to serum parathyroid hormone (PTH), phosphorus (P) levels and tissue properties of the parathyroid gland is unknown in primary hyperparathyroidism cases. Revealing this relationship may be useful for understanding the etiopathogenesis of primary hyperparathyroidism and determining the time of treatment.Methods:Ninety patients (71 females, 19 males, age range; 27–73 years, average age; 46) who underwent single gland excision with the diagnosis of primary hyperparathyroidism were studied. The patients were divided into 2 groups as serum Ca level <12 and serum Ca level ≥12. Age and sex of the patients, mean cell number of the gland, mean volume of the gland, serum levels of PTH, P, and histopathologic type of hyperplasia were evaluated.Results:The mean cell number per cubic centimeter is 22.9 (10–220 range) million in all glands. Serum Ca level was <12 in 82 (91.1%) of the patients, and ≥12 in 8 (8.9%) cases. Mean cell number of the gland, mean volume of the gland, existence of cystic hyperplasia of the gland, serum levels of PTH and P were statistically significant between the 2 groups (P < .001, P < .001, P < .05, P < .001, P < .05 respectively).Conclusion:In primary hyperparathyroidism cases serum Ca level is not related to age and sex but directly related to proportionals to the cell number and volume of the gland and serum levels of PTH, inversely related to cystic hyperplasia and serum levels of P. Early surgical intervention should be planned since the serum Ca level will be high in large adenomas with a noncystic radiological appearance.

Physiological Repairing of Postpartum Rat Uterus Treated with Pelawan (Tristaniopsis obovata Benn.) Extract

The study examined the effectiveness of ethyl-acetate fraction of Pelawan extract on the activities of superoxide dismutases in physiological repairing of uterus in postpartum rat based on SOD activity, MDA concentration, and Cu, Zn SOD expressions. The experiment was arranged with a factorial, completely randomized design. Seventy-two female rats were divided into 4 groups, namely, control 1 (P01= 9), control 2 (P02= 9), giving birth once (P1= 27), and giving birth twice (P2= 27). The experimental rats were given treatments orally with 4 doses, namely 0 mg/kg BW, 50 mg/kg BW, 100 mg/kg BW, and 150 mg/kg BW. Observation data were taken on days 0, 3, and 5 after parturition. Variables measured were SOD activity, MDA levels, and the number of cells in the uterine tissue expressing Cu, Zn SOD. The results of studies on the condition of P01, P02, P1, and P2 showed significant changes in Cu, Zn SOD expressions. The low of SOD and MDA activities were found at doses of 100 mg and 150 mg. The high number of cells expressing Cu, Zn SOD in the epithelium cells and gland cells were found at a dose of 100 mg. Antioxidant plant extracts and their dosages determine the activities of SOD, MDA, and Cu, Zn SOD expressions in the uterus that will affect the process of repairing and involution of uterine tissue. Allegedly, the bioactive compounds extracted at certain doses effectively affect the physiological repair of cells in the uterine tissue of postpartum mothers.

Fluoride induced down-regulation of IKBKG Gene expression inhibits hepatocytes senescence

BackgroundAccumulating evidences have confirmed that liver is one of the more severely damaged organs during chronic fluorosis. However, the detail mechanism is unclear to data. At present, the objective of this study was to investigate the relationship between down-regulation of IKBKG gene expression and hepatocyte senescence induced by sodium fluoride (NaF). MethodsChronic fluorosis rats and NaF-exposure human liver L02 cells were reproduced the model of hepatocyte senescence in vivo and in vitro. The mRNA and protein levels of p16, p21 and IKBKG, the IL-8 level were determined. The role of IKBKG in fluoride-induced senescence of hepatocytes was explored by knock down in hepatocytes in vivo and in vitro. ResultsThe number of senescence-positive cells in rat liver tissues was increased as well as the level of IL-8 and the expression levels of p16, p21 and IKBKG in fluoride exposure to rat depending on the fluoride concentration. The similar results were obtained in NaF treated liver L02 cells, and the number of cells that stagnated in the G2 phase increased significantly. Further, our results confirmed that decreasing the expression of IKBKG in hepatocytes could reduce fluoride-induced hepatocyte senescence and the changes of senescence-related indicators both in vivo and in vitro. ConclusionThese results indicated that the elevated expression of IKBKG was positive relation with the fluoride-induced senescence in hepatocytes, suggesting the hepatocyte senescence might have a special relationship with fluoride-caused liver damage. Because of the present results limitation, the mechanism of fluoride induced senescence in hepatocytes should be concentrated in the future in detail, especially the novel targets for fluoride induced liver injury.

Shikonin regulates autophagy via the AMPK/mTOR pathway and reduces apoptosis of human umbilical cord mesenchymal stem cells to improve survival in tissues surrounding brain contusion.

Shikonin has been reported to regulate autophagy via the AMP-activated protein kinase (AMPK)/mTOR signalling pathway and decrease apoptosis in transplanted human umbilical cord mesenchymal stem cells (HUMSCs). In the present study, HUMSCs were exposed to oxygen glucose deprivation (OGD) in vitro for 12 h, and TUNEL fluorescence staining was used to detect apoptosis. Differences in autophagy and AMPK/mTOR pathway-related protein expression following treatment with shikonin were quantitatively analyzed by western blotting. Green fluorescent protein-labelled stem cells were implanted into traumatic brain injury-model mice and the survival of HUMSCs was observed after 7 days. Shikonin increased the number of cells in brain tissue surrounding the contusion 7 days after transplantation. Furthermore, shikonin treatment decreased apoptosis, increased the expression of autophagy-related proteins, increased phosphorylated AMPK expression and downregulated phosphorylated mTOR expression. In addition, the autophagy inhibitor 3-methyladenine attenuated these effects and aggravated apoptosis. Subsequently, shikonin upregulated autophagy and protected HUMSCs in the area surrounding contused brain tissue. Shikonin may regulate autophagy via the AMPK/mTOR signalling pathway and protect transplanted HUMSCs from apoptosis induced by hypoxia/ischemia.

Effects of ferulic acid on muscle development and intestinal microbiota of zebrafish.

Ferulic acid (FA) is one of a common ingredients in Chinese herbal medicine. FA has the interesting property of promoting growth and improving meat quality in livestock, but the mechanism is not understood. This study evaluated both safety and mechanism of efficacy in zebrafish model. At 15μg/mL or above, FA led to pericardial oedema and delayed growth in zebrafish embryos. Dietary FA promoted growth and feed assimilation in male adult zebrafish. Genes related to myogenic development (myod1, myog and myf5) were significantly upregulated by FA and muscle fibre width in skeletal muscle was increased. At 20µg/g, FA significantly increased number of goblet cells in zebrafish intestinal tissue, and gut microbiota composition also changed. Based on 16s rRNA gene sequences, 20μg/g FA decreased Firmicutes and increased Bacteroides. 20μg/g FA also stimulated the expression of PPAR-α, a gene associated with fat metabolism, and decreased the expression of PPAR-β and PPAR-γ. These gene expression changes were beneficial to fatty acid synthesis and metabolism and decreased fat deposition. Our overall results indicated that FA can be a safe growth promotor in fish particularly in skeletal muscles.

Multi-Omics Reveals the Immunological Role and Prognostic Potential of Mitochondrial Ubiquitin Ligase MARCH5 in Human Breast Cancer.

E3 ubiquitin-linked enzyme MARCH5, also known as membrane-associated circular finger 5, is an enzyme encoded by the human MARCH5 gene. The main objective of this study was to visualize the prognosis of MARCH5 in breast cancer and to determine the relationship between MARCH5 expression and tumor immunity. MARCH5 expression was significantly higher in several cancers, including breast cancer (BRCA), compared with corresponding normal tissues. Not only was high MARCH5 expression associated with poorer overall survival, but also MARCH5 expression was positively correlated with the number of tumor-infiltrating immune cells in BRCA malignant tissues. Furthermore, MARCH5 expression showed a strong correlation with various immune markers of BRCA, suggesting its role in regulating tumor immunity. MARCH5 is a useful prognostic biomarker in several cancers, and its expression is highly correlated with tumor immune cell infiltration, and increased MARCH5 expression may serve as a new biomarker for BRCA diagnosis and prognosis.

The Potential Roles of Mucosa-Associated Invariant T Cells in the Pathogenesis of Gut Graft-Versus-Host Disease After Hematopoietic Stem Cell Transplantation.

Gut acute graft-versus-host disease (aGVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is associated with high mortality. Mucosa-associated invariant T (MAIT) cells are a group of innate-like T cells enriched in the intestine that can be activated by riboflavin metabolites from various microorganisms. However, little is known about the function or mechanism of action of MAIT cells in the occurrence of gut aGVHD in humans. In our study, multiparameter flow cytometry (FCM) was used to evaluate the number of MAIT cells and functional cytokines. 16S V34 region amplicon sequencing analysis was used to analyze the intestinal flora of transplant patients. In vitro stimulation and coculture assays were used to study the activation and function of MAIT cells. The number and distribution of MAIT cells in intestinal tissues were analyzed by immunofluorescence technology. Our study showed that the number and frequency of MAIT cells in infused grafts in gut aGVHD patients were lower than those in no-gut aGVHD patients. Recipients with a high number of MAITs in infused grafts had a higher abundance of intestinal flora in the early posttransplantation period (+14 days). At the onset of gut aGVHD, the number of MAIT cells decreased in peripheral blood, and the activation marker CD69, chemokine receptors CXCR3 and CXCR4, and transcription factors Rorγt and T-bet tended to increase. Furthermore, when gut aGVHD occurred, the proportion of MAIT17 was higher than that of MAIT1. The abundance of intestinal flora with non-riboflavin metabolic pathways tended to increase in gut aGVHD patients. MAIT cells secreted more granzyme B, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ under the interleukin (IL)-12/IL-18 stimulation [non-T-cell receptor (TCR) signal] and secreted most of the IL-17 under the cluster of differentiation (CD)3/CD28 stimulation (TCR signal). MAIT cells inhibited the proliferation of CD4+ T cells in vitro. In conclusion, the lower number of MAIT cells in infused grafts was related to the higher incidence of gut aGVHD, and the number of MAIT cells in grafts may affect the composition of the intestinal flora of recipients early after transplantation. The flora of the riboflavin metabolism pathway activated MAIT cells and promoted the expression of intestinal protective factors to affect the occurrence of gut aGVHD in humans.

Aromatic Bis[aminomethylidenebis(phosphonic)] Acids Prevent Ovariectomy-Induced Bone Loss and Suppress Osteoclastogenesis in Mice.

Osteoporosis is a skeletal disease associated with excessive bone turnover. Among the compounds with antiresorptive activity, nitrogen-containing bisphosphonates play the most important role in antiosteoporotic treatment. In previous studies, we obtained two aminomethylidenebisphosphonates—benzene-1,4-bis[aminomethylidene(bisphosphonic)] (WG12399C) acid and naphthalene-1,5-bis[aminomethylidene(bisphosphonic)] (WG12592A) acid—which showed a significant antiproliferative activity toward J774E macrophages, a model of osteoclast precursors. The aim of these studies was to evaluate the antiresorptive activity of these aminobisphosphonates in ovariectomized (OVX) Balb/c mice. The influence of WG12399C and WG12592A administration on bone microstructure and bone strength was studied. Intravenous injections of WG12399C and WG12592A bisphosphonates remarkably prevented OVX-induced bone loss; for example, they sustained bone mineral density at control levels and restored other bone parameters such as trabecular separation. This was accompanied by a remarkable reduction in the number of TRAP-positive cells in bone tissue. However, a significant improvement in the quality of bone structure did not correlate with a parallel increase in bone strength. In ex vivo studies, WG12399C and WG12592A remarkably bisphosphonates reduced osteoclastogenesis and partially inhibited the resorptive activity of mature osteoclasts. Our results show interesting biological activity of two aminobisphosphonates, which may be of interest in the context of antiresorptive therapy.

Restoration of Intestinal Mucosa in Euphorbia kansui-treated Severe Acute Pancreatitis Rats based on HMGB1/MFG-E8 Expression.

The pathogenesis of Severe Acute Pancreatitis (SAP) is mediated substantially by dysfunctions in the intestinal barrier. Euphorbia kansui (EK) is a medicinal plant used widely in traditional Chinese medicine to treat inflammation; however, its efficacy and mechanism of action in SAP treatment are not yet well understood. To investigate the role of EK in intestinal barrier tissue repair and in the pathogenesis and development of SAP. The rat SAP model was established by a retrograde injection of sodium taurocholate into the pancreatic bile duct. The SAP model group and the SAP + EK treatment groups were divided into 6 subgroups according to timing: 2, 6, 12, 24, 48, or 72h after inducing SAP. The progression of the SAP rats and of the rats receiving the EK treatment was evaluated using the ascites volume, serum amylase and plasma endotoxin levels, and histological grading of intestinal mucosal damage. In addition, serum inflammatory factor contents were measured using Enzyme-Linked Immunosorbent Assay (ELISA) tests and apoptotic cells in damaged ileum tissue were detected using TUNEL staining. Apoptosis markers and other signaling proteins in intestinal mucosal cells were detected by immunohistochemical assays and then validated by combining these data with quantitative polymerase chain reactions and western blotting. Compared with the results of the SAP model rats, the results of the rats that received EK treatment demonstrated that EK could effectively reduce the ascites volume and serum amylase and plasma endotoxin levels. EK treatment also greatly reduced the abnormal intestinal morphological alterations in the rat SAP model and significantly downregulated the serum contents of Interleukin (IL)-1β, IL-6, and tumor necrosis factor-α. EK treatment inhibited the elevation of capapse-3, inhibited the decrease of the Bcl-2 protein, and decreased the number of apoptotic cells in rat ileum tissue. Finally, EK treatment abrogated the increase of HMGB1 and the suppression of MFG-E8 protein expression in the SAP + EK rat ileum tissue. EK suppresses SAP pathogenesis by restoring the intestinal barrier function and modulating the HMGB1/MFG-E8 signaling axis.

Study on the characterization of grouper (Epinephelus coioides) immunoglobulin T and its positive cells

Immunoglobulins (Igs) play a vital role in the adaptive immunity of gnathostomes. IgT, a particular Ig class in teleost fishes, receives much attention concerning the mucosal immunity. While, the characteristic and function of Epinephelus coioides IgT is still unknown. In our study, a polyclonal antibody was first prepared with grouper IgT heavy chain recombinant protein. IgT was revealed to be polymeric in serum and mucus. In normal groupers, IgT had high expression level in head kidney and spleen, while little amount in gills, thymus, gut and liver. The number of IgT-positive cells in different tissues was in line with their IgT expression. Furthermore, IgT could coat fractional bacteria in the mucus. In conclusion, this research revealed the protein characteristic, basal expression and bacterial coverage of grouper IgT. This is the first study to identify the characteristic of grouper IgT and demonstrate the capacity of coating microbes.

Cells with Cancer-associated Mutations Overtake Our Tissues as We Age.

To shed light on the earliest events in oncogenesis, there is growing interest in understanding the mutational landscapes of normal tissues across ages. In the last decade, next-generation sequencing of human tissues has revealed a surprising abundance of cells with what would be considered oncogenic mutations. We performed meta-analysis on previously published sequencing data on normal tissues to categorize mutations based on their presence in cancer and showcase the quantity of cells with cancer-associated mutations in cancer-free individuals. We analyzed sequencing data from these studies of normal tissues to determine the prevalence of cells with mutations in three different categories across multiple age groups: 1) mutations in genes designated as drivers, 2) mutations that are in the Cancer Gene Census (CGC), and 3) mutations in the CGC that are considered pathogenic. As we age, the percentage of cells in all three levels increase significantly, reaching over 50% of cells having oncogenic mutations for multiple tissues in the older age groups. The clear enrichment for these mutations, particularly at older ages, likely indicates strong selection for the resulting phenotypes. Combined with an estimation of the number of cells in tissues, we calculate that most older, cancer-free individuals possess at least a 100 billion cells that harbor at least one oncogenic mutation, presumably emanating from a fitness advantage conferred by these mutations that promotes clonal expansion. These studies of normal tissues have highlighted the specific drivers of clonal expansion and how frequently they appear in us. Their high prevalence throughout cancer-free individuals necessitates reconsideration of the oncogenicity of these mutations, which could shape methods of detection, prevention and treatment of cancer, as well as of the potential impact of these mutations on tissue function and our health.

Mechanism of lncRNA-ANRIL/miR-181b in autophagy of cardiomyocytes in mice with uremia by targeting ATG5.

ObjectivesThis study is to investigate whether the cardiac microvascular endothelial cells (CMECs) can regulate the autophagy of cardiomyocytes (CMs) by secreting lncRNA-ANRIL/miR-181b exosomes, thus participating in the occurrence of uremic cardiovascular disease (CVD).MethodsA 5/6 nephrectomy uremia model was established, with the mice injected with ANRIL-shRNA lentivirus vector, miR-181b agomir, and related control reagents, containing the serum creatinine and urea nitrogen measured. The renal tissue sections of mice were stained with Periodic Acid-Schiff (PAS), TUNEL, and Hematoxylin-Eosin (HE) performed on myocardial tissue sections of mice. ANRIL-shRNA, miR-181b mimics, and related control reagents were transfected into CMECs, in which the exosomes were extracted and co-cultured with CMs. The expressions of ANRIL, miR-181b and ATG5 were detected by qRT-PCR, and the expressions of autophagy related proteins by Western blot, as well as the binding of ANRIL and miR-181b by the double luciferase reporter gene experiment.ResultsANRIL down-regulation or miR-181b up-regulation can increase the weight of mice with uremia, as well as the expressions of p62 and miR-181b, and reduce the content of serum creatinine and urea nitrogen, the damage of kidney and myocardial tissues, the number of apoptotic cells in myocardial tissues, as well as the expressions of ANRIL, ATG5, Beclin1, and LC3. CMs can absorb the exosomes of CMECs. Compared with IS+ CMEC-Exo group, the expressions of ANRIL and ATG5 in CMs of IS+ CMEC-Exo + sh lncRNA ANRIL and IS+CMEC-Exo+miR-181b mimics groups was down-regulated, as well as the expressions of ATG5, Beclin1, and LC3, while miR-181b expression was up-regulated as well as P62 expression.ConclusionsCMECs can regulate autophagy of CMs by releasing exosomes containing ANRIL and miR-181b.

Ameliorative effects of hesperidin and N-acetylcysteine against formaldehyde-induced-hemato- and genotoxicity.

This study investigated the hemato- and genotoxic effects of formaldehyde (FA) and the possible mitigating role of hesperidin (HP) and N-acetylcysteine (NAC), each alone and in combination. Sixty-four adult male albino rats were divided into eight equal groups; the study was conducted for 8weeks; Group I (negative control: received no medication), Group II (positive control: received distilled water), Group III (received HP 50mg/kg/day), Group IV (received NAC 50mg/kg/day), Group V (received FA 10mg/kg/day), Group VI (FA+HP), Group VII (FA+NAC), and Group VIII (FA+HP+NAC). Groups VI, VII, VIII received the same previously mentioned doses and for the same duration. All treatments were given by intraperitoneal administration. At the end of the study, complete blood count, oxidative stress, histopathological changes, immunohistochemical staining of inducible nitric oxide synthase, and proliferating cell nuclear antigen and genotoxicity by comet assay in the bone marrow of treated rats were assessed. FA administration caused significant hematotoxicity represented by elevated white blood cell numbers and serum malondialdehyde levels and reduced red blood cell numbers, platelets, and serum superoxide dismutase values. Histologically, it induced an increase in fat cell numbers in bone marrow tissue with a widening of marrow spaces and decreased cellularity of hematopoietic cells, megakaryocytes, and granulocytes. FA exposure significantly decreased immunoreactivity for proliferating cell nuclear antigen, whereas the immunoreactivity for inducible nitric oxide synthase was increased. Genotoxicity, as measured by comet assay, revealed a significant increase in comet% and tail length in FA-treated group when compared with other groups. The cotreatment with HP and NAC revealed their ability to protect against hematological changes, oxidative damage, histopathological, and immunohistochemical changes, and genotoxicity induced by FA.

O-190 Comparison between effects of exposure to platinum-based chemotherapeutics (cisplatin and carboplatin) on Sertoli cell number and functions in immature human testicular tissues

Abstract Study question Does exposure to either cisplatin or carboplatin have a damaging effect on the Sertoli cell population in the immature human testicular tissues? Summary answer Exposure to cisplatin or carboplatin did not appear to have a major effect on Sertoli cell number or function in the immature human testicular tissues What is known already Long-term survival rates for children with cancer are more than 80%. However, childhood cancer treatment may result in subsequent infertility. Cisplatin is one of the most commonly used drugs for childhood cancers. Carboplatin, a second generation platinum drug, is administered at 10-times the dose of cisplatin and is believed to be less gonadotoxic. In our recent publication we have shown that exposure to both cisplatin and carboplatin acutely reduce the germ cell number in immature human testicular tissues. However, it is not known how cisplatin and carboplatin affect Sertoli cell number and function. Study design, size, duration In-vitro culture of human fetal and pre-pubertal testicular tissues was utilised. Tissue pieces were cultured for 1-3 days prior to exposure to clinically-relevant doses of chemotherapeutics or vehicle control for 24hrs in two sets of experiments: 1) 0.5 or 1 μg/ml cisplatin and culture ended at 24 and 96hrs post-exposure (fetal only); 2) 0.5 μg/ml cisplatin or 5 ­μg/ml carboplatin until 72 (both fetal and pre-pubertal) and 240hrs post-­exposure (fetal only). Participants/materials, setting, methods Testicular tissue fragments from second trimester human fetal (14-22 gestational weeks; n = 3-6) or pre-pubertal patients (1-8years old; n = 5) were cultured in a ‘hanging drop’ system.Quantification of Sertoli cell number (cells per cord/tubular area (mm2)) was performed on sections stained for expression of SOX9. Culture medium was collected to measure levels (ng/ml) of Anti-Mullerian hormone (AMH) and Inhibin B using ELISA. Statistical analysis was performed using two-way ANOVA to account for inter-individual variation between fetuses/patients. Main results and the role of chance Quantification of positively stained Sertoli cells showed that exposure to both doses of cisplatin had no effect on Sertoli cell number at 24 and 96hrs post-exposure. No changes in AMH and inhibin B levels were observed at these time-points. Comparison between cisplatin- or carboplatin-exposed human fetal testicular tissues showed no difference in Sertoli cell numbers at either 72hrs or 240hrs post-exposure. No difference in Sertoli cell number was observed in pre-pubertal testicular tissues exposed to either cisplatin or carboplatin at 72hrs post-exposure. Limitations, reasons for caution Human fetal and pre-pubertal testis tissue is of limited availability, thus, sample sizes used in this study were relatively low. ‘Hanging drop’ culture might not recapitulate all in-vivo aspects of immature testis microenvironment. Wider implications of the findings Exposure to cisplatin or carboplatin did not affect Sertoli cell number in the immature human testicular tissues. Taken together with our recent publication, this suggests that these two platinum-based chemotherapeutic agents cause direct damage to germ cells. Functionality of Sertoli cells in chemotherapy-exposed tissues need to be further investigated. Trial registration number not applicable

P–744 The features expression of some lymphocyte markers in the pelvic peritoneal adhesions’ tissue at reproductive age women

Abstract Study question To study the expression of CD4, CD8, CD20, CD 138 in the tissue of the pelvic peritoneal adhesions at women of reproductive age. Summary answer Immunohistochemical study of pelvic adhesions revealed the CD8-positive cells is directly involved in the formation of the immune response at the late stages of adhesiogenesis. What is known already One of the reason identifies the high frequency of adhesion formation is the presence of inflammation in the abdominal cavity with different severity and origin. It is known that Insufficiency of the fibrinolytic system, increased levels of a number of cytokines, including transforming growth factor- β1, and tissue hypoxia induce neoangiogenesis and fibrotization of the fibrin matrix, which leads to the formation of adhesions. Data on expression of CD4, CD8, CD20, CD138/syndecan–1 in the pelvic peritoneal adhesions in connection with their prescription, localization and origin is absent at accessible literature. Study design, size, duration Two hundred infertile women (aged 19–49 yrs) with pelvic peritoneal adhesions, who were underwent operative laparoscopy and adhesiolysis. Participants/materials, setting, methods The material for this study was the fragments of surgical material (adhesions and their parts) n = 200, taken from the women of reproductive age who suffered with infertility during operative laparoscopy.The morphological and immunohistochemical study of adhesions were carried out by standard techniques using paraffin blocks, reagents of Dako and monoclonal antibodies to CD4 (Clone 4B12 Ready-to-Use),CD8 (Clone C8/144B Ready-to-Use), CD20 (Clone L26 Ready-to-Use),CD138/syndecan–1 (Clone MI15 Ready-to-Use) of Abcam with automatic coloring Dako Cytomation. Main results and the role of chance To assess the population composition of these cell infiltrates, as well as individual diffusely located inflammatory cells, an immunohistochemical method with the main lymphocytic markers (CD4, CD8, CD20, CD138) was used. First of all, it is necessary to note the complete absence of CD20-positively colored cells in all observations, which indicates that at the final stage of the formation of adhesions, there is no element of the B-lymphocytic immune response. In an immunohistochemical study with syndecan–1 (CD138) antibodies, we identified a small number of positively colored cells that were located mainly perivascular, as part of mononuclear infiltrates. Quantitative analysis showed that the number of such cells is 0.8±0.2. When studying CD4–positive T-lymphocytes, it was found that they are usually located in the form of band-shaped infiltrates and focal perivascular clusters. The number of CD4-positive cells in the spike tissue is 5.6±0.2. CD8-positive cells were located mainly submesothelial, and in the form of perivascular clusters, the number of such cells was 9.2±0.6. Limitations, reasons for caution Age limitation, only women aged 19–49 yrs took part in this study. Exclusion criteria were the following for the groups: acute gynecological diseases, malignant diseases of female genitalia and ovarian tumors. Wider implications of the findings: The absence of B-cells in the “mature” adhesions’ tissue was found.The number of CD8-positive cells in our study was 1.5 times higher than the number of CD4-positive T-lymphocytes.CD4-positive T-lymphocytes play an important role and their number significantly prevails over the number of CD8-positive T-lymphocytes at the initial stages of adhesiogenesis. Trial registration number Case control study

Pathogenicity of Porcine Circovirus Type 2d (PCV2d) in Pigs Infected with PCV2d or Co-infected with Mycoplasma hyopneumoniae and PCV2d or with Porcine Reproductive and Respiratory Syndrome Virus and PCV2d

The objective of this study was to determine the pathogenicity of porcine circovirus type 2d (PCV2d) in pigs inoculated intranasally with PCV2d alone, PCV2d in combination with Mycoplasma hyopneumoniae or PCV2d in combination with porcine reproductive and respiratory syndrome virus (PRRSV). Pigs infected with PCV2d alone were asymptomatic. All pigs inoculated with either M. hyopneumoniae and PCV2d or with PCV2d and PRRSV developed porcine circovirus-associated disease (PCVAD), as characterized by a sudden onset of clinical signs and disseminated granulomatous inflammation. Inflammation was mainly present in lymph nodes and spleen, and occasionally in liver and kidney. Pigs in both of these dually infected groups also had significantly higher (P<0.05) microscopic lymphoid lesion scores and a significantly higher (P<0.05) number of PCV2-positive cells in lymph node tissue than did pigs inoculated with PCV2d alone. The M. hyopneumoniae and PRRSV combination potentiated the PCV2d load in the blood. Co-infection with PRRSV and PCV2d resulted in a significantly higher blood load of PCV2d compared with the M. hyopneumoniae and PCV2d combination. Successful reproduction of PCVAD in pigs appears to require PCV2d with at least one additional infectious agent, such as M. hyopneumoniae or PRRSV, for the full manifestation of disease.