Ve Cells Research Articles

CHRONIC PERICARDITIS SECONDARY TO COXSACKIE VIRAL INFECTION PRESENTING AS FIBROSING MEDIASTINITIS

SESSION TITLE: Fellows Chest Infections Posters SESSION TYPE: Fellow Case Report Posters PRESENTED ON: October 18-21, 2020 INTRODUCTION: Fibrosing mediastinitis (FM) is a rare fibroinflammatory disease classified as idiopathic or secondary. Most common infectious cause in the United states is histoplasma. Additional causes include malignancy, autoimmune, and sarcoidosis. CASE PRESENTATION: 45-year-old female presented with chest pain. A transthoracic echocardiogram (TTE) showed pericardial effusion with tamponade physiology and pericardial window with biopsy was done. Pathology showed fibrovascular tissue, adipose tissue with perivascular chronic inflammation. Cytology; negative for malignancy. Weakly positive Coxsackie titers were 1:16 and 1:8 for B1 & B2 respectively, negative bacterial, fungal, mycobacterial, and viral cultures. Negative Lyme antibody titers, mycoplasma IgG, cryptococcal and histoplasma antigens. Normal IgG levels. Negative rheumatological work up apart from weakly positive anti-nuclear antibody. Progressive symptoms of dyspnea despite treatment with anti-inflammatory medications. Subsequent CT showed mediastinal mass encasing the right pulmonary artery (PA) trunk (figure 1). Moderate to severe narrowing of the right PA seen on cardiac MRI. Bronchoscopy with EBUS guided TBNA revealed lymphocytes but no definite diagnosis. Endovascular biopsy of the mediastinal mass showed fibrillar sclerosing lesion with plasma cells and necrotizing granuloma. IgG staining positive for IgG4+ plasma cells but not enough to meet criteria for IgG4-related disease. Abundance of CD20+ve cells on histology. DISCUSSION: Patient did not meet criteria for IgG4-RD diagnosis. She tested negative for all secondary etiologies. Coxsackie virus infection causing chronic pericarditis leading to FM is our hypothesis. CONCLUSIONS: This represents the first report of FM caused by chronic pericarditis probably due to coxsackie viral infection. Reference #1: RadioGraphics 2019; 39:651-667 Reference #2: Ann Thorac Med. 12(3):209-212 DISCLOSURES: No relevant relationships by Sammar Alsunaid, source=Web Response no disclosure on file for Allen Burke; no disclosure on file for Joseph Friedberg; No relevant relationships by Van Holden, source=Web Response Consultant relationship with Medtronic Please note: $1001 - $5000 Added 06/11/2020 by Edward Pickering, source=Web Response, value=Consulting fee Consultant relationship with Merit Endotek Please note: $1-$1000 Added 04/06/2020 by Ashutosh Sachdeva, source=Web Response, value=Consulting fee Consultant relationship with Medtronic Please note: $1001 - $5000 Added 04/06/2020 by Ashutosh Sachdeva, source=Web Response, value=Consulting fee Speaker/Speaker's Bureau relationship with COOK Please note: $1001 - $5000 Added 04/06/2020 by Ashutosh Sachdeva, source=Web Response, value=Honoraria Consultant relationship with Medtronic Please note: $1001 - $5000 by Ashutosh Sachdeva, source=Web Response, value=Consulting fee Unrestricted education grant relationship with Olympus Please note: $1001 - $5000 by Ashutosh Sachdeva, source=Web Response, value=Grant/Research Support Consultant relationship with COOK Please note: $1001 - $5000 Added 04/27/2020 by Ashutosh Sachdeva, source=Web Response, value=Honoraria

Calcitriol attenuates TLR2/IL-33 signaling pathway to repress Th9 cell differentiation and potentially limits the pathophysiology of rheumatoid arthritis.

There is limited information regarding the TLR2 signaling pathway involved in Th9 cell differentiation. The role of calcitriol in regulating TLR2-mediated Th9 cell development is unknown. Thus, we aimed to unravel the TLR2 signaling pathway in Th9 cells and its regulation by calcitriol. We have used n = 5-6 animals for each murine experiment. Human studies involved five healthy volunteers. Moreover, ten healthy individuals and ten RA patients were included in the study. Murine and human Th9 cells were treated with Calcitriol (100nM) and Pam3CSK4 (2µg/mL). The number of IL-9+ve cells was determined by flow cytometry. Real-time PCR was used to assess the gene expression. Serum 25(OH)D3 levels were determined by HPLC. We observed that TLR2 signals via IL-33/ST2 in Th9 cells. Increased TLR2 expression associated with increased IL9 expression and augmented disease severity in RA patients. Calcitriol attenuated TLR2 signaling in murine and human Th9 cells. Low serum vitamin D3 level negatively associated with increased IL-9 and TLR2 expression and disease severity in RA patients. Our data suggest a potential role of calcitriol to ameliorate the disease severity of RA patients.

Abstract CT166: TRX-E-002-1 in treatment-refractory ovarian cancer - 3-month follow-up results from a phase I study dose escalation phase

Abstract Introduction: The poor outcomes associated with current therapies for recurrent epithelial ovarian cancer (EOC) are thought to reflect the existence of drug-resistant ovarian cancer stem cells (OCSCs). The introduction of emerging therapies such as anti-angiogenic monoclonal antibodies and PARP inhibitors have added to the treatment armamentarium but have generated new resistance mechanisms to contend with. TRX-E-002-1 is a novel, 3rd generation benzopyran molecule that induces caspase-dependent and -independent apoptosis in CD44 +ve ovarian cancer stem-like cells and CD44 -ve ovarian somatic cancer cells. The dose escalation phase of a progressive design trial investigating intraperitoneal (IP)-administered TRX-E-002-1 monotherapy and in combination with standard of care for recurrent EOC (NCT02903771) has been completed with a Maximum Tolerated Dose (MTD) of 5 mg/kg established. Methods: Women with platinum-resistant relapsed disease, platinum refractory disease, or documented intolerance to platinum therapy were enrolled into the study. Patients had completed at least 2 or more prior regimens (including adjuvant therapy) prior to enrolment. Patients were dosed weekly with single agent TRX-E-002-1 IP for the first 2 cycles after which addition of pre-defined standard intravenous chemotherapy regimens was allowed cycles 3-8. Patients were followed up for 3 months after the end of treatment. Assessments included disease response, based on CA-125 and Response Evaluation Criteria in Solid Tumours or GCIG criteria along with circulating epithelial tumour cells (CETC) and stem cell markers. Results: For the 11 evaluable patients, on average, 4 prior lines of therapy, across a range of current and emerging treatments were completed. A total of 9 subjects were evaluable for MTD analysis with 5 patients showing stable disease on monotherapy per RECIST 1.1 criteria and 4 completing 8 cycles (6 months) of treatment; 2 remained progression-free at the 3-month endpoint post-treatment. Preliminary data on activity includes 2 partial responses (PR) per RECIST 1.1 criteria observed at follow-up. With no measurable disease per RECIST 1.1 criteria, 1 patient demonstrated a complete response (CR) during combination treatment which was maintained through-out follow-up, as per GCIG criteria in patients without initial measurable disease. Median progression free survival shown to be 5.5 months. CETC data for responders shows growth of circulating cancer stem cells no longer detected during cultivation of epithelial-antigen +ve cells indicating that the epithelial antigen cells present are not capable of clonal expansion. Conclusion: IP administered TRX-E-002-1 as a first-in-class, dual acting, anti-cancer therapy has preliminary activity for the treatment of platinum resistant ovarian cancer for patients who have demonstrated resistance to a range of prior treatments. Citation Format: Jermaine Coward, Ganessan Kichenadasse, Paul Harnett, Kathleen Moore, Minal Barve, Daniel Berg, James Garner, Don Dizon. TRX-E-002-1 in treatment-refractory ovarian cancer - 3-month follow-up results from a phase I study dose escalation phase [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT166.

Alterations in the epidermis of the carp, Labeo rohita (Cyprinidae: Cypriniformes), infected by the bacteria, Aeromonas hydrophila: A scanning electron microscopic, histopathological and immunohistochemical investigation.

This study was carried out to comprehend the pathogenicity of the bacteria in the epidermis of Labeo rohita inoculated with Aeromonas hydrophila. Alterations in the histopathology of the epidermis were examined using scanning electron microscopy, light microscopy and the localization of iNOS and caspase 3+ve cells by means of immunohistochemical methods. Skin samples obtained from infected fish at different intervals 2, 4, 6, 8 and 10days showed significant changes in the cellular components of the epidermis. Epithelial cells often appeared hypertrophied with fragmented and loosely arranged microridges, and in the process of exfoliation. Mucous goblet cells increased significantly in density. Club cells showed degenerative changes, often with simultaneous confluence of adjacent cells and release of their contents. Increase in density of iNOS and caspase 3+ve cells indicates inflammatory response and apoptosis. This study could provide valuable information on the pathogenesis of the disease, and disease outbreaks in farmed fish. Further, it could provide useful guidelines for fish farmers to take preventive measures for the control of the disease.

FRI0575 BIOMARKER ANALYSIS FROM THE RISE-SSC STUDY OF RIOCIGUAT IN EARLY DIFFUSE CUTANEOUS SYSTEMIC SCLEROSIS (DCSSC)

FRI0575 BIOMARKER ANALYSIS FROM THE RISE-SSC STUDY OF RIOCIGUAT IN EARLY DIFFUSE CUTANEOUS SYSTEMIC SCLEROSIS (DCSSC)

Histological and immunohistochemical study on the effect of Aflatoxin B1 on the ileum of adult male albino rat and possible protective effect of sodium selenite

Background: Aflatoxin B1 is one of dangerous mycotoxins which is produced by Aspergillus fungi. Small intestine especiallyileum is important site for absorption and immunity.Aim: To study histological, ultrastructure and immunohistochemical changes by Aflatoxin B1 on ileum of adult male albinorat and the possible protective effect of sodium selenite.Materials and Methods: Forty adult male albino rats were used, they were divided into four groups, control group (GI),sodium selenite group (GII), Aflatoxin B1 group (GIII), Aflatoxin B1 and sodium selenite group (GIV). Sections were stainedby H&E, Mallory, Alcain blue and proliferating cell nuclear antigen (PCNA). Ultrathin sections were prepared for electronmicroscopic study. Morphometric study for different measurement in ileum were done.Results: Aflatoxin group show marked destruction, disorganization of most villi and crypts, necrosis and vacuolation of coveringand lining cells. Hemorrhage, hyalinization and necrosis in submucosa and muscle layers with heavy lymphocytic infiltrationare seen. Alcain blue stain show significant decrease in intensity. Goblet cells and PCNA + ve cells reveal significant decreasein their numbers. All sections show marked improvement with selenite treatment, ultrastructure changes are in harmony tolight microscope findings.Conclusion: Aflatoxin B1 has destructive histopathological changes on all layers of ileum and sodium selenite has protectiveeffect against this damage.

Immunohistochemical study of morphology and distribution of CD163+ve macrophages in the normal adult equine gastrointestinal tract

Intestinal macrophages are the largest group of mononuclear phagocytes in the body and play a role in intestinal innate immunity, neuroimmune interactions and maintaining intestinal homeostasis. Conversely, they also are implicated in numerous pathologies of the gastrointestinal tract, such as postoperative ileus and inflammatory bowel disease. As a result, macrophages could be potential therapeutic targets. To date, there are limited studies on the morphology and distribution of macrophages in the equine gastrointestinal tract (GIT). The aim of this study was to identify the location and abundance of resident macrophages in the equine GIT using CD163 as an immunohistochemical marker. Tissue samples were obtained post-mortem from 14 sites along the gastrointestinal tracts of 10 horses free from gastrointestinal disease; sample sites extended from the stomach to the small colon. CD163+ve cells were present in all regions of the equine GIT from stomach to small colon. CD163+ve cells were also identified in all tissue layers of the intestinal wall, namely, mucosa, submucosa, muscularis externa (ME), myenteric plexus and serosa. Consistent with a proposed function in regulation of intestinal motility, CD163+ve cells were regularly distributed within the ME, with accumulations closely associated with the myenteric plexus and effector cells such as neurons and the interstitial cells of Cajal (ICC).

OR28-03 Drug Repurposing Identifies Inhibitors of the Proteostasis Network to Augment Radioiodine Uptake in Combinatorial Approaches Targeting Thyroid Cancer

New combinatorial drug strategies are urgently needed to improve radioiodine (RAI) uptake and efficiently ablate thyroid cancer cells, thereby reducing the risk of recurrent disease. Drug repurposing offers the promise of identifying already approved compounds capable of inducing sodium iodide symporter (NIS) function to enhance iodide uptake. However, a lack of thyroid cell-based assays amenable to high-throughput screening has limited progress. We utilised the mutated yellow fluorescent protein (YFP) as a surrogate biosensor of intracellular iodide and screened the Prestwick Chemical Library (1200 drugs; 95% approved) for quenching of YFP fluorescence. This allowed us to identify putative candidate drugs which increased iodide uptake >2 SD above mean. Categorisation of these revealed a high proportion of drugs that modulate the proteostasis network (19/48; ~40%), including key processes in protein homeostasis such as endoplasmic reticulum-associated protein degradation (ERAD) and autophagy. Secondary screening validated the activity of proteostasis modulators in enhancing iodide uptake after ranking 73 leading compounds based on their pharmacologic (AUC, EMAX and EC50) and specificity of response (NIS+ve vs NIS-ve YFP-thyroid cells) at ten different drug doses (0.1 to 50 μM). Of importance, several repurposed drugs (e.g. ebastine, Prestwick N, Prestwick C and clotrimazole) in combination with the HDAC inhibitor vorinostat induced a robust enhancement in RAI uptake in thyroid cancer cells (TPC-1 and 8505C NIS+ve cells, up to 11-fold vs DMSO, P<0.001), which was significantly greater than using vorinostat alone (up to 3-fold, P<0.01). For clotrimazole, we designed 7 new chemical derivatives, 3 of which showed enhanced aqueous solubility and retained the ability to significantly enhance RAI uptake. TaqMan RT-PCR revealed that, in contrast to vorinostat, our repurposed drugs failed to alter NIS mRNA expression, highlighting post-transcriptional mechanisms. Critically, 11 repurposed drugs induced significant gains in RAI uptake in human primary thyroid cells (up to 4.1-fold; P<0.05), the most physiological setting for NIS function. In conclusion, we performed high-throughput screening and identified proteostasis modulators, as well as other repurposed drugs, that markedly enhance radioiodine uptake. Further clinical investigation of these drugs might offer new combinatorial approaches, especially with existing therapies, to improve the treatment of thyroid cancer.

Protective Role Of Estrogen Against Excessive Erythrocytosis In Monge’s Disease

RationaleChronic mountain sickness (CMS), or Monge’s disease, is an advancing and debilitating syndrome caused by chronic (years) exposure to high altitude hypoxia, as experienced by people living in the Andean region. Excessive erythrocytosis (EE) is one of the critical traits of CMS and this excessive pathobiologic response has deleterious effects since a high hematocrit would increase blood viscosity and reduce blood flow to hypoxia‐sensitive organs (brain and heart), often resulting in myocardial infarction or stroke in young adults. Within the population of Cerro de Pasco in the Andes, CMS prevalence is vastly different between males and females, being rare in females. Of interest is that there is a sharp increase in CMS incidence in females after menopause. This suggests the role of gender and sex hormones in altering disease manifestation.MethodsIn order to understand the molecular basis for polycythemia of high altitude, we have generated a disease in‐the dish‐model by re‐programming fibroblasts and native CD34+ve cells from CMS and non‐CMS subjects and converting them to RBC. Using this in‐vitro platform, we are now studying the interactions between genetic factors and sex hormones (Testosterone, Estrogen and Progesterone) and their effect on RBC production in the CMS and non‐CMS subjects at high altitude.ResultsWhile we found that testosterone increased RBC production somewhat, estrogen in physiologic concentrations (10nM), in sharp contrast, reduced the CD235a (Glycophorin A‐marker of mature RBC) remarkably (from 65.4% in the untreated CMS sample to 11% in the treated CMS) and eliminated the CMS EE phenotype. There was also stage‐specificity since we found that the impact was most severe when estrogen was present at the EB stage. This strongly suggests that estrogen has a protective role against the polycythemia phenotype. To probe further, we studied the effect of estrogen (10nM) on GATA1, a critical link with erythropoiesis. There was a sharp 5 fold decrease in GATA1 (p&lt;0.01) and its target genes such as Alas2 (14 fold, p&lt;0.001) in erythroid cells. This decreased expression resulted in marked reduction (&gt;50%) in RBC production, demonstrating that estrogen constrains RBC production in females.ConclusionOur results demonstrate that estrogen inhibits GATA1 expression and prevents excessive erythropoiesis and Monge’s disease in females at high altitude. We believe that these studies not only shed light on role of sex hormones on hypoxia‐induced excessive erythropoiesis but have a broader application in their regulation of erythropoiesis and related blood disorders.

Collagen Hydrolysate against Fluvoxamine Maleate-Induced Osteoporosis in Albino Rats: A Histological and Immunohistochemical Study

Background: Osteoporosis (OP) is a prevalent degenerative bone disease among patients receiving selective serotonin reuptake inhibitors as fluvoxamine maleate (FM). Collagen hydrolysate (CH) is a nutritional component that has antiresorptive effect.Aim of Work: Evaluate the possible protective effect of CH against FM-induced OP in adult male albino rats.Material and Methods: Thirty six rats were divided into 4 groups; group I (control), group II (OP group): injected with FM daily for 4 weeks, group III (CH group): received FM concomitant with oral CH for 4 weeks, group IV (recovery group]: received only FM for 4 weeks & were left without taking any drugs for another 4 weeks. Total serum alkaline phosphatase (ALP) and calcium (ca+2) were measured. Bone specimens from the right femurs and first lumbar vertebrae were processed for HE osteocytes with pyknotic nuclei inside widened lacuna, widened haversian canals. Bone matrix showed fain areas, cavitations & multiple resorption cavities with osteoclasts. There was significant reduction in the mean thickness of compact bone, the mean area of trabecular bone, area % of OPN & mean number of PCNA +ve cells compared to control. Group III exhibited significant reduction in ALP & elevation in Ca+2. The bone showed preserved histological architecture almost as the control. There was significant increase in the mean thickness of compact bone, the mean area of trabecular bone, area % of OPN & mean number of PCNA +ve cells compared to groups II & IV.Conclusion: CH has a potential osteoprotective effect against FM-induced osteoporosis

Effect of Stem Cells, Ascorbic Acid and SERCA1a Gene Transfected Stem Cells in Experimentally Induced Type I Diabetic Myopathy.

Background and ObjectivesSarco/endoplasmic reticulum Ca2＋-ATPase (SERCA) inhibition was proved in streptozotocin (STZ)-diabetic rats. The present study aimed at investigating and comparing the therapeutic effect of bone marrow mesenchymal stem cells (BMMSCs), BMMSCs combined with ascorbic acid (AA) and SERCA1a gene transfected BMMSCs in induced type I diabetic myopathy of male albino rat.Methods and Results54 rats were divided into donor group of 6 rats for isolation, propagation and characterization of BMMSCs and SERCA1a transfected BMMSCs, groups I∼V 48 rats. Group I of 8 control rats, group II (Diabetic) of 10 rats given STZ 50 mg/kg intraperitoneal, group III (BMMSCs) of 10 rats given STZ and BMMSCs intravenous (IV), group IV (BMMSCs and AA) of 10 rats given STZ, BMMSCs IV and AA 500 mg/kg and group V (SERCA 1a transfected BMMSCs) of 10 rats given STZ and SERCA1a transfected BMMSCs IV. The rats were sacrificed after 8 weeks. Gastrocnemius specimens were subjected to biochemical, histological, morphometric and statistical studies. Diabetic rats revealed inflammatory and degenerative muscle changes, a significant increase in blood glucose level, mean DNA fragmentation and mean MDA values and a significant decrease in mean GSH and catalase values, area of pale nuclei, area% of CD105 and CD34 ＋ve cells, SERCA1a protein and gene values. The morphological changes regressed by therapy. In group III significant decrease in DNA fragmentation and MDA, significant increase in GSH and catalase, significant increase in the mean area of pale nuclei, area % of CD105 and CD34 ＋ve cells versus diabetic group. In group IV, same findings as group III versus diabetic and BMMSCs groups. In group V, same findings as group IV versus diabetic and treated groups. Western blot and PCR proved a mean value of SERCA1a protein and gene comparable to the control group. Mean calcium concentration values revealed a significant increase in the diabetic group, in BMMSCs and AA group versus control and SERCA1a group.ConclusionsSERCA1a transfected BMMSCs proved a definite therapeutic effect, more remarkable than BMMSCs combined with AA. This effect was evidenced histologically and confirmed by significant changes in the biochemical tests indicating oxidative stress, muscle calcium concentration, morphometric parameters and PCR values of SERCA1a.

3:27 PM Abstract No. 230 Comparison of embolic-associated immune response in a translational model of hepatocellular carcinoma and transarterial embolization

Transarterial chemoembolization and transarterial embolization (TAE) induce tumor necrosis and expose the immune system to tumor debris with a demonstrated potential for immune activation; however, there remains limited data regarding differences in the immune response to different embolic agents. The goal of this study was to characterize the local immune response adjacent to two different embolics commonly used in the clinic. TAE was performed in a translational rat model (n = 20) of DEN-induced autochthonous HCC using either 40-120 um tris-acryl gelatin microspheres (TAGM) - Embosphere Microspheres (Merit Medical Systems) or 70-150 um polyvinyl alcohol 2-acrylamido-2-methylpropanesulfonate sodium salt (PVA-AMPS) - DC Bead LUMI (Biocompatibles Ltd). After embolization, paraffin embedded tumors were processed for Hematoxylin and Eosin (H&E) and immunohistochemical (IHC) staining with anti-CD3, anti-CD8a and anti-FoxP3. H&E samples were analyzed by an attending pathologist. Quantitation of immune cell populations on IHC was performed using BZ-X Analyzer (Keyence corp. Osaka, Japan). Data are reported as means and standard deviations with statistical comparisons made using a student’s t-test. H&E analysis demonstrated a foreign body reaction adjacent to TAGM that was nearly or fully absent when using PVA-AMPS beads. IHC staining revealed a quantitative difference in the number of Tumor Infiltrating Lymphocytes (TILs) around the beads. This difference was present in CD3+ T-cells (7.06 ± 5.1 vs. 3.16 ± 3.9, N = 36 and 19 respectively, P <0.005), CD8+ T-cells (5.71 ± 4.0 vs. 2.05 ± 1.9, N = 35 and 21 respectively, P <0.0005), and FoxP3+ve cells (3.15 ± 2.7 vs. 1.14 ± 1.7, N = 34 and 21 respectively, P <0.005). Additionally, TAGM beads demonstrated a bead dependent increase in the number of CD3 (P <0.005) and CD8 (P <0.005) infiltrating cells while PVA-AMPS did not. Embolization using TAGM demonstrated a prominent foreign body reaction adjacent to embolics in embolized tumors, associated with an increase in CD3+, CD8+ and FoxP3+ T-cells as compared to embolization performed with PVA-AMPS. Further studies are required to understand the underlying mechanism and therapeutic implications.

Fibrin Hydrogel Aided Cardiac Progenitor Cell Delivery Enhances Regenerative Tendency in Myocardial Infarct Model

Attempts to regenerate myocardial infarct (MI) using stem cells have been achieving random success rates. This study considered delivering cardiac progenitors (rCPs), developed from rat adipose-derived mesenchymal stem cells (rADMSCs), into rat MI tissue. The study aimed to employ fibrin-based insoluble matrix as a niche for in vitro differentiation of rADMSCs to rCPs and specific composition of fibrin hydrogel to deliver the cells into MI tissue. PKH26 tracker dye labelled-rCPs/rADMSCs suspended in fibrinogen/thrombin was injected to peri-infarct zone in rat heart created by coronary artery ligation. The MI tissues were harvested after 28d, imaged to locate transplanted cells, and analyzed to detect markers of differentiation, angiogenesis, and immune response. Co-localization of Connexin 43/TroponinT with PKH26 indicated differentiation of transplanted progenitors into cardiomyocyte like cells. Additionally, the presence of CD31+ve cells, fewer CD68+ve cells, and reduced collagen deposition are appreciable in tissues transplanted with rCP in fibrin, as compared to those that received rADMSC. The results obtained indicate that fibrin acts as a favorable niche for in vitro priming of rADMSCs into transplantable lineagecommitted rCPs. The in situ clottable fibrin aided the retention and differentiation of transplanted rCPs into cardiomyocytes. Also by 28d, the tissues showed better angiogenic/ immunomodulatory effects and reduced the calcification tendency.

Phenotype changes of oral epithelial stem cells after in vitro culture.

The aim of our study was to isolate populations of keratinocyte stem cells based on the expression of cell surface markers and to investigate whether the culture could affect their phenotype. keratinocytes from human oral mucosa were sorted based on the expression of the epithelial stem cell markers p75NTR and CD71. We also examined the co-expression of other epithelial stem markers such as integrins β1 and α6 and their stem cell-like proprieties in in vitro assays. Three passages after being sorted by MACS, more than 93% of the p75NTR+ve cells lost the expression of p75NTR, while 5.46% of the p75NTR-ve gained it. Within the small population of the p75NTR+ve cells, 88% co-expressed other epithelial stem cell markers such as integrins β1 and α6, while only 28% of p75NTR-ve cells co-expressed these markers. These results were confirmed by sorting cells by FACS. Additionally, when double staining was used for sorting cells, 99% of the p75NTR+veCD71-ve and 33% of the p75NTR-veCD71+ve cells expressed both integrins, but just one week after culture, only 1.74% of the p75NTR+veCD71-ve cells still expressed p75NTR and only 0.32% still expressed CD71. Similar results were obtained when co-culturing p75NTR+ve and p75NTR-ve populations before analysis. Our results suggest that phenotype changes may be part of an intrinsic cellular mechanism to conserve levels of protein expression as they may found in the human body. In addition, in vitro culture may not offer ideal conditions for epithelial stem cell maintenance due to phenotype changes under standard culture conditions.

Muscle type from which satellite cells are derived plays a role in their damage response.

The aim of this study was to evaluate the response of satellite cells to muscular atrophies which possess different pathological characteristics and which were induced by distinct damages. Right lower limbs of rats were exposed to denervation or disuse and later its tibialis anterior (TA) or soleus (SOL) muscles were analyzed. After confirming their functional impairments indicated by common but distinct pathological and electrophysiological characteristics, the quantitative polymerase chain reaction analysis of Pax7 and Pax3 expressions and the number of Pax7+ve and Pax3+ve cells were analyzed sequentially at day 0, day 7, and day 14. TA muscles of both denervation- and disuse-induced atrophy models showed persisted low level of Pax7 expression from day 7 (0.91 ± 0.23 and 0.31 ± 0.07, P = 0.06, n = 6) through day 14 (1.09 ± 0.15 and 0.4 ± 0.09 [P < 0.05]). On the other hand, significant elevations were observed in Pax3 expression in both atrophy models (2.73 ± 0.46 and 2.75 ± 0.26 [P < 0.05]) at day 7. Similar to TA muscle, resembled pattern of Pax7 and Pax3 expression changes were observed between the SOL muscles of denervation- and disused-atrophy models. These trends were further confirmed by the changes in Pax7+ve and Pax3+ve cell numbers of TA and SOL muscles in both atrophy models. Despite the distinct pathological findings, similar patterns in the changes of Pax3 and Pax7 expressions and the changes of Pax7+ve and Pax3+ve cell numbers were observed between the denervation- and disuse-induced atrophy models and this commonality was admitted among the muscle type. Therefore, we claim that the muscle regeneration orchestrated by satellite cells was governed by the muscle type in which satellite cells reside.

Marjoram and sage oils protect against testicular apoptosis, suppressed Ki‐67 expression and cell cycle arrest as a therapy for male infertility in the obese rats

Alterations in testicular apoptosis, cell cycle progression and proliferation rate in dietary-induced obese male rats and role of oral administration of marjoram (0.16ml/kg BW) and sage (0.05ml/kg BW) oils were evaluated. Results showed increased body weight, serum leptin, testicular lipid peroxidation, protein oxidation and nitric oxide, with reduction in serum testosterone, sperm count and endogenous enzymatic and non-enzymatic antioxidants in testis of the obese rats. Flow cytometry results revealed increased number of annexin-V (+ve) cells with activation of apoptotic proteins (Bax, caspase-3) and reduction of anti-apoptotic Bcl-2. Cell cycle arrest at phases S and G2/M with decline in expression of Bcl-2 and germ cell proliferation marker ki-67 was also validated, indicating lowered spermatogenesis in the obese rats. Supplementation of marjoram or sage oils displayed normalized body weight, sperm count, germ cells apoptosis and proliferation, suggesting the two oils as a new therapeutic approach against obesity promoted male infertility. PRACTICAL APPLICATIONS: Obese men have a greater chance of fertility problems compared to those with normal weight. Obesity-associated oxidative stress and free radicals production have shown to adversely affect sperm quality with activation of pro-apoptotic pathways, allowing germ cell death. Marjoram and sage essential oils are now being widely studied due to their antioxidant and radical scavenging properties. Our findings indicated effectiveness of the two oils for combating body weight gain, testicular oxidative stress, and apoptosis, which seemed to aid in increasing sperm count. The outcomes of this study may help scientists to formulate novel medications for improving fertility problems in men.

Preemptive plerixafor based mobilization strategy in multiple myeloma patients for autologous stem cell transplantation

Optimum mobilization of CD34+ve cells following G-CSF is critical for engraftment in multiple myeloma (MM) patients undergoing autologous stem cell transplantation (ASCT). Approximately 10% of patients are poor mobilizers. We compared use of pre-emptive plerixafor based on day 4 circulating CD34+ve cells with patients mobilized with G-CSF alone.

1418P - Liquid biopsy and array comparative genomic hybridization (aCGH)

BackgroundCancer of unknown primary origin (CUP) represents a metastatic cancer with unidentified primary origin. An efficient cancer treatment algorithm is based on detection and characterization of the tumor’s origin. CUP is characterized by chromosomal instability; therefore the detection of chromosomal aberrations might contribute in tumor characterization. aCGH combines DNA microarray with CGH providing better detection rates than conventional cytogenetic methods. The present study aimed to evaluate aCGH as a technique for detection the origin of tumor, based on liquid biopsy and particular in circulating tumor cells. MethodsBlood samples were collected from five patients suffering from prostate (2), lung (2) and breast (1) cancer and five healthy individuals. CTCs isolated using enrichment protocols while CD45 (-ve) cells isolated from healthy individuals. In addition to patients’ samples, six commercial cancer cell lines provided by ECACC were used, representing prostate and lung cancer (DU145, 22Rv1, LNcaP, COLO699N, COR0L 105 and MOR). Genomic DNA extracted and aCGH experiments followed with Sureprint G3 platform (Agilent). The genes located in chromosomal aberrations were literately analyzed and potential cancer type suggested by another researcher. The researcher performed the analysis based only on aCGH raw data, ignoring the identity and medical history of all samples. Samples firstly analyzed based on normal vs cancer prediction and secondly whether the predicted type of cancer was correct. ResultsThe sensitivity was around 90% while the specificity was 80%. Among eleven cancer samples only one predicted as normal, as well as one normal sample predicted as cancer. As far as the cancer type prediction the positive predictive value was 90.1%. Only one sample categorized wrongly in total cancer samples. ConclusionsaCGH is a powerful technique with potential of discrimination of cancer and healthy samples, but most important with ability to distinguish the type of cancer. The identification of primary origin of cancer is very important in CUP, since it is correlated with more efficient treatment algorithm. The above encouraging data from the combination of aCGH and liquid biopsy need to be validated in more samples and types of cancer so to be used at clinical level. Legal entity responsible for the studyResearch Genetic Cancer Centre S.A. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Application of multi-dimensional (0D, 1D, 2D) nanostructures for the cytological evaluation of cancer cells and their bacterial response

The size of nanomaterial is a significant parameter, which influences the physicochemical characteristic.These materials also have a part to affect various biological activities in cells and bacterial solution. Because of the larger applicability and enough potential of nanostructures, the current work was displyed the dissimilar shaped nano structures such as quantum dots (QDs), rods (NRs) and sheets (NSs). The structures were processed through solution method and applied to evaluate the efficacy against cancer (C2C12) and bacterial (Gram ˗ Ve) cells. The nanostructures were analysed via the X-ray diffrection (XRD), scanning electron, transmission electron and and high resoultion trans- mission electron microscopy (SEM, TEM and HR-TEM) correspondingly. The low doses of nanostructures (100, 500 and 1000 ng/mL) were applied against cancer cells for to evaluvate the % activity of sustained and non-sustained cells through MTT assay. The general microscopy was operated for to observe the morphology of treated and non-treated cells with nanostructures respectively. The cancer cells treated with nanostructures were also examined via confocal microscopy to access the density and efficacy of nanostructures through staining, whereas the apoptotic studies were also scrutinized with caspase 3 and 7 with control gene, revealed that cells were up-regulated in all conditions.The morphological and quantitative study of bacterial cells with nanostructures were studies through UV visible spectroscopy individually and also describes possible mechanism.

The possible ameliorative role of mesenchymal stem cells and curcumin on the lung in experimentally induced type I diabetes: Histological and immunohistochemical study

Background: Type 1 diabetes mellitus affects lungs due to poor glycemic control. Improvement in both lung function andarchitecture in experimentally-induced lung injury that received exogenous AMSCs by different mechanisms was found.Curcumin has a potent antioxidant and antifibrotic function.Objective: The current study was designed to elucidate the possible ameliorative role of mesenchymal stem cells and curcuminon the lung in experimentally induced type I diabetes by histological and immunohistochemical study.Materials and Methods:Twenty six adult albino rats were divided into five groups: donor, control group, curcumin group andadipose derived mesenchymal stem cell (AMSCs) group. Group II (diabetic group) received a single dose STZ 50 mg/kg/day,group III received STZ and powdered diet supplemented with 0.05% curcumin, group IV received STZ and 1x106 of culturedand labelled rat AMSCs. Lung sections were subjected to histological, morphometric, biochemical and statistical studies.Results: Diabetes induced histological changes in lung in the form of inflammatory changes in bronchiole, a sig increase inthe thickness of interalveolar septa, thickness of the vessels wall, area % of collagen and count CD105+ve cells. Sig decreasein antioxidant activity and sig increase in oxidant enzymes. Sig increase in L-hydroxyproline(L-hyp) level as well STAT andSMAD genes. Administration of curcumin and AMSCs improved sig the previous changes.Conclusion: Diabetes induced lung injuries in rats that was ameliorated by by curcumin, but AMSCs have more pronouncedeffect than curcumin therapy.