Translational studies of electrophoretic mobility and phase picture of erythrocytes with consideration of development of stress response during a pathological process

Rationale:Modern cell diagnostic methods are in high demand during the development of new approaches in personalized medicine. Coherent phase interferometry and cell microelectrophoresis are among such methods that are being actively introduced into the diagnostic process in medical institutions.Aim:To substantiate the potential use of biophysical and morphodensitometrical erythrocytes parameters as criteria of treatment efcacy and course of adaptation process in patients with gastrointestinal tract disorders.Materials and methods:The study included 25 patients aged from 40 to 54 years (11 males and 14 females), among them 9 (36%) with gastric peptic ulcer, 3 (12%) with duodenal ulcer, 8 (32%) with acute gastritis, and 5 (20%) with acute pancreatitis. Biophysical and morphological particulars of peripheral blood erythrocytes were assessed before and after treatment using cell diagnostic techniques, such as microelectrophoresis and laser modulation interference microscopy. Also, we evaluated changes over time in routine clinical laboratory tests, such as red and white blood cell counts, hemoglobin levels, and erythrocyte sedimentation rate (ESR), and diﬀerential leukocyte counts. The control group included 10 healthy donors aged from 36 to 52 years.In vitroexperiments were performed to assess the erythrocyte electrophoretic mobility (EEPM) and morphology of erythrocytes treated with epinephrine or cortisol.Results:After the treatment, the patients demonstrated a decrease in their leukocyte counts (by 27%), a 2-fold increase in monocyte counts and an ESR decrease (by 10%), compared to the corresponding baseline values before treatment (p < 0.05 for all comparisons). EEPM increased by 12% (1.37 vs. 1.22 mcm × cm/V × s, p < 0.05). The erythrocyte pool of the patients before treatment, had a decreased proportion of discocytes, compared to that in the control group (85.2 vs. 95.4%, р < 0.05), increased proportions of echinocytes, stomatocytes and degenerative forms (11, 2.8 and 1%, respectively, р < 0.05). After the treatment, the discocytes counts increased virtually up to their physiological normal range (91.3%). However, the surface of the discoid cells remained heterogeneous with multiple microspicules; this resulted in changes of electrokinetic and morphological properties of erythrocyte response to stress reaction occurring in the body. The impact of the stress eﬀectors was confrmed inin vitroexperiments assessing the eﬀects of epinephrine (1 × 10-9 g/mL) and cortisol (5 × 10-7 g/mL) on erythrocytes. At 120 minutes of the experiment, epinephrine decreased EEPM (1.14 vs. 1.24 mcm × cm/V × s at baseline, р < 0.05) and increased cell sphericity. On the contrary, cortisol increased EEPM (1.72 vs. 1.36 mcm × cm/V × s, р < 0.05), with non-signifcant echinocytic transformation.Conclusion:Biophysical and morphodensitometric parameters of red blood cells obtained with the use of current express methods of cell microelectrophoresis and coherent interference microscopy help to objectivize the intensity of stress response during a pathological process and activation of adaptation mechanisms during the treatment.

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Analysis of the intervention effect and self-satisfaction of sports dance exercise on the psychological stress of college students

Work ◽

10.3233/wor-213505 ◽

2021 ◽

pp. 1-13

Author(s):

Changliang Zheng ◽

Hongmei Ji

Keyword(s):

College Students ◽

Stress Response ◽

Psychological Stress ◽

Rating Scale ◽

Response Characteristic ◽

Control Group ◽

Observation Group ◽

Excessive Pressure ◽

Psychological Pressure ◽

The Impact

BACKGROUND: College students are a high-risk subpopulation of psychological disorders. The problem of various adverse phenomena and consequences caused by excessive pressure on college students has gradually become the focus of social and psychological academic circles. However, studies related to individual self-concept and psychological pressure are rare. OBJECTIVE: To explore the impact of sports dance exercises on college students’ psychological pressure and improve the psychological effects of their self-satisfaction. METHODS: College students were taken as research objects, randomly divided into a control group and an observation group. The observation group is intervened with sports dance exercises. The observation group was intervened with sports dance exercises. The stress response characteristic questionnaire and multidimensional self-satisfaction rating scale were utilized to measure college students’ conditions before and after the intervention. Finally, the obtained data were statistically analyzed. RESULTS: Generally, the psychological stress response of college students was mild, with self-satisfaction and various dimensions at a moderately higher level. No significant differences were discovered in the psychological stress response and self-satisfaction level between the control group and the observation group before intervention (P > 0.05), which were homogeneous. Compared with the data obtained before the intervention, after the intervention, the control group scores were slightly reduced in all dimensions of the psychological stress response and self-satisfaction. In contrast, the scores were significantly increased in the experimental group (P < 0.05). Psychological stress response could reliably explain 30.4%of the total self-satisfaction variance (P < 0.01). The negative self-evaluation was the most important variable affecting self-satisfaction, followed by poor interpersonal communication and poor sleeping quality. CONCLUSIONS: Sports dance exercises could alleviate the psychological stress of college students and improve their self-satisfaction. Colleges and universities should include sports dance in the content of optional public courses and encourage more college students to actively participate in sports dance exercises to improve their mental health.

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Effects of Vitrification on the Blastocyst Gene Expression Profile in a Porcine Model

International Journal of Molecular Sciences ◽

10.3390/ijms22031222 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1222

Author(s):

Cristina Cuello ◽

Cristina A. Martinez ◽

Josep M. Cambra ◽

Inmaculada Parrilla ◽

Heriberto Rodriguez-Martinez ◽

...

Keyword(s):

Gene Expression ◽

Gene Expression Analysis ◽

Survival Rates ◽

Control Group ◽

P Value ◽

Transcriptome Profile ◽

Embryo Viability ◽

The Impact

This study was designed to investigate the impact of vitrification on the transcriptome profile of blastocysts using a porcine (Sus scrofa) model and a microarray approach. Blastocysts were collected from weaned sows (n = 13). A total of 60 blastocysts were vitrified (treatment group). After warming, vitrified embryos were cultured in vitro for 24 h. Non-vitrified blastocysts (n = 40) were used as controls. After the in vitro culture period, the embryo viability was morphologically assessed. A total of 30 viable embryos per group (three pools of 10 from 4 different donors each) were subjected to gene expression analysis. A fold change cut-off of ±1.5 and a restrictive threshold at p-value < 0.05 were used to distinguish differentially expressed genes (DEGs). The survival rates of vitrified/warmed blastocysts were similar to those of the control (nearly 100%, n.s.). A total of 205 (112 upregulated and 93 downregulated) were identified in the vitrified blastocysts compared to the control group. The vitrification/warming impact was moderate, and it was mainly related to the pathways of cell cycle, cellular senescence, gap junction, and signaling for TFGβ, p53, Fox, and MAPK. In conclusion, vitrification modified the transcriptome of in vivo-derived porcine blastocysts, resulting in minor gene expression changes.

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Abstract 420: Delineating the Caspase-dependent Targets and Signal Pathways That Promote Pathological Cardiac Hypertrophy

Circulation Research ◽

10.1161/res.117.suppl_1.420 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Charis Putinski ◽

Mohammad Abdul-Ghani ◽

Rebecca Stiles ◽

Steve Brunette ◽

Sarah A Dick ◽

...

Keyword(s):

Cardiac Hypertrophy ◽

Pathological Process ◽

Signal Pathways ◽

Caspase Cleavage ◽

Substrate Protein ◽

Cell Death Pathway ◽

Effector Caspase ◽

The Impact

Although cardiac hypertrophy is initially an adaptive response, chronic stress on the heart is a maladaptive process that inevitably leads to end-stage heart failure. Interestingly, this pathological process is also characterized by cell behaviors associated with apoptosis. We previously demonstrated the essential role of the intrinsic cell death pathway during cardiac hypertrophy; however, the caspase-dependent pathways and cleavage targets remain elusive. To this aim, we evaluated a myocyte enhancer factor 2 (MEF2) transcription factor inhibitor, histone deacetylase 3 (HDAC3), and gelsolin as potential caspase cleavage substrates involved in the induction and/or maintenance of cardiac hypertrophy. In vitro cleavage assays were completed with effector caspase and recombinant substrate protein which demonstrated caspase-dependent cleavage. HDAC3 cleavage was observed during early stages of hypertrophy and reduced in the presence of a caspase inhibitor. Luciferase assays demonstrated that the transcriptional activity of MEF2 is dependent on intact caspase function suggesting caspase-directed HDAC3 cleavage may serve as a novel regulatory mechanism to alleviate MEF2 suppression to engage the hypertrophy gene expression program. Unlike HDAC3, caspase mediated gelsolin cleavage occurs at latter stages and is coincident with the cytoskeletal alterations that occur during this process. As gelsolin is a potent actin capping/severing enzyme, we hypothesize that caspase-mediated gelsolin activation acts as a key regulatory step in the structural rearrangements that allow for hypertrophy to occur. We have generated adenoviral vectors containing caspase cleavage mutants and cleaved forms of HDAC3 and gelsolin and will discuss the impact of these modified substrates on the hypertrophy process in vitro and in vivo. Collectively, this work suggests that caspase signalling acts to engage both the transcriptional program and cytoskeletal accommodations that characterize cardiac hypertrophy. Importantly, these observations suggest that identification of inhibitors that suppress caspase activity and/or activity of its cognate substrates may offer novel therapeutic targets to limit the development of pathological hypertrophy.

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The Impact of Traditional Medicine-Based Lifestyle and Diet on Infertility Treatment in Women Undergoing Assisted Reproduction: A Randomized Controlled Trial

Complementary Medicine Research ◽

10.1159/000505016 ◽

2020 ◽

Vol 27 (4) ◽

pp. 230-241

Author(s):

Zeinab Alibeigi ◽

Effat Jafari-Dehkordi ◽

Soleiman Kheiri ◽

Maryam Nemati ◽

Gholamreza Mohammadi-Farsani ◽

...

Keyword(s):

Traditional Medicine ◽

Controlled Trial ◽

Intervention Group ◽

Infertility Treatment ◽

Control Group ◽

Lifestyle Modifications ◽

Mature Ovum ◽

The Impact ◽

Diet And Lifestyle

The problem of infertility is growing rapidly in the world. Traditional medicine with thousands of years of history has claimed that it can treat some kinds of infertility using nutritional and lifestyle modifications and interventions. The purpose of this study was to evaluate the effect of a traditional medicine-oriented diet and lifestyle on infertility treatment. Based on a clinical trial study, 180 infertile women who were 20–40 years old and candidates for in vitro fertilization (IVF) were randomly assigned to 2 groups: an intervention group and a control group. The intervention group used diet and lifestyle recommendations based on Iranian traditional medicine for at least 3 months. The number of ova, mature ovum number, embryo number, embryo quality, and fertilization rate were significantly higher in the intervention group than in the control group (for all items; p < 0.05). Overall pregnancy rate was significantly higher in the intervention group (35.2 vs. 12.4%; odds ratio [OR], 3.8; 95% CI, 1.8–8.3). The intervention group had a higher rate of getting spontaneous pregnancy than the control group (20.9 vs. 2.2%; OR, 11.5; 95% CI, 2.6–50.9). Chemical pregnancy was significantly higher in the intervention group (64 vs. 27.5%; OR, 4.7; 95% CI, 1.9–11.6). Diet and lifestyle modifications based on traditional medicine can contribute greatly to the infertility treatment. Thus, many infertility cases can be treated without the need to use advanced methods. In case of using assisted reproductive techniques, traditional medicine can enhance the efficiency of these methods.

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153 SUPPLEMENTATION WITH LINOLENIC ACID AND L-CARNITINE DURING IVM REDUCED THE EXPRESSION OF GENES RELATED TO LIPOGENESIS BUT DID NOT ALTER THE LIPID CONTENT AND CRYOTOLERANCE OF IN VITRO-PRODUCED EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab153 ◽

2017 ◽

Vol 29 (1) ◽

pp. 185 ◽

Cited By ~ 1

Author(s):

B. C. S. Leao ◽

N. A. S. Rocha Frigoni ◽

P. C. Dall'Acqua ◽

M. Ambrogi ◽

G. B. Nunes ◽

...

Keyword(s):

Gene Expression ◽

Linolenic Acid ◽

Lipid Content ◽

Control Group ◽

Intracellular Lipid ◽

Chi Square ◽

Sudan Black B ◽

Expression Of Genes ◽

The Impact

This study was conducted to evaluate the impact of supplementation during in vitro maturation (IVM) with linolenic acid (ALA), l-carnitine (L-car), or the combination of both supplements on the embryo intracellular lipid content and cryotolerance, as well as in the embryo expression of genes involved in lipid metabolism (lipogenesis regulation: SCD1, FASN, and SREBP1; and β-oxidation pathway: CPT1B and CPT2). Cumulus-oocyte complexes (n = 1076) were IVM for 22 h at 38.5°C and 5% CO2 in air, in TCM-199 medium with bicarbonate, hormones, and 10% FCS (control group), supplemented with 100 μM ALA (ALA group), 5 mM L-car (L-car group), or a combination of 100 μM ALA + 5 mM L-car (ALA + L-car group). After IVF, presumptive zygotes were in vitro cultured in SOFaa medium supplemented with 5 mg mL−1 BSA and 2.5% FCS, at 38.5°C and 5% CO2 in air during 7 days. Cleavage and blastocyst rates were evaluated on Day 3 and 7, respectively (IVF = Day 0). At Day 7, the blastocysts were stained with the lipophilic dye Sudan Black B (n = 60), vitrified/warmed (n = 260; Ingámed® protocol, Maringa-PR, Brazil), or collected for analysis of gene expression (n = 180). Embryonic development were analysed by ANOVA and the multiple comparisons of means were determined by Tukey’s test. The embryonic re-expansion data were subjected to chi-square test and the differences in gene expression among groups were evaluated by Duncan’s multiple range test (P < 0.05). Data are presented as means ± standard error means. There was no effect (P > 0.05) of the supplements used during IVM on cleavage (79.54 ± 2.76% to 82.16 ± 1.13%) and blastocyst rates (29.03 ± 3.07% to 30.46 ± 2.01%). Similarly, the intracellular lipid content in Day-7 blastocysts (1.03 ± 0.04 to 1.15 ± 0.07 pixels) and the embryonic cryotolerance, assessed by the re-expansion rates after 24 h (67.3 to 78.3%) hatching rates after 48 h (11.5 to 25.5%) of post-warming culture, were unaffected (P > 0.05) by the supplements of IVM medium. Although the treatments did not alter (P > 0.05) the expression of CPT1B and CPT2 genes, the expression of FASN gene was decreased (P < 0.05) in the ALA group and the expression of SREBP1 gene was decreased (P < 0.05) in the ALA and L-car groups. The expression of the gene SCD1 was reduced (P < 0.05) in all treatments compared with the control group. Thus, despite the lack of effects of the treatments performed during IVM on the intracellular lipid content and cryotolerance of the embryos derived from the treated oocytes, a reduction in the expression of genes related to lipogenesis was observed in Day-7 blastocysts. These results suggest that treatments performed in the oocytes during IVM may have prolonged effects, affecting the subsequent expression of genes in embryos. Further studies are needed to determine the mechanisms related to the differentiation of the oocyte machinery during maturation. Financial support was provided by FAPESP (#2012/10084–4 and #2013/07382–6).

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Bovine Babesiosis in Turkey: Impact, Current Gaps, and Opportunities for Intervention

Pathogens ◽

10.3390/pathogens9121041 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1041

Author(s):

Sezayi Ozubek ◽

Reginaldo G. Bastos ◽

Heba F. Alzan ◽

Abdullah Inci ◽

Munir Aktas ◽

...

Keyword(s):

Vaccine Development ◽

Point Of Care ◽

Animal Health ◽

Current Control ◽

Diagnostic Methods ◽

Control Measures ◽

Bovine Babesiosis ◽

Research Programs ◽

The Impact

Bovine babesiosis is a global tick-borne disease that causes important cattle losses and has potential zoonotic implications. The impact of bovine babesiosis in Turkey remains poorly characterized, but several Babesia spp., including B. bovis, B. bigemina, and B. divergens, among others and competent tick vectors, except Rhipicephalus microplus, have been recently identified in the country. Bovine babesiosis has been reported in all provinces but is more prevalent in central and highly humid areas in low and medium altitude regions of the country housing approximately 70% of the cattle population. Current control measures include acaricides and babesicidal drugs, but not live vaccines. Despite the perceived relevant impact of bovine babesiosis in Turkey, basic research programs focused on developing in vitro cultures of parasites, point-of-care diagnostic methods, vaccine development, “omics” analysis, and gene manipulation techniques of local Babesia strains are scarce. Additionally, no effective and coordinated control efforts managed by a central animal health authority have been established to date. Development of state-of-the-art research programs in bovine babesiosis to address current gaps in knowledge and implementation of long-term plans to control the disease will surely result in important economic, nutritional, and public health benefits for the country and the region.

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Clinical utility of complex assessment with evoked potentials in acute lymphoblastic leukemia survivors: comparison of various treatment protocols

BMC Cancer ◽

10.1186/s12885-021-07873-x ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Slawomir Kroczka ◽

Konrad Stepien ◽

Izabela Witek-Motyl ◽

Kinga Kwiecinska ◽

Eryk Kapusta ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Evoked Potentials ◽

Lymphoblastic Leukemia ◽

Objective Assessment ◽

Harmful Effect ◽

Diagnostic Methods ◽

Joint Analysis ◽

Control Group ◽

The Impact

Abstract Background One of the greatest success of pediatric hematology is a prominent improvement of survival in acute lymphoblastic leukemia (ALL). Therefore, special attention needs to be paid to long-term side effects of the treatment such as neurotoxicity. One of the few diagnostic methods that allow an objective assessment of sensory systems are evoked potentials (EP). Methods The analyzed group consisted of 167 ALL long-term survivors, aged 4.9–28.4 years, without auditory, visual and sensory deviations. Patients were treated with New York (NY, n = 35), previous modified Berlin-Frankfurt-Münster (pBFM, n = 47) and BFM95 (n = 85) protocols. In order to assess the impact of radiotherapy on recorded EP, a joint analysis of NY and pBFM groups was performed. The control group consisted of 35 patients, aged 6–17 years. The analyzed patients underwent a complex assessment with visual EP (VEP), somatosensory EP (SEP) and brainstem auditory EP (BAEP) in accordance with current standards. Results ALL treatment contributed to the shortening of wave I latency (1.59 vs 1.90, P = 0.003) and prolongation of I-III (2.23 vs 2.04, P = 0.004) and I-V (4.57 vs 4.24, P = 0.002) interwave latencies of BAEP. A significant effect was also noticed in P100 (106.32 vs 101.57, P < 0.001) and N135 (151.42 vs 138.22, P < 0.001) latencies of VEP and N18 amplitude (3.24 vs 4.70, P = 0.007) and P25 latency (21.32 vs 23.39, P < 0.001) of SEP. The distribution of abnormalities between protocols was similar in BAEP (NY - 68.6%, pBFM - 61.7%, BFM95–69.4%, P = 0.650), VEP (NY - 68.6%, pBFM - 42.5%, BFM95–58.3%, P = 0.053) and significantly different for SEP (NY - 62.9%, pBFM - 36.2%, BFM95–53.0%, P = 0.045). The harmful effect of radiotherapy was most clearly marked in numerous disturbances of SEP parameters. Conclusions The presented analysis indicates a high frequency of subclinical abnormalities in EP regardless of the analyzed protocol. To our knowledge current study is the largest and one of the most complex research examining the role of EP in ALL patients. The obtained results indicate the possibility of using a single, objective and non-invasive measurement of EP in ALL survivors in order to stratify the risk of developing sensory abnormalities in adulthood.

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Grb10 Mediated Akt Activation Is Required for Induction of CML Like Myeloproliferative Disease in Mice by BCR-ABL.

Blood ◽

10.1182/blood.v110.11.1012.1012 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1012-1012

Author(s):

Corinna Albers ◽

Anna L. Illert ◽

Cornelius Miething ◽

Christian Peschel ◽

Justus Duyster

Keyword(s):

Bone Marrow ◽

Tyrosine Kinases ◽

Chromosomal Translocation ◽

Control Group ◽

Myeloproliferative Disease ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Vitro Analysis ◽

The Impact

Abstract Chronic myelogenous leukaemia (CML) results from the neoplastic transformation of hematopoietic stem cells (HSC) and is characterized by a chromosomal translocation t(9;22)(q34;q11). This aberration leads to the expression of the oncogenic tyrosine kinase BCR-ABL, which mediates signals for proliferation, transformation and anti-apoptosis via various signalling pathways. Grb10, a member of the growth factor bound proteins, is known to bind activated tyrosine kinases like BCR-ABL and might be involved in the activation of the Akt signalling pathway. Here we report the impact of Grb10 for BCR-ABL mediated transformation. We exerted a siRNA based approach in combination with a murine bone marrow transplantation model. To this end we designed a MSCV based retrovirus encoding both a Grb10 microRNA and the BCR-ABL oncogene on a single construct. This approach ensured knockdowns of more than 90% in every BCR-ABL transformed cell. Methylcellulose assays demonstrated that bone marrow coexpressing Grb10 microRNA and BCR-ABL had a 4-fold decreased colony forming ability compared to control cells. We then transduced bone marrow (BM) with retrovirus coexpressing Grb10 microRNA and p185 BCR-ABL and transplanted lethally irradiated recipient Balb/C mice. The onset and progression of leukaemia was significantly delayed in mice transplanted with Grb10 microRNA and BCR-ABL compared with the BCR-ABL transduced control microRNA group. However, we were not able to completely avoid the development of leukaemia by Grb10 knockdown. Mice transplanted with the Grb10 knockdown construct showed a delayed lymphoblastic disease, positive for B220, whereas the control group developed a rapid myeloproliferative disease, characterized by CD11b and Gr-1. In vitro analysis of BaF/3 and 32D cells showed that Grb10 knockdown in combination with BCR-ABL expression leads to a reduced phosphorylation of Akt. Taken together our data demonstrate that Grb10 is required for the development of a myeloproliferative disease by BCR-ABL in mice. Hereby, Grb10 seems to be critical for the BCR-ABL induced activation of the Akt pathway. In addition, this study describes a novel approach to express an oncogene and a microRNA using a single retroviral construct. This tool can be used to systematically screen for drugable signalling targets involved in oncogenesis.

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Prolonged Thrombocytopenia After Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) and Its Association with an Increase in CD8+ CX3CR1+ Cells in Bone Marrow.

Blood ◽

10.1182/blood.v120.21.3077.3077 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3077-3077

Author(s):

Xiao-hui Zhang ◽

Guo-xiang Wang ◽

Yan-rong Liu ◽

Lan-Ping Xu ◽

Kai-Yan Liu ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Surface Expression ◽

T Cell Subsets ◽

Control Group ◽

Hematopoietic Stem ◽

Cell Counts

Abstract Abstract 3077 Background: Since prolonged thrombocytopenia (PT) is an independent risk factor for poor clinical outcome after allogeneic hematopoietic stem cell transplantation (allo-HSCT), the underlying mechanisms need to be understood in order to develop selective treatments. Previous studies1–4 have suggested that abnormalities in B cells may play a role in the pathogenesis of PT. However, abnormalities in B cells alone do not fully explain the complete pathogenic mechanisms of PT. Our previous studies5 showed that the frequency of megakaryocytes with a ploidy value ≤ 8N was significantly increased in patients who developed PT after allo-HSCT compared to the control group. Mechanisms concerning the megakaryocyte hypoplasia in PT after allo-HSCT are not well understood. Design and Methods: PT was defined as a platelet count ≤80 × 109/L for more than 3 months after HSCT, recovery of all other cell counts, and no apparent cause for thrombocytopenia, such as aGVHD, disease recurrence, CMV infection, or antiviral drug treatment at three months post-HSCT when all other blood cell counts had return to normal.5 We analyzed T cell subsets in bone marrow (BM) and peripheral blood (PB) from allo-HSCT recipients with and without PT (n = 23 and 17, respectively) and investigated the expression characteristics of homing receptors CX3CR1, CXCR4 and VLA-4 by flow cytometry. Futhermore, Mononuclear cells (MNCs) from PT patients and controls were cultured with and without autologous CD8+ T cells in vitro, and clarify the effect of activated CD8+ T cells on the ploidy and apoptosis of megakaryocytes in the bone marrow. Results: The results demonstrated that the percentage of CD3+ T cells in the BM was significantly higher in PT patients than the experimental controls (76.00 ± 13.04% and 57.49 ± 9.11%, respectively, P < 0.001), whereas this difference was not significant for the PB (71.01 ± 11.49% and 70.49 ± 12.89%, respectively, P = 0.911). While, some T cell subsets in the BM and PB from allo-HSCT recipients with PT were not significantly different from that of the experimental control group, such as CD8+ T cells, CD4+ T cells, CD4+ CD25bright T cells (regulatory T cells), CD44hi CD62Llo CD8+ T cells and naive T cells (CD11a+ CD45RA+). Furthermore, the surface expression of homing receptor CX3CR1 on BM T cells (64.16 ± 14.07% and 37.45 ± 19.66%, respectively, P < 0.001) and CD8+ T cells (56.25 ± 14.54% and 35.16 ± 20.81%, respectively, P = 0.036), but not in blood, were significantly increased in PT patients compared to controls. For these two groups of patients, the surface expression of CXCR4 and VLA-4 on T cells and CD8+ T cells from both BM and PB did not show significant differences. Through the study in vitro, we found that the activated CD8+ T cells in bone marrow of patients with PT might suppress apoptosis (MNC group and Co-culture group: 18.02 ± 3.60% and 13.39 ± 4.22%, P < 0.05, respectively) and Fas expression (MNC group and Co-culture group: 21.10 ± 3.93 and 15.10 ± 2.33, P <0.05, respectively) of megakaryocyte. In addition, megakaryocyte with a ploidy value ≤ 8N (MNC group: 40.03 ± 6.42% and 24.54 ± 4.31%, respectively, P < 0.05) was significantly increased in patients with PT compared to the control group. Conclusions: In conclusion, an increased surface expression of CX3CR1 on T cells may mediate the recruitment of CD8+ T cells into the bone marrow in patients with PT who received an allo-HSCT. Moreover, CD8+CX3CR1+ T cells, which can have significantly increased numbers in bone marrow of patients with PT, likely caused a reduction in the megakaryocyte ploidy, and suppressed megakaryocyte apoptosis via CD8+ T cell-mediated cytotoxic effect, possibly leading to impaired platelet production. Therefore, treatment targeting CX3CR1 should be considered as a reasonable therapeutic strategy for PT following allo-HSCT. Disclosures: No relevant conflicts of interest to declare.

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Effect ofAllium cepaL. on Lipopolysaccharide-Stimulated Osteoclast Precursor Cell Viability, Count, and Morphology Using 4′,6-Diamidino-2-phenylindole-Staining

International Journal of Cell Biology ◽

10.1155/2014/535789 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 11

Author(s):

Tatiane Oliveira ◽

Camila A. Figueiredo ◽

Carlos Brito ◽

Alexander Stavroullakis ◽

Anuradha Prakki ◽

...

Keyword(s):

Escherichia Coli ◽

Cell Viability ◽

Allium Cepa ◽

Porphyromonas Gingivalis ◽

Precursor Cells ◽

Osteoclast Precursor ◽

Control Group ◽

Cell Counts ◽

Affect Cell Viability

Allium cepaL. is known to possess numerous pharmacological properties. Our aim was to examine thein vitroeffects ofAllium cepaL. extract (AcE) onPorphyromonas gingivalisLPS andEscherichia coliLPS-stimulated osteoclast precursor cells to determine cell viability to other future cell-based assays. Osteoclast precursor cells (RAW 264.7) were stimulated byPgLPS (1 μg/mL) andE. coliLPS (1 μg/mL) in the presence or absence of different concentrations of AcE (10–1000 μg/mL) for 5 days at 37°C/5% CO2. Resazurin reduction and total protein content assays were used to detect cell viability. AcE did not affect cell viability. Resazurin reduction assay showed that AcE, at up to 1000 μg/mL, did not significantly affect cell viability and cellular protein levels. Additionally a caspase 3/7 luminescence assay was used to disclose apoptosis and there was no difference in apoptotic activity between tested groups and control group. Fluorescence images stained by DAPI showed no alteration on the morphology and cell counts of LPS-stimulated osteoclast precursor cells with the use of AcE in all tested concentrations when compared to control. These findings suggest thatAllium cepaL. extract could be used forin vitrostudies onPorphyromonas gingivalisLPS andEscherichia coliLPS-stimulated osteoclast precursor cells.

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