Average Number Of Cells Research Articles

Clinical Cohort Study in Canine Patients, to Determine the Average Platelet and White Blood Cell Number and Its Correlation with Patient's Age, Weight, Breed and Gender: 92 Cases (2019-2020).

Due to its easy preparation and that it is well tolerated, the use of autologous platelet-rich plasma (PRP) has become increasingly popular in regenerative medicine. However, there are still no clear guidelines on how it should be classified or whether the individual canine patient’s clinical status can influence its quality. Objective: This study aims to show if the weight, age, sex, neutered status or breed of canine patients have any correlation with the composition of PRP. Design: A blinded count of the platelets and white blood cells (WBC) was performed from 111 samples from 92 client owned dogs undergoing treatment for degenerative joint disease (DJD). The results were analysed using Pearson correlation test, ANOVA test or Student T-test. Results: There is a positive correlation between the number of platelets and WBC in canine patients of different breeds, but there was no significant difference on the platelet number and WBC number among the different breeds. The weight of the patient is also directly correlated to the platelet number (p = 0.003) but not WBC number. WBC number was negatively correlated to the weight of the patient. The sex and age of the patient did not affect platelets and WBC number, although WBC number is increased in non-neutered male population (p = 0.003). However, it would be interesting to investigate whether the growth factors released from the platelet granules are affected by patient variables in a canine population. Conclusions: Our results show that it is possible to obtain good quality autologous PRP, irrespective of age, sex, neutered status or weight of the patient, for PRP regenerative therapy.

May Argyrophilic Nucleolar Organizer Regions Be Used as a Biomarker for the Detection of the Degree of Ischemic Damage Instead of Tunel in Testicular Torsion?

Background and Objectives: It is of great importance to obtain information about the severity of ischemic damage and duration of testicular torsion for an effective treatment strategy. Nucleolar-organizing regions (NORs) are sites of the ribosomal genes composed of ribosomal DNA and proteins. Post-silver staining NORs are termed “AgNOR”. Since AgNORs clearly reveals the self-renewal potential of cells damaged in ischemic events, we performed the current study. Materials and Methods: The study was carried out in four groups as control, sham, early, and late T/D. In the surgical groups, testes were corrected after a 4-h ischemia period. Testicular tissue samples were taken on the third day after detorsion in group 1, 2, 3, and on the tenth day after detorsion in group 4. TUNEL and silver stainings were applied to all samples. Results: The differences were significant among the groups for both mean AgNOR number and total AgNOR area/total nuclear area (TAA/TNA). Moreover, the differences between control and early torsion-detorsion (T/D), between control and late T/D, between sham and early T/D, between sham and late T/D, and between early T/D and late were statistically significant for AgNOR amount. Furthermore, statistically significant differences among the groups for an average number of apoptotic cells per tubule and the percentage of apoptotic tubule values were detected. Discussion: The apoptotic index gives the ratio of cells that are damaged and will die in a programmed way and cells that remain intact, rather than show the viability of the returning testicle. However, by measuring cells that regenerate with AgNOR, we can show not only those that survive but also cells that can repair themselves. Conclusion: AgNOR proteins are usable for the early observation of ischemic injury levels. The amount of AgNOR protein can enlighten us about the extent of testicular damage after T/D treatment. It may also help the physician in the development of effective treatment strategies for cases.

Intravenous injection of mesenchymal stem cell spheroids improves the pulmonary delivery and prolongs in vivo survival.

Because of the excellent therapeutic potential, mesenchymal stem cells (MSCs) have been used as cell therapeutics for various diseases. However, the survival rate and duration of MSCs after transplantation are extremely low and short, respectively. To solve these problems, in this study, we prepared multicellular spheroids of MSCs and investigated their survival and function after intravenous injection in mice. The murine adipose-derived MSC line m17.ASC was cultured in agarose-based microwell plates to obtain size-controlled m17.ASC spheroids of an average diameter and cell number of approximately 170μm and 1100 cells/spheroid, respectively. The intravenously injected m17.ASC spheroids mainly accumulated in the lung and showed a higher survival rate than suspended m17.ASC cells during the experimental period of 7 days. m17.ASC spheroids efficiently reduced the lipopolysaccharide-induced increase in plasma concentrations of interleukin-6 and tumor necrosis factor-α. These results indicate that spheroid formation improved the pulmonary delivery and survival of MSCs, as well as their therapeutic potential against inflammatory pulmonary diseases.

Identification of fetal aneuploidy with dual-probe fluorescence in situ hybridization analysis in circulating trophoblasts after enrichment using a high-sensitivity microfluidic platform.

To evaluate a microfluidics-based positive selection technology for isolating circulating trophoblasts (CTs) from peripheral blood of women whose pregnancies are affected by aneuploidy and to evaluate fetal karyotype using fluorescence in situ hybridization (FISH). Ten 18-ml samples of peripheral blood were collected consecutively from pregnant women whose fetus was affected by aneuploidy. A preservation buffer was added, and the specimens were shipped overnight to the testing laboratory at ambient temperature. The specimen was infused into the fully automated microfluidics-based LiquidScan® instrument without pre-processing. This instrument contains microfluidic chips, which are coated with antibodies (anti-huEpCAM and a proprietary antibody mixture) specific to CT surface epitopes. FISH analysis was performed on the enriched cells. Fetal aneuploidy evaluated included trisomy 21 (n=3), trisomy 18 (n=1), trisomy 13 (n=1), monosomy X (n=3), and triploidy (n=1). CTs for analysis by FISH were identified in all samples. The average number of mononucleate cells per 1ml of whole blood was 2.11 (range 0.38-4.63) overall and was 2.67 (range 1.13-4.63) using the proprietary combination of antibodies. FISH results were concordant with the aneuploidy based on other testing in all cases. Multinucleate cells were searched for and identified in the last seven samples (average number: 0.84/1ml). Our study demonstrates that the LiquidScan® , a high-sensitivity microfluidic platform, can enrich circulating trophoblasts (mononucleate and multinucleate). FISH can then be used to detect fetal aneuploidy.

Changes in the cellular composition of guinea pig’s distal airways epithelium in the dynamics of experimental ovalbumin-induced allergic inflammation

The problem of studying the processes of restructuring of airways epithelium of humans and animals of adaptive nature under the influence of various factors on the body remains the subject of scientific discussions. The aim of this work is to study morphometric changes in the cellular composition guinea pig’s distal airways epithelium in the dynamics of experimental ovalbumin-induced allergic inflammation. We studied lung of 48 male guinea pigs, using histological, morphometric and statistical methods, under conditions of experimental ovalbumin-induced allergic inflammation, simulated by subcutaneous sensitization and subsequent intranasal inhalation with ovalbumin. The average number of epithelial cells of small bronchi and terminal bronchioles was determined: basal epithelium cells, ciliated cells, goblet cells and exocrine bronchiolar cells per unit area of 10000 μm2. We have shown the most significant reactive morphometric changes on the 23rd and 30th days of the experiment. We demonstrated a decrease of the number of basal cells (by 1.5 times compared to the control, p*/**<0.01) and ciliated cells (by 1.6 times compared to the control, p*/**<0.001) and an elevation of the average number of goblet cells (by 2.6 times compared to the control, p*/**<0.01) in bronchioles, a decrease of the average number of exocrine bronchiolar cells (by 1.6 times compared to the control, p*/**<0.01) in terminal bronchioles. These changes are the morphological confirmation of the development of bronchial hyperreactivity as a result of the action of the allergen. Sensitization and allergization with ovalbumin cause statistically significant morphological changes in the cellular composition of small bronchi and terminal bronchioles of an alterative nature in the early period and adaptive – in the late period of allergic inflammation, corresponding to the main morphological manifestations of allergic inflammation.

Применение регенеративных клеточных технологий при лечении тяжёлых местных лучевых поражений у пострадавших в чрезвычайных ситуациях: из опыта работы специалистов Федерального медицинского биофизического центра им. А.И.Бурназяна ФМБА России

It has been noted that one of the most common types of radiation injuries when a person is exposed to ionizing radiation is radiation burns — severe local radiation injuries. The aim of the study is to apply stromal and vascular fraction of adipose tissue to increase the efficiency of complex therapy for local radiation lesions. Materials and methods of the study. In 2017-2019, 7 patients (all male; mean age — (54.83±9.41) years) with local radiation lesions of the skin — ulcerative-necrotic lesions of the skin and underlying tissues — were treated at the Federal Medical Biophysical Center named after A.I. Burnazyan of FMBA of Russia. For more than 6 months the patients received conventional conservative therapy of local radiation lesions and a single injection of cell suspension of autologous cells of stromal-vascular fraction of adipose tissue — the average number of cells was (60.33×106±64.04). Results of the study and their analysis. All patients had no serious adverse events and reactions associated with the introduction of autologous regenerative cells of adipose tissue. During the whole period of observation after stromal-vascular fraction of adipose tissue was injected, late radiation ulcers remained without signs of inflammation and infiltration. The patients were discharged from the hospital in satisfactory condition. According to the authors, the use of stromal-vascular fraction of adipose tissue in local radiation lesions provides favorable conditions: to increase the effectiveness of complex therapy; to reduce healing time of the wound surface; to regulate and activate immune and reparative processes in the dermis; to restore the damaged vascular network, lost skin without severe scarring changes; to heal and achieve a satisfactory result, decent quality of life of patients.

Intravitreally Injected Methylene Blue Protects Retina against Acute Ocular Hypertension in Rats

ABSTRACT Purpose To assess the neuroprotective effects of methylene blue (MB) in a rat model of acute ocular hypertension (AOH) and explore its possible mechanisms. Methods Our AOH rat model was obtained with anterior chamber perfusion for 60 min. After that, 100 μM MB was injected into the vitreous cavity immediately after injury. Electroretinogram, fundus photography, optical coherence tomography (OCT) and retina morphology examination were utilized to quantify retinal damage before surgery, as well as 7, 14 and 28 days after. The average number of surviving retinal ganglion cells (RGCs) was counted after fluorescent retrograde labelling with 4% DiI. And TUNEL assay was used to investigate retinal cell apoptosis at 24 hours after AOH. Nrf2 and BACE1 in the retina were determined by RT-qPCR analysis. Results AOH did produce a severe degeneration effect on the whole retinal layer. Intravitreally injected MB maintained certain retinal thickness after AOH, reduced the destruction of electroretinograms, and enhanced RGCs survival. The average number of TUNEL-labelled cells statistically reduced in the MB-treated retina tissue compared with retina treated with normal saline. The relative mRNA level of Nrf2 was also much higher in the MB-treated retinas after AOH, and the expression of BACE1 had a decline in the AOH + MB group. Conclusions MB can protect the retina from AOH injury and the possible mechanism might involve the inhibition of BACE1 expression and the activation of Nrf2 antioxidant pathway.

Defining Substantial Lymphovascular Space Invasion in Endometrial Cancer.

Lymphovascular space invasion (LVSI) occurs in a minority of endometrial cancer (EC) cases, and the extent of LVSI is an important risk factor for recurrence and/or metastases. Our aim was to improve the reproducibility of measuring clinically meaningful LVSI by performing a quantitative analysis of the correlation between LVSI and the risk of pelvic lymph node recurrence in EC. EC samples from PORTEC-1 and PORTEC-2 trials were retrieved and used to collect quantitative data, including the number of LVSI-positive vessels per H&E-stained slide. Using a predefined threshold for clinical relevance, the risk of pelvic lymph node recurrence risk was calculated (Kaplan-Meier method, with Cox regression) using a stepwise adjustment for the number of LVSI-positive vessels. This analysis was then repeated in the Danish Gynecological Cancer Database (DGCD) cohort. Among patients in PORTEC-1 and PORTEC-2 trials who did not receive external beam radiotherapy, the 5-yr pelvic lymph node recurrence risk was 3.3%, 6.7% (P=0.51), and 26.3% (P<0.001), respectively when 0, 1 to 3, or ≥4 vessels had LVSI involvement; similar results were obtained for the DGCD cohort. Furthermore, both the average number of tumor cells in the largest embolus and the number of LVSI-positive H&E slides differed significantly between focal LVSI and substantial LVSI. On the basis of these results, we propose a numeric threshold (≥4 LVSI-involved vessels in at least one H&E slide) for defining clinically relevant LVSI in EC, thereby adding supportive data to the semiquantitative approach. This will help guide gynecologic pathologists to differentiate between focal and substantial LVSI, especially in borderline cases.

EFFICACY OF COMBINED IMMUNOTHERAPY WITH PROPES AND INFLAMAFERTIN IN SELECTIVE DEFICIENCY OF NK AND NKT CELLS IN CHILDREN WITH AUTISM SPECTRUM DISORDERS ASSOCIATED WITH GENETIC DEFICIENCY OF THE FOLATE CYCLE

Objectives. The results of previous small clinical trials indicate the potential benefit of combination immunotherapy with Propes and Inflamafertin to compensate for NK and NKT cell deficiency due to genetic deficiency of the folate cycle in children with autism spectrum disorders. The purpose of the research was to study the effectiveness of combined immunotherapy with Propes and Inflamafertin in NK and NKT cell deficiency in children with autism spectrum disorders associated with genetic deficiency of the folate cycle. Material and methods. This single-center, prospective, controlled, nonrandomized clinical trial included 96 children aged 2 to 10 years with autism spectrum disorders associated with a genetic folate deficiency (study group, SG). Children of SG received Propes at a dose of 2 ml IM every other day for 3 consecutive months (45 injections), and Inflamafertin at a dose of 2 ml IM every other day for 3 months in a row, alternating with Propes (45 injections). The control group (CG) consisted of 32 children of similar age and gender distribution who suffered from autism spectrum disorders associated with genetic deficiency of the folate cycle, but who did not receive immunotherapy. Outcomes. The number of NK cells reached the lower limit of normal in 39 out of 53 patients (74% of cases), with the resulting deficiency of these lymphocytes, and the average number of NK cells in the blood in SG almost doubling during the 3-month course of immunotherapy (р ˂ 0.05; Z ˂ Z0.05). However, it returned to almost initial level in the 2 months following the discontinuation of immunotherapeutic agents (р˃0.05; Z˃Z0.05). The number of NKT cells was normalized in 78 out of 87 patients (89% of cases) with an initial deficiency of these cells, and the average number of NKT cells in the blood in the DG increased during the course of immunotherapy by half (р ˂ 0.05; Z ˂ Z0.05) and continued to grow for the next 2 months after the discontinuation of immunotropic drugs (р ˂ 0.05; Z ˂ Z0.05). There was a link between immunotherapy and normalization of NK - (χ2 = 18.016; OR = 13.929; 95%CI = 3.498-55.468) and NKT-cells (χ2 = 60.65; OR = 46.800; 95%CI = 14.415-151.937) in the blood with a strong association between these processes (criterion φ = 0.504 and 0.715 respectively; С = 0.450 and 0.581 respectively). Conclusions. Combination immunotherapy with Propes and Inflamafertin is an effective strategy for the treatment of immunodeficiency caused by genetic deficiency of the folate cycle in children with autism spectrum disorders.

Pre-Clinical Modeling of Concomitant Therapy with Azacytidine and Eltrombopag in MDS/AML

Hemorrhage-related morbidity and mortality frequently arises in patients with myelodysplastic syndromes (MDS). A major therapeutic goal in patients with MDS is the alleviation commonly occurring thrombocytopenia. Low platelet counts increase bleeding risk, limit tolerated doses and efficacy of standard therapies, and are associated with an increased risk of leukemic transformation. Preclinical and phase I/II studies on eltrombopag (EPAG), a megakaryopoiesis-stimulating thrombopoietin receptor (TPO-R) agonist, demonstrated safety and efficacy of the small molecule as monotherapy in MDS. However, a phase III study testing the ability of EPAG to increase platelet numbers in patients with intermediate or higher-risk MDS under azacytidine (AZA) therapy (NCT02158936) was prematurely terminated due to inferior performance of EPAG-treated vs. placebo-receiving patients. While the study did not find a difference in overall survival rates between the two groups, concerns pertaining to disease progression under concomitant treatment with EPAG and azacytidine have been raised.We assessed whether EPAG exerts antagonistic physiological activities in the context of anti-leukemia treatment with AZA exploiting a set of complementary pre-clinical ex vivo assays measuring aberrant cell proliferation, differentiation and self-renewal. MDS and AML patient-derived cell lines (n=4) and primary cell specimen (n=6) were treated with non-cytotoxic, clinically-relevant doses of EPAG and AZA in short-term liquid cultures, myeloid colony-forming assays, and long-term serial-replating assays. To elucidate functional effects of the combination treatment, we determined cell growth, apoptosis, differentiation and long-term colony initiation as a surrogate for self-renewal upon treatment. We also quantified cell surface presence of TPO-R, and assessed pathway activation downstream of the receptor using phosphoflow.Consistent with previous observations, continuous single agent treatment with either >0.3μM AZA, or 3-20mg/ml EPAG were found to be cytotoxic for leukemic cells. Simultaneous treatment with a non-cytotoxic functionally-relevant dosage of AZA (0.3μM) and EPAG at concentrations above 3mg/ml resulted in a striking decrease of cell viability compared to AZA single agent treatment in short-term cell line cultures. Lower-dose EPAG treatment (0.3 and 0.5mg/ml) along with AZA (0.3μM) did not lead to any significant changes in cell viability and growth in short term cultures (vs. single agent treated controls) of cell lines or primary cells. Apoptosis, cell surface differentiation marker analysis, and morphological assessment revealed no discernable differences upon combination treatment compared to AZA single agent treatment in cell lines. Characterization of aberrant self-renewal using serial replating colony assays revealed a significant decrease in the number of colony-initiating cells upon continuous single agent treatment with AZA, which was not diminished by combination treatment with EPAG. An AZA-resistant cell line also did not show altered long-term colony-forming cell numbers in the presence of both drugs. We found no evidence of increased leukemic self-renewal upon simultaneous treatment using primary cells in 5/6 specimen tested. One sample showed a transient increase in colony formation and average cell numbers per colony in the presence of both drugs, compared to AZA-treated controls, albeit the number of serial replatings was unchanged compared to AZA controls. We tested whether TPO-R/CD110-expressing aberrant cells would expand under EPAG single agent, or combination treatment with AZA, and found no significant changes in the frequency of CD110-positive cells compared to AZA single agent controls. TPO-R downstream signaling in primary mononuclear cells comprised a marked, yet transient impairment in STAT5 and AKT phosphorylation upon combination treatment, compared to single EPAG treatment.Togehter, our data show that simultaneous treatment of leukemic cells with non-cytotoxic, but clinically-relevant dosages of EPAG and AZA does not lead to a stimulation of leukemic cell growth, even in the presence of TPO-R, in several pre-clinical models. These results do not support a generalized mechanistic antagonism between the two drugs in leukemic cells. Further investigations are underway to understand the impact on non-leukemic hematopoiesis. DisclosuresSteidl:Bayer Healthcare: Consultancy; Novartis: Research Funding; Celgene: Consultancy; GlaxoSmithKline: Research Funding; Aileron Therapeutics: Consultancy, Research Funding. Marques Ramos:Novartis Pharmaceuticals: Employment. Pallaud:Novartis Pharmaceuticals: Employment. Will:Novartis Pharmaceuticals: Consultancy, Research Funding.

Vitrification yields higher cryo-survival rate than slow freezing in biopsied bovine in vitro produced blastocysts

Vitrification and slow freezing are the two commonly used embryo cryopreservation methods. In most studies, vitrification of intact embryos has proven superior in several respects, including cell and embryo survival and pregnancy rate. However, there is a lack of data for comparing these two methods in in vitro produced (IVP) bovine blastocysts, which have been subjected to the retrieval of trophectoderm (TE) biopsy. Day 7 IVP blastocysts were pooled and randomized into four groups: 1) non-biopsy (NB), 2) biopsy (B), 3) biopsy-vitrification (BV), 4) biopsy-slow freeze (BSF). The blastocysts in the B, BV, and BSF groups were subjected to TE biopsy. For the B group, this was followed by 5 hours (h) incubation and subsequent scoring of the biopsy-survival (re-expansion) rate before processing for further analyses. For the BV and BSF groups, the biopsy procedure was followed by 2 h incubation, allowing for a quick re-expansion, after which the blastocysts were subjected to vitrification and slow freezing, respectively. After warming and thawing, respectively, they were then incubated for 5 h followed by scoring the cryo-survival (re-expansion) rates before processing for further analyses. These included quantification of ICM and TE cells, cleaved caspase-3- and TUNEL-positive cells, quantitative PCR on cellular stress markers (SOD1 and PRDX1), and ultrastructural analysis.The biopsy-survival rate in the B group was 94% (307/326). The cryo-survival rate in BV (86%, 138/161) was higher than that in BSF (57%, 81/142; P < 0.001). No differences were noted between the average ICM, TE, and total cell numbers of the groups. The percentages of cleaved caspase-3-positive cells were higher in BV vs. NB (P < 0.05), in BSF vs. NB (P < 0.001), and in BSF vs. B (P < 0.001). The percentages of TUNEL-positive cells were higher in BV vs. NB (P < 0.05) and in BSF vs. NB (P < 0.001). The levels of mRNA abundance for SOD1 and PRDX1 in B, BV, and BSF were not different from that in NB. The ultrastructural analysis of blastocysts in the BV and BSF groups showed distension of extracellular spaces and appearance of intracellular vacuoles in the ICM, distension of mitochondria, and disorganization of mitochondrial cristae in both ICM and TE, and weakened tight junctions between adjacent TE cells. In summary, our findings demonstrate that vitrification yields a higher cryo-survival rate than slow freezing in biopsied bovine IVP blastocysts. However, biopsy-vitrification and biopsy-slow-freeze values are comparable in terms of ICM, TE, and total blastocyst cell numbers, as well as cleaved caspase-3- and TUNEL-positive cell rates. Moreover, biopsy and cryopreservation performed alone had no effect on ICM, TE, total blastocyst cell numbers, or TUNEL-positive cell rates. Biopsy and vitrification performed alone had no effect on the cleaved caspase-3 positive cell rates, whereas slow freezing resulted in an increased rate. Furthermore, double traumatization with a combination of biopsy and cryopreservation, either vitrification or slow freezing, resulted in increased rates of cleaved caspase-3- and TUNEL-positive cells.

Thymoquinone Increased Expression of CD4CD25Treg in Sprague-Dawley Rats Induced Dimethylbenzanthracene

BACKGROUND: The carcinogen dimethylbenzanthracene (DMBA) is immunotoxic. Thymoquinone, meanwhile, is known to have antioxidant and anti-inflammatory effects. AIM: This study aims to determine the effect of thymoquinone and tamoxifen on the CD4CD25Treg count in Sprague-Dawley (SD) rats induced by DMBA. METHODS: The 50 SD rats were divided into five groups. Group I (normal control) was given standard drinking and food. Group II was given thymoquinone, Group III was given tamoxifen, Group IV was given DMBA, and Group V was given solvent control. Thymoquinone, tamoxifen, and solvent control administration started 2 weeks before DMBA administration and continued during DMBA induction. In the 3rd week, except for the normal group, all groups were created to be induced with 10 × 20 mg/kg body weight of DMBA for 5 weeks. In the 21st week, surgery and data collection were performed. The hematology profile and CD4CD25Treg number were carried out employing a flow cytometer. The difference in the average number of CD4CD25Treg and blood cells between groups was analyzed with one-way analysis of variance RESULTS: The results revealed that DMBA induction reduced the number of erythrocytes, HB levels, platelet counts, and leukocyte counts (p &lt; 0.05). The administration of thymoquinone and tamoxifen reduced the hematopoiesis effect of DMBA. The thymoquinone and tamoxifen group had a higher number of CD4CD25Treg and leukocytes than the DMBA group (p &lt; 0.05). CONCLUSION: There was no difference in the average CD4CD25Treg, leukocyte count, lymphocyte count, and monocyte count between the thymoquinone and the tamoxifen groups (p &gt; 0.05).

Development of Porcine Somatic Cell Nuclear Transfer Embryos Following Treatment Time of Endoplasmic Reticulum Stress Inhibitor.

We examine the effect of endoplasmic reticulum (ER) stress inhibitor treatment time on the in vitro development of porcine somatic cell nuclear transfer (SCNT) embryos. Porcine SCNT embryos were classified by four groups following treatment time of ER stress inhibitor, tauroursodeoxycholic acid (TUDCA; 100 μM); 1) non-treatment group (control), 2) treatment during micromanipulation process and for 3 h after fusion (NT+3 h group), 3) treatment only during in vitro culture after fusion (IVC group), and 4) treatment during micromanipulation process and in vitro culture (NT+IVC group). SCNT embryos were cultured for six days to examine the X-box binding protein 1 (Xbp1) splicing levels, the expression levels of ER stress-associated genes, oxidative stress-related genes, and apoptosis-related genes in blastocysts, and in vitro development. There was no significant difference in Xbp1 splicing level among all groups. Reduced expression of some ER stress-associated genes was observed in the treatment groups. The oxidative stress and apoptosis-related genes were significantly lower in all treatment groups than control (p<0.05). Although blastocyst development rates were not different among all groups (17.5% to 21.7%), the average cell number in blastocysts increased significantly in NT+3 h (48.5±2.3) and NT+IVC (47.7±2.4) groups compared to those of control and IVC groups (p<0.05). The result of this study suggests that the treatment of ER stress inhibitor on SCNT embryos from the micromanipulation process can improve the reprogramming efficiency of SCNT embryos by inhibiting the ER and oxidative stresses that may occur early in the SCNT process.

Inflammatory Cell Numbers in the Stomach of Japanese Subjects with Endoscopically Normal Mucosa without Helicobacter pylori Infection

Introduction: Since inflammatory cells, such as lymphocytes and plasma cells, normally inhabit the stomach, the border between normal and mild inflammation is difficult to visually determine using the updated Sydney system scale of gastritis. Additionally, eosinophils in the gastric mucosa must be counted to diagnose eosinophilic gastritis. We aimed to determine the normal number of inflammatory cells in patients with endoscopically normal mucosa and without Helicobacter pylori infections. Methods: We assessed patients aged 20–79 years, who had undergone upper gastrointestinal endoscopy at Kawasaki Medical School Hospital between January 2010 and December 2014. Inflammatory cells were counted in 1,000 μm² fields of pyloric and fundic gland mucosal biopsy specimens. We finally included 325 (male, n = 141; female, n = 184; average age = 49.3 years) patients without inflammation who had H. pylori-negative endoscopic results and negative histological findings interpreted based on the updated Sydney System and the Kyoto classification of gastritis. Results: The average numbers of nucleated cells were 83.3 ± 14.2 and 65.4 ± 12.6/mm² in the pyloric and fundic gland mucosae, respectively. Inflammatory cells were significantly more abundant in the pyloric mucosa than in the fundic gland mucosa (p < 0.05). Age and sex distribution did not significantly differ. Eosinophils were absent or scanty in the gastric mucosae of both glands in all patients. Conclusion: We determined the absolute values of inflammatory cells, including eosinophils, in normal mucosae of pyloric and fundic glands. These findings could be important in defining gastric mucosal inflammation, including eosinophilic gastritis diagnosis.

PARTICULARITIES ASSOCIATED WITH THE EXPRESSION OF GLIAL ACIDIC FIBRILLARY PROTEIN ON THE STRUCTURAL COMPONENTS OF CEREBELLUM OF THE RATS INFLUENCED BY THE FOOD ADDITIVES COMPLEX

The aim: To define the degree for glial acidic fibrillary protein expression on the structural components of cerebellum of the rats in health and when rats influenced by the food additives complex. Materials and methods: In order to determine the degree of expression of the immunohistochemical marker GFAP on the structural components of the cerebellum of rats we applied immunohistochemical, morphometric and statistical methods in our study. Results: In histological specimens at the end of 1st week of observation in the gray matter of the cerebellum there occurred a gradual increase in 1.16 times of the average number of GFAP-positive cells. At the end of 4th week of the experimental study, the average number of GFAP-positive cells increased accurately (at p<0.05 compared to the control group) in 1.27 times, at the end of 8th week it has increased in 1.99 times, at the end of 12th week in 2.25, and at the end of 16th week in 2.39 times. Conclusions: The outcomes of our study are as follows the increase in the average number of GFAP-positive cells is directly related to the decrease in the average number of major neurons of the gray matter of the brain, while the fluctuations in the average number of astrocytic glia cells represent a compensatory mechanism in the recovery of gray matter neurons of the brain from neural stem cells with the subsequent development of reactive astrogliosis and, thereafter the possible development of neuropathology.

Efficacy of combined immunotherapy with propes and inflamafertin in selective deficiency of NK and NKT cells in children with autism spectrum disorders associated with genetic deficiency of the folate cycle.

The purpose of the research was to study effectiveness of combined immunotherapy with Propes and Inflamafertin in NK and NKT cell deficiency in children with autism spectrum disorders associated with genetic deficiency of the folate cycle. This single-center, prospective, controlled, nonrandomized clinical trial included 96 children aged 2 to 10 years with autism spectrum disorders associated with a genetic folate deficiency (study group, SG). Children of SG received Propes at a dose of 2 ml IM every other day for 3 consecutive months (45 injections), and Inflamafertin at a dose of 2 ml IM every other day for 3 months in a row, alternating with Propes (45 injections). The number of NK cells reached the lower limit of normal in 39 out of 53 patients (74% of cases), with the resulting deficiency of these lymphocytes, and the average number of NK cells in the blood in SG almost doubling during the 3- month course of immunotherapy (??0.05; Z?Z0.05). The number of NKT cells was normalized in 78 out of 87 patients (89% of cases) with an initial deficiency of these cells, and the average number of NKT cells in the blood in the DG increased during the course of immunotherapy by half (??0.05; Z?Z0.05) and continued to grow for the next 2 months after the discontinuation of immunotropic drugs (??0.05; Z?Z0.05). Combination immunotherapy with Propes and Inflamafertin is effective strategy for the treatment of immunodeficiency caused by genetic deficiency of the folate cycle in children with autism spectrum disorders.

Disentangling the abundance and structure of Vibrio communities in a semi-enclosed Bay with mariculture (Dongshan Bay, Southern China)

The genus Vibrio contains a diverse group of heterotrophic bacteria, which are members of ubiquitous and abundant microbial communities in coastal ecosystems. Vibrio has been frequently found in a wide range of marine environments either by employing Vibrio-specific 16S rRNA sequencing or culturing methods. A combination of molecular and cultivation-dependent methods was developed to more precisely discriminate between different members of the genus Vibrio in seawater. This newly developed assay was subsequently applied to characterize Vibrio community composition in surface water at 18 mariculture sites. It Substantially improved the taxonomic resolution of Vibrio species when compared to traditional 16S rRNA analysis. Our qPCR and cultivation analyses revealed that average Vibrio abundance (Vibrio 16S rRNA gene copy numbers: 3.46 × 106 to 6.70 × 106 copies L−1) and live cell numbers (5.65 × 104–5.75 × 105 cfu mL−1) are significantly related to pH. Total bacteria and Vibrio-specific 16S rRNA metabarcode sequenceing resulted in a total of 10 and 32 operational taxonomic units (OTUs), respectively, and 15 Vibrio species were identified by targeted cultivation of Vibrio strains, with Vibrio fortis and V. brasiliensis dominating in the mariculture areas. The purpose of this study was to combine several analytical methods to improve current sequence-based Vibrio community surveys, and to prove for the effectiveness of this methodological approach comprehensively testing for Vibrio dynamics in different coastal environments.

Persistence of Epstein-Barr virus antigens in gastric cancer: characteristic of inflammatory cellular reactions in tumor

Is to establish the relationship between the persistence of viral antigens of the Epstein-Barr virus (EBV) and the cellular composition of the immune microenvironment of tumor tissue and the mucous membrane of peritumoral area in gastric cancer. We used samples of surgical material from 55 patients with a verified diagnosis of gastric cancer. The expression of CD4, CD8, CD68, CD1a and LMP-1 was assessed. The results were assessed by the morphometric method. We selected three fields of view (magnification x200) in tumor tissue and in peritumoral areas separately and counted an absolute number of cells with positive staining with further calculation of the average number of cells and the median. LMP-1-negative tumors with LMP-1 expression in epithelium of peritumoral area were characterized by the highest density of CD4+ lymphocyte infiltration in the central part of the tumor; the highest density of CD8+ lymphocyte infiltration in the mucous membrane of peritumoral area (p=0.0190); the highest density of infiltration by macrophages in the mucous membrane of peritumoral area (p=0.2492); the highest density of infiltration by CD1a+ cells in the mucous membrane of peritumoral area (p=0.1503). The highest density of infiltration with CD1a+ cells was characteristic for LMP-1-positive and LMP-1-negative tumors (p=0.0813). The persistence of the LMP-1 viral antigen in the glandular epithelium of the peritumoral area in our sample does not have a statistically significant effect on the prognosis of the disease (RR=1.7718; p=0.0885) but there is a tendency towards a negative predictive value. High density of infiltration of glandular epithelium of peritumoral area with the expression of LMP-1 by CD4+ and CD8+ lymphocytes is most likely associated with the activation of the cellular immune response and may be one of the signs of the persistence of viral antigens. It was shown for the first time that the phenomenon of persistence of the LMP-1 viral antigen is characterized by a trend towards negative predictive value for patients with gastric cancer.

PENGARUH GEL EKSTRAK BAWANG PUTIH (ALLIUM SATIVUM L.) TERHADAP JUMLAH SEL MAKROFAG PADA PENYEMBUHAN LUKA INSISI GINGIVA MARMUT (CAVIA PORCELLUS)

Surgical with an incision or tissue incision process can cause injury or tissue damage to the oral cavity, especially the gingiva. Wound healing can last a long time, this condition can increase the potential for complications such as infections and less blood supply. Garlic contains several active substances including flavonoids, tannins, phenols and alkaloids, and organosulfur compounds namely allicin. The content is believed to stimulate macrophage cells and help the work of macrophages in cleaning the wound area so that it accelerates into the phase of proliferation and wound healing. The research method was experimental laboratory research with a post-test design group. The results showed that the average number of macrophage cells in the control group CMC-Na 2% 97,50 cells, mean group of 40% garlic extract gel was 58,83 cells, mean group of 50% garlic extract gel was 18,00 cells, mean 60% garlic extract gel group was 7,83 cells. The results showed that there are significant differences (ρ &lt;0,05) in the four study groups. Garlic extract gel concentration of 60% was more effective in reducing the number of macrophage cells in guinea pig gingival incision because of the percentage of active content.

Effects of 6-dimethylaminopurine, cytochalasin B and cycloheximide on in vitro development of I pig cloned embryos

The present study was conducted to evaluate the effects of 6-dimethylaminopurine (6-DMAP), cytochalasin B (CyB) and cycloheximide (CHX) on in vitro development of “I” pig somatic cell nuclear transfer embryos. Oocytes after SCNT were activated with 2 mM 6-DMAP; 7.5 µg/ml CyB and 10 µg/ml CHX. The results indicated that the rate of oocytes no lysis in group activated by CHX was significantly lower compared with those in group activated by 6-DAMP and CyB (90.91% and 88.01%, respectively, P &lt; 0,05). The blastocyst formation rate did not differ between group actvated by 6-DAMP and CyB (24.93% vs 24.4%, P &gt; 0.05). Meanwhile the blastocyst formation rate in group activated by CHX was 15.01%, significantly lower than that in group activated by 6-DAMP and CyB (P &lt; 0.05). The average number of cells in the blastocyst in group activated by 6-DAMP and CyB were higher than that in group activated by CHX (47.78, 44.57 vs 39.42, respectively). The use of 6-DMAP and CyB in the activation of pig oocytes after SCNT was more effective than CHX. The results of this study show that for the first time in Vietnam, we have created “I” pig SCNT embryo without zona pellucida with the rate of blastocyst formation from 15.01% to 24.93%.