Search results for: cytometry analysis

Authors: Santhita Tungkajiwangkoon, Yoshikazu Hoshi

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Three Japaneses Drosera species are the good model to study genome organization with highly specialized morphological group for insect trapping, and has revealed anti-inflammatory, and antibacterial effects, so there must be a reason for botanists are so appealing in these plants. Cytology and Flow cytometry were used to investigate the genetic stability and ploidy estimation in three related species. The cytological and Flow cytometry analysis were done in Drosera rotundifolia L., Drosera spatulata Labill and Drosera tokaiensis. The cytological studies by fluorescence staining (DAPI) showed that D. tokaiensis was an alloploid (2n=6x=60, hexaploid) which is a natural hybrid polyploids of D. rotundifolia and D. spatulata. D. rotundifolia was a diploid with the middle size of metaphase chromosomes (2n=2x=20) as a paternal origin and D. spatulata was a tetraploid with small size of metaphase chromosome (2n=4x=40) as a maternal origin. We confirmed by Flow cytometry analysis to determine the ploidy level and DNA content of the plants. The 2C-DNA values of D. rotundiflolia were 2.8 pg, D. spatulata was 1.6 pg and D. tokaiensis was 3.9 pg. However, 2C- DNA values of D. tokaiensis should be related from their parents but in the present study the 2C-DNA values of D. tokaiensis was no relation from the theoretical of hybrids representing additive parental. Possibility of D. tokaiensis is a natural hybrid, which is also hybridization in natural evolution can cause the genome reduction in plant.

Keywords: drosera, hybrid, cytology, flow cytometry

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Authors: Fatimah A. Alhomaid

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The present study was planned to investigate the role of molecular changes and immunohistochemical in early detection of liver cancer in Saudi patients. our results were carried out on 54 patients liver cancer. We obtained our data from laboratory in King Khalid University Hospital. The specimens were taken (54) patients with liver cancer 34 male and 14 female and 2 control. The average age of varied from 37-85 years. The tumor was diagnosed as grade I in tow patients (male and female) and grade 2 in 45 patients (28 male and 17 female) while the grade 3 in 4 patients (all males). The specimens were processed for haematoxylin and eosin staining, immunohistochemical technique and flow cytometry analysis. Our study noted that most patients had adenocarcinoma which characterized by presence of signet-ring cells were very clear in advanced patients with adenocarcinoma. Our sections in adenocarcinoma in grade 2 and stage 3 had an increase in signet ring cells,an increase in the acini of glands and an increase in number of lymphocytes which spread to the muscular layer. With advancing the disease, there were haemorrhage in blood and increase in lymphocytes and increase in the number of nuclei in the tubular glands. Our study was carried on 48 patients, immunohistochemical diagnosis (CK20, PCNA, P53) and the analysis of DNA content by flow cytometry technique. Our study indicated that the presence of correlation between the immunohistochemical analysis for P53 and the grades. The reaction of P53 appeared as strong in nucleus in grades &stage 3 and appeared in other sections as dark brown pigment. Our study indicated that the absence of correlation between the immunohistochemical analysis for PCAN and the grades. In our sections there were strong reaction in the more 80% of nuclei in grade 1& stage 2. Our study indicated that the presence of correlation between the immunohistochemical analysis for CK20 and the grades. Our results indicated the presence of positive reaction in cytoplasm varied from weak to moderate in grade 3 & stage 4. Concerning the Flow cytometry technique our results indicated that the presence of correlation between the DNA and different stages of liver cancer.

Keywords: cancer, CK20, DNA, cytometry analysis, liver, immunohistochemical, molecular changes, PCNA, p53

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Authors: Yener Tekeli, Hayri Baba

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The natural active compounds found in medicinal plants are belong to various chemical structures including polyphenolic compounds, flavonoids, essential oils, and vitamins and some of these compounds have anticancer, antioxidant, and antimicrobial activity. However, these compounds have been little known about mechanisms to confer antibacterial drug resistance. In this study; some macrofungi extracts (Pholiota lucifera, Gnaoderma applanatum and Pleurotus ostreatus) were investigated for their abilities to enhance bacterial permeability by flow cytometry. This experiments exhibited enhancement of these extracts to disrupt the cytoplasmic membrane of living bacterial (Listeria innocua and Escherichia coli) cells. These experiments were designed to detect uptake of PI&SYT by enhancing with a ranged concentration of herb extracts.

Keywords: antimicrobial activity, flow cytometry, macrofungi, multidrug resistant

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Authors: William E. Bauta, Jennifer Rebeles, Reggie Jacob

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Porphyrins are noteworthy in biomedical science for their cancer tissue accumulation and photophysical properties. The preferential accumulation of some porphyrins in cancerous tissue has been known for many years. This, combined with their characteristic photophysical and photochemical properties, including their strong fluorescence and their ability to generate reactive oxygen species in vivo upon laser irradiation, has led to much research into the application of porphyrins as cancer diagnostic and therapeutic agents. Porphyrins have been used as dyes to detect cancer cells both in vivo and, less commonly, in vitro. In one example, human sputum samples from lung cancer patients and patients without the disease were dissociated and stained with the porphyrin TCPP (5,10,15,20-tetrakis-(4-carboxyphenyl)-porphine). Cells were analyzed by flow cytometry. Cancer samples were identified by their higher TCPP fluorescence intensity relative to the no-cancer controls. However, quantitative analysis of fluorescence in cell suspensions stained with multiple fluorophores requires particles stained with each of the individual fluorophores as controls. Fluorescent control particles must be compatible in size with flow cytometer fluidics and have favorable hydrodynamic properties in suspension. They must also display fluorescence comparable to the cells of interest and be stable upon storage amine-functionalized spherical polystyrene beads in the 5 to 20-micron diameter range that was reacted with TCPP and EDC in aqueous pH six buffer overnight to form amide bonds. Beads were isolated by centrifugation and tested by flow cytometry. The 10-micron amine-functionalized beads displayed the best combination of fluorescence intensity and hydrodynamic properties, such as lack of clumping and remaining in suspension during the experiment. These beads were further optimized by varying the stoichiometry of EDC and TCPP relative to the amine. The reaction was accompanied by the formation of a TCPP-related particulate, which was removed, after bead centrifugation, using a microfiltration process. The resultant TCPP-functionalized beads were compatible with flow cytometry conditions and displayed a fluorescence comparable to that of stained cells, which allowed their use as fluorescence standards. The beads were stable in refrigerated storage in the dark for more than eight months. This work demonstrates the first preparation of porphyrin-functionalized flow cytometry control beads.

Keywords: tetraaryl porphyrin, polystyrene beads, flow cytometry, peptide coupling

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Authors: Fatimah Alhomaid

Abstract:

The present study was planned to investigate the role of molecular changes and immunohistochemical in early detection of colon cancer in Saudi patients. Our results were carried out on 48 patients colon cancer. We obtained our data from laboratory in King Khalid university hospital. The specimens were taken (48) patients with colon cancer 34 male and 14 female and 2 control. The average age of varied from 37-85 years. The tumor was diagnosed as I in tow patients (male and female) and grade 2 in 42 patients (29 male and 13 female) while the grade 3 in 4 patients (all males). The specimens were processed for haematoxylin and eosin staining , immunohistochemical technique and flow cytometry analysis. Our study noted that most patients had adenocarcinoma which characterized by presence of signet-ring cells were very clear in advanced patients of adenocarcinoma. Our sections in adenocarcinoma in grade 2 and stage 3 had an increase in signet ring cells,an increase in the acini of glands and an increase in number of lymphocytes which spread to the muscularis layer. With advancing the disease, there were haemorge in blood and increase in lymphocytes and increase number of nuclei in the tubular glands. Our study was carried on 48 patients, immunohistochemical diagnosis (CK20,PCNA,P53) and the analysis of DNA content by flow cytometry technique. Our study indicated that the presence of correlation between the immunohistochemical analysis for P53 and the grades. The reaction of P53 appeared as strong in nucleus in grades &stage 3 and appeared in other sections as dark brown pigment. Our study indicated that the absence of correlation between the immunohistochemical analysis for pcan and the grades. In our sections, there were strong reactions in the more 80% of nuclei in grade 1& stage 2. Our study indicated that the presence of correlation between the immunohistochemical analysis for CK20 and the grades. Our results indicated the presence of positive reaction in cytoplasm varied from weak to moderate in grade 3 & stage 4. Concerning the Flow cytometry technique our results indicated that the presence of correlation between the DNA and different stages of colon cancer.

Keywords: DNA-CK20, PCNA, P53, colon cancer

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Authors: Sana Abbasa, Shahzad Bhattiab, Nadir Khan

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Sargassum siliquastrum is brown seaweed with traditional claims for some medicinal properties. This research was done to investigate the methanol extract of S. siliquastrum for antiproliferative activity against human cervical cancer cell line, HeLa and its mode of cell death. From methylene blue assay, S. siliquastrum exhibited antiproliferative activity on HeLa cells with IC50 of 3.87 µg/ml without affecting non-malignant cells. Phase contrast microscopy indicated the confluency reduction in HeLa cells and changes on the cell shape. Nuclear staining with Hoechst 33258 displayed the formation of apoptotic bodies and fragmented nuclei. S. siliquastrum also induced early apoptosis event in HeLa cells as confirmed by FITC-Annexin V/propidium iodide staining by flow cytometry analysis. Cell cycle analysis indicated growth arrest of HeLa cells at G1/S phase. Protein study by flow cytometry indicated the increment of p53, slight increase of Bax and unchanged level of Bcl-2. In conclusion, S. siliquastrum demonstrated an antiproliferative activity in HeLa cell by inducing G1/S cell cycle arrest via p53-mediated pathway.

Keywords: sargassum siliquastrum, cervical cancer, P53, antiproleferation

Procedia PDF Downloads 578

Authors: Claude Takenga, Rolf-Dietrich Berndt, Erling Si, Markus Diem, Guohui Qiao, Melanie Gau, Michael Brandstoetter, Martin Kampel, Michael Dworzak

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In patients with acute lymphoblastic leukaemia (ALL), treatment response is increasingly evaluated with minimal residual disease (MRD) analyses. Flow Cytometry (FCM) is a fast and sensitive method to detect MRD. However, the interpretation of these multi-parametric data requires intensive operator training and experience. This paper presents a pipeline-software, as a ready-to-use FCM-based MRD-assessment tool for the daily clinical practice for patients with ALL. The new tool increases accuracy in assessment of FCM-MRD in samples which are difficult to analyse by conventional operator-based gating since computer-aided analysis potentially has a superior resolution due to utilization of the whole multi-parametric FCM-data space at once instead of step-wise, two-dimensional plot-based visualization. The system developed as a telemedical network reduces the work-load and lab-costs, staff-time needed for training, continuous quality control, operator-based data interpretation. It allows dissemination of automated FCM-MRD analysis to medical centres which have no established expertise for the benefit of an even larger community of diseased children worldwide. We established a telemedical network system for analysis and clinical follow-up and treatment monitoring of Leukaemia. The system is scalable and adapted to link several centres and laboratories worldwide.

Keywords: data security, flow cytometry, leukaemia, telematics platform, telemedicine

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Authors: P. Jost, L. Muckova, M. Matula, J. Pejchal, D. Jun, R. Stetina

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Sulfur mustard (bis(2-chlorethyl) sulfide) is highly toxic, chemical warfare agent that has been used in the past in several armed conflicts. Except for the skin, respiratory tract is one of the important routes of exposure. The elucidation and understanding of the mechanism of toxicity of SM have been effort intensive research. The multiple targets character of SM caused cellular damage resulted in activation of many different mechanisms which contribute to cellular response and participate in the final cytopathology effect. In our present work, we compared time-dependent changes in sulfur mustard exposed adult human lung fibroblasts NHLF and lung epithelial alveolar cell line A-549. Cell viability (MTT assay, Calcein-AM assay, and xCELLigence - real-time cell analysis), apoptosis (flow cytometry), mitochondrial membrane potential (Δψm, flow cytometry), reactive oxygen species induction (DC and cell cycle distribution (flow cytometry) were studied. We observed significantly decreased mitochondrial membrane potential and subsequent induction of apoptosis correlating with decreased cellular viability in the sulfur mustard exposed cells. In low concentrations, sulfur mustard-induced S-phase cell cycle arrest, on the other hand, high concentrations, cell cycle phase distribution of sulfur mustard exposed cells resembled cell cycle phase distribution of control group, which implies nonspecific cell cycle inhibition. Epithelial cells A-549 was found as more sensible to sulfur mustard toxicity. Acknowledgements: This work was supported by a long-term organization development plan Medical Aspects of Weapons of Mass Destruction of the Faculty of Military Health Sciences, University of Defence.

Keywords: apoptosis, cell cycle, cytotoxicity, sulfur mustard

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Authors: Aliaa M. Issa, Mahmoud N. ElRouby, Sahar A. S. Ahmad, Mahmoud M. El-Merzabani

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Sider honey is a type of honey produced by bees feeding on the nectar of Sider tree, Ziziphus spina-christi (L) Desf . Honey is an effective agent for preventing, inhibiting and treating the growth of human and animal cancer cell lines in vitro and in vivo. The aim of the present study was to evaluate the impact of different dilutions from crude Sider honey and different duration times of exposure on the growth of six tumor cell lines (human cervical cancer cell line, HeLa; human hepatocellular carcinoma cell line, HepG-2; human larynx carcinoma cell line, Hep-2; brain tumor cell line, U251) as well as one animal cancerous cell line (Ehrlich ascites carcinoma cells line, EAC) and one normal cell line, Homo sapiens, human, (WISH) CCL-25. Different concentrations and treatment durations with Sider honey were tested on the growth of several cancer cell lines types. Histopathological changes in the tumor masses, animal survival, apoptosis and necrosis of the used cancer cell lines (using flow cytometry) were evaluated. Sider honey was administers either to the tumor mass itself by intratumoral injection or via drinking water. One-way ANOVA test was used for the analysis of (the means + standard error) of the optical density obtained from the Elisa reader and flow cytometry. The study revealed that different concentrations of Sider honey affected the growth patterns of all the studied cancer cell lines as well as their histopathological changes, and it depended on the cell line nature and the concentration of honey used. It is obvious that the relative animal survival percentage (bearing Ehrlich ascites carcinoma, EAC cells) was proportionally increased with the increase in the used honey concentrations. The study of apoptosis and necrosis using the flow cytometry technique emphasized the viability results. In conclusion, Sider honey was effective as antitumor agent, in the used concentrations.

Keywords: antitumor, honey, sider, tumor cell lines

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Authors: Ankush M. Dewle, Suditi Bhattacharya, Prachi R. Abhang, Savita Datar, Ajay J. Jog, Rupesh K. Srivastava, Geetanjali Tomar

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Objectives: The aim of this study was to investigate the quality of mesenchymal stem cells (MSCs) obtained from ageing gingival tissues, in order to suggest their potential role in autologous stem cell therapy for old individuals. Methods: MSCs were isolated from gingival tissues of young (18-45 years) and old (above 45 years) donors by enzymatic digestion. MSCs were analysed for cfu-f, surface marker expression by flow-cytometry and multilineage differentiation potential. The angiogenic potential was compared in a chick embryo yolk sac membrane model. The aging and differentiation markers including SA-β-galactosidase and p21 respectively were analysed by staining and flow-cytometry analysis. Additionally, osteogenic markers such as glucocorticoid receptor (GR), vitamin D receptor (VDR) were measured by flow-cytometry and RT-qPCR was performed for quantification of osteogenic gene expression. Alizarin Red S and alkaline phosphatase (ALP) activity were also quantitated. Results: Gingival MSCs (GMSCs) from both the age groups were similar in their morphology and displayed cfu-f. They had similar expression of MSC surface markers and p21, comparable rate of proliferation and differentiated to all the four lineages. GMSCs from young donors had a higher adipogenic differentiation potential as compared to the old GMSCs. Moreover, these cells did not display a significant difference in ALP activity probably due to comparable expression of GR, VDR, and osteogenic genes. Conclusions: Ageing of GMSCs occurs at a much slower rate than stem cells from other sources. Thus we suggest GMSCs as an excellent candidate for autologous stem cell therapy in degenerative diseases of elderly individuals. Clinical Significance: GMSCs could help overcome the setbacks in clinical implementation of autologous stem cell therapy for regenerative medicine in all age group of patient.

Keywords: bone regeneration, cell therapy, senescence, stem cell

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Authors: Nivaldo Ferreira do Nascimento, Matheus Pereira-Santos, Nycolas Levy-Pereira, José Augusto Senhorini, George Shigueki Yasui, Laura Satiko Okada Nakaghi

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Tetraploid individuals, which could produce diploid gametes, can be used for production of 100% triploid fish. Therefore, the aim of this study was to develop a tetraploidization protocol for A. altiparanae. We tested the effect of heat shock (40 °C; 2 min) at 16, 18, 20, 22, 24 and 26 minutes post fertilization (mpf). Untreated eggs were used as control. After hatching, ploidy status of the larvae was checked by flow cytometry. No difference were observed for the hatching rate between all treatments (P = 0.5974). However, we observed an increase in the larval abnormality in the heat shock treatments, in special at 22 (82.17 ± 6.66%) 24 (78.31 ±7.28%) and 26 mpf (79.01 ± 7.85%) in comparison with the control group (12.87 ± 4.46%). No tetraploid was observed at 16 and 18 mpf. The higher number of tetraploid individuals (52/55) was observed at 26 mpf. Our results showed that high percentages of tetraploids are obtained by heat shock (40°C; 2min) at 26 mpf, which could enable the mass production of triploid individuals in A. altiparanae.

Keywords: chromosome manipulation, polyploidy, flow cytometry, tetraploidization

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Authors: Aftab Ali

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Cryopreservation of semen is one of the key factors for a successful breeding business along with other factors. To achieve high fertility in boar, one should know about spermatozoa response to different treatments proceeds during cryopreservation. The running project is highly focused on cryopreservation and its effects on sperm quality parameters in both boar and bull semen. Semen sample from A, B, C, and D, were subjected to different thawing conditions and were analyzed upon different treatments in the study. Parameters like sperm cell motility, viability, acrosome, DNA integrity, and phospholipase C zeta were detected by different established methods. Different techniques were used to assess different parameters. Motility was detected using computer assisted sperm analysis, phospholipase C zeta using luminometry while viability, acrosome integrity, and DNA integrity were analyzed using flow cytometry. Thawing conditions were noted to have an effect on sperm quality parameters with motility being the most critical parameter. The results further indicated that the most critical step during cryopreservation of boar semen is when sperm cells are subjected to freezing and thawing. The findings of the present study provide insight that; boar semen cryopreservation is still suboptimal in comparison to bull semen cryopreservation. Thus, there is a need to conduct more research to improve the fertilizing potential of cryopreserved boar semen.

Keywords: cryopreservation, computer assisted sperm, flow cytometry, luminometry

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Authors: Aliya Sekenova, Vyacheslav Ogay

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The derivation of human induced pluripotent stem cells (iPSCs) from somatic cells by direct reprogramming opens wide perspectives in the regenerative medicine. It means the possibility to develop the personal and, consequently, any immunologically compatible cells for applications in cell-based therapy. The purpose of our study was to develop the technology for the production of NK cells from T cell-derived induced pluripotent stem cells (TiPSCs) for subsequent application in adoptive cancer immunotherapy. Methods: In this study iPSCs were derived from peripheral blood T cells using Sendai virus vectors expressing Oct4, Sox2, Klf4 and c-Myc. Pluripotent characteristics of TiPSCs were examined and confirmed with alkaline phosphatase staining, immunocytochemistry and RT-PCR analysis. For NK cell differentiation, embryoid bodies (EB) formed from (TiPSCs) were cultured in xenogeneic serum-free medium containing human serum, IL-3, IL-7, IL-15, SCF, FLT3L without using M210-B4 and AFT-024 stromal feeder cells. After differentiation, NK cells were characterized with immunofluorescence analysis, flow cytometry and cytotoxicity assay. Results: Here, we for the first time demonstrate that TiPSCs can effectively differentiate into functionally active NK cells without M210-B4 and AFT-024 xenogeneic stroma cells. Immunofluorescence and flow cytometry analysis showed that EB-derived cells can differentiate into a homogeneous population of NK cell expressing high levels of CD56, CD45 and CD16 specific markers. Moreover, these cells significantly express killing activation receptors such as NKp44 and NKp46. In the comparative analysis, we observed that NK cells derived using feeder-free culture system have more high killing activity against K-562 tumor cells, than NK cells derived by feeder-dependent method. Thus, we think that our obtained data will be useful for the development of large-scale production of NK cells for translation into cancer immunotherapy.

Keywords: induced pluripotent stem cells, NK cells, T cells, cell diffentiation, feeder cell-free culture system

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Authors: Sharmi Mukherjee, Anindita Chakraborty

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Cellular irradiation incites complex responses including arrest of cell cycle progression. This article accentuates the effects of radiation on cell cycle status of radiation non-targeted cells. Human Hepatoma HepG2 cells were exposed to increasing doses of γ radiations (1, 2, 4, 6 Gy) and their cell culture media was transferred to non-targeted HepG2 cells cultured in other Petri plates. These radiation non-targeted cells cultured in the ICCM (Irradiated cell conditioned media) were the bystander cells on which cell cycle analysis was performed using flow cytometry. An apparent decrease in the distribution of bystander cells at G0/G1 phase was observed with increased radiation doses upto 4 Gy representing a linear relationship. This was accompanied by a gradual increase in cellular distribution at G2/M phase. Interestingly the number of cells in G2/M phase at 1 and 2 Gy irradiation was not significantly different from each other. However, the percentage of G2 phase cells at 4 and 6 Gy doses were significantly higher than 2 Gy dose indicating the IC50 dose to be between 2 and 4 Gy. Cell cycle arrest is an indirect indicator of genotoxic damage in cells. In this study, bystander stress signals through the cell culture media of irradiated cells disseminated the radiation induced DNA damages in the non-targeted cells which resulted in arrest of the cell cycle progression at G2/M phase checkpoint. This implies that actual radiation biological effects represent a penumbra with effects encompassing a larger area than the actual beam. This article highlights the existence of genotoxic damages as bystander effects of γ rays in human Hepatoma cells by cell cycle analysis and opens up avenues for appraisal of bystander stress communications between tumor cells. Contemplation of underlying signaling mechanisms can be manipulated to maximize damaging effects of radiation with minimum dose and thus has therapeutic applications.

Keywords: bystander effect, cell cycle, genotoxic damage, hepatoma

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Authors: Ainur Mukhambetova, Miras Karzhauov, Vyacheslav Ogay

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Introduction: Mesenchymal stem cells (MSCs) have the capacity to be differentiated into several cell lineages and are a promising source for cell therapy and tissue engineering. However, currently most MSCs culturing protocols use media supplemented with fetal bovine serum (FBS), which limits their application in clinic due to the possibility of zoonotic infections, contamination and immunological reactions. Consequently, formulating effective serum free culture medium becomes one of the important problems in contemporary cell biotechnology. Objectives: The aim of this study was to define an optimal serum-free medium for culturing of periosteum derived MSCs. Materials and methods: The MSCs were extracted from human periosteum and transferred to the culture flasks pretreated with CELLstart™. Immunophenotypic characterization, proliferation and in vitro differentiation of cells grown on STEM PRO® MSC SFM were compared to the cells cultured in the standard FBS containing media. Chromosome analysis and flow cytometry were also performed. Results: We have shown that cells were grown on STEM PRO® MSC SFM retained all the morphological, immunophenotypic (CD73, CD90, CD105, vimentin and Stro-1) and cell differentiation characteristics specific to MSCs. Chromosome analysis indicated no anomalies in the chromosome structure. Flow cytometry showed a high expression of cell adhesion molecules CD44 (98,8%), CD90 (97,4%), CD105 (99,1%). In addition, we have shown that cell is grown on STEM PRO® MSC SFM have higher proliferation capacity compared to cell expanded on standard FBS containing the medium. Conclusion: We have shown that STEM PRO® MSC SFM is optimal for culturing periosteum derived human MSCs which subsequently can be safely used in cell therapy.

Keywords: cell technologies, periosteum-derived MSCs, regenerative medicine, serum-free medium

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Authors: B. Gonzalez-Yebra, H. Barajas, P. Palomares, M. Hernandez, O. Torres, M. Ayala, A. L. González, G. Vazquez-Ortiz, M. L. Guzman

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Leukemia arises by molecular alterations of the normal hematopoietic stem cell (HSC) transforming it into a leukemic stem cell (LSC) with high cell proliferation, self-renewal, and cell differentiation. Chronic myeloid leukemia (CML) originates from an LSC-leading to elevated proliferation of myeloid cells and acute lymphoblastic leukemia (ALL) originates from an LSC development leading to elevated proliferation of lymphoid cells. In both cases, LSC can be identified by multicolor flow cytometry using several antibodies. However, to date, LSC levels in peripheral blood (PB) are not established well enough in ALL and CML patients. On the other hand, the detection of the minimal residue disease (MRD) in leukemia is mainly based on the identification of the mRNA bcr-abl gene in CML patients and some other genes in ALL patients. There is no a properly biomarker to detect MDR in both types of leukemia. The objective of this study was to determine mRNA bcr-abl and the percentage of LSC in peripheral blood of patients with CML and ALL and identify a possible association between the amount of LSC in PB and clinical data. We included in this study 19 patients with Leukemia. A PB sample was collected per patient and leukocytes were obtained by Ficoll gradient. The immunophenotype for LSC CD34+ was done by flow cytometry analysis with CD33, CD2, CD14, CD16, CD64, HLA-DR, CD13, CD15, CD19, CD10, CD20, CD34, CD38, CD71, CD90, CD117, CD123 monoclonal antibodies. In addition, to identify the presence of the mRNA bcr-abl by RT-PCR, the RNA was isolated using TRIZOL reagent. Molecular (presence of mRNA bcr-abl and LSC CD34+) and clinical results were analyzed with descriptive statistics and a multiple regression analysis was performed to determine statistically significant association. In total, 19 patients (8 patients with ALL and 11 patients with CML) were analyzed, 9 patients with de novo leukemia (ALL = 6 and CML = 3) and 10 under treatment (ALL = 5 and CML = 5). The overall frequency of mRNA bcr-abl was 31% (6/19), and it was negative in ALL patients and positive in 80% in CML patients. On the other hand, LSC was determined in 16/19 leukemia patients (%LSC= 0.02-17.3). The Novo patients had higher percentage of LSC (0.26 to 17.3%) than patients under treatment (0 to 5.93%). The amount of LSC was significantly associated with the amount of LSC were: absence of treatment, the absence of splenomegaly, and a lower number of leukocytes, negative association for the clinical variables age, sex, blasts, and mRNA bcr-abl. In conclusion, patients with de novo leukemia had a higher percentage of circulating LSC than patients under treatment, and it was associated with clinical parameters as lack of treatment, absence of splenomegaly and a lower number of leukocytes. The mRNA bcr-abl detection was only possible in the series of patients with CML, and molecular detection of LSC could be identified in the peripheral blood of all leukemia patients, we believe the identification of circulating LSC may be used as biomarker for the detection of the MRD in leukemia patients.

Keywords: stem cells, leukemia, biomarkers, flow cytometry

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Authors: M. H. Moreira da Silva, L. Valadao, F. Moreira da Silva

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In the present study, the fertilizing capacity of bovine spermatozoa was evaluated before and after its cryopreservation. For this, the testicles of 100 bulls slaughtered on Terceira Island were dissected, the epididymal tails were separated, and semen was recovered by the flotation method and then evaluated by phase contrast microscopy and by flow cytometry. For phase contrast microscopy, a drop of semen was used to evaluate the percentage of motile spermatozoa (from 0 to 100%) and motility (from 0 to 5). After determining the concentration and the abnormal forms, semen was diluted to a final concentration of 50 x 106 spz/ml and evaluated by flow cytometer for membrane and acrosome integrity using the conjugation of fluorescent probes propidium iodide (PI) and Arachis hypogea agglutinin (FITC-PNA). Freezing was carried out in a programmable semen freezer, using 0.25 ml straws, in a total of 20 x 106 viable sperm per straw with glycerol as a cryoprotectant in a final concentration of 0.58 M. It was observed that, on average, a total of 7.25 ml of semen was collected from each bull. The viability and vitality rates were respectively 83.22 ± 7.52% and 3.8 ± 0.4 before freezing, decreasing to 58.81 ± 11.99% and 3.6 ± 0.6, respectively, after thawing. Regarding cytoplasmic droplets, it was observed that a high percentage of spermatozoa had medial cytoplasmic droplets (38.47%), with only 3.32% and 0.15% presenting proximal and distal cytoplasmic drops, respectively. By flow cytometry, it was observed that before freezing, the percentage of sperm with the damaged plasma membrane and intact acrosome was 3.61 ± 0.99%, increasing slightly to 4.21 ± 1.86% after cryopreservation (p<0.05). Regarding spermatozoa with damaged plasma membrane and acrosome, the percentage before freezing was 3.37±1.87%, increasing to 4.34 ±1.16% after thawing, and no significant differences were observed between these two values. For the percentage of sperm with the intact plasma membrane and damaged acrosome, this value was 2.04 ± 2.34% before freezing, decreasing to 0.89 ± 0.48% after thawing (p<0.05). The percentage of sperm with the intact plasma membrane and acrosome before freezing was 90.99±2.75%, with a slight decrease to 90.57±3.15% after thawing (p<0.05). From this study, it can be clearly concluded that, after the slaughtering of bulls, the spermatozoa can be recovered from the epididymis and cryopreserved, maintaining an excellent rate of sperm viability and quality after thawing.

Keywords: bovine semen, epididymis, cryopreservation, fertility assessment

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Authors: Sweta Agrawal, Sachin Chaugule, Gargi Rane, Shashank More, Madhavi Indap

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Angiogenesis and metastasis are two of the most important hallmarks of cancer. Hence, most of the cancer therapies nowadays are multi-targeted so as to reduce resistance and have better efficacy. As synthetic molecules arise with a burden of their toxicities and side-effects, more and more research is being focussed on exploiting the vast natural resources of drugs, in the form of plants and animals. Although, the idea of using marine organisms as a source of pharmaceuticals is not new, the pace at which marine drugs are being discovered, has definitely up surged! In the present study, we have assessed the anti-angiogenic and in vitro anti-metastatic activity of aqueous fraction from the extract of marine gastropod Euchelus asper. The soft body of Euchelus Asper was extracted with methanol and named EAME. Partition chromatography of EAME gave three fractions EAME I, II and III. Biochemical analysis revealed the presence of proteins in EAME III. Preliminary analysis had revealed the anti-angiogenic activity was exhibited by EAME III out of the three fractions. Hereafter, EAME III (concentration 25µg/ml-400µg/ml) was tested on chick chorioallantoic membrane (CAM) model for the detailed analysis of its potential anti-angiogenic effect. In vitro testing of the fraction (concentration 0.25µg/ml - 1µg/ml), involved cytotoxicity by SRB assay, cell cycle analysis by flow cytometry and anti-proliferative effect by scratch wound healing assay on A549 lung carcinoma cells. Apart from this, a portion of treated CAM as well as conditioned medium from treated A549 were subjected to gelatin zymography for assessment of matrix metalloproteinases MMP-2 and MMP-9 levels. Our results revealed that EAME III exhibited significant anti-angiogenic activity on CAM which was also supported by histological observations. During histological studies of CAM, it was found that EAME III caused reduction in angiogenesis by altering the extracellular matrix of the CAM membrane. In vitro analysis disclosed that EAME III exhibited moderate cytotoxic effect on A549 cells and its effect was not dose-dependent. The results of flow cytometry confirmed that EAME III caused cell cycle arrest in A549 cell line as almost all of the treated cells were found in G1 phase. Further, the migration and proliferation of A549 was significantly reduced by EAME III as observed from the scratch wound assay. Moreover, Gelatin zymography analysis revealed that EAME III caused suppression of MMP-2 in CAM membrane and reduced MMP-9 and MMP-2 expression in A549 cells. This verified that the anti-angiogenic and anti-metastatic effects of EAME III were correlated with the suppression of MMP-2 and -9. To conclude, EAME III shows dual anti-tumour action by reducing angiogenesis and exerting anti-metastatic effect on lung cancer cells, thus it has the potential to be used as an anti-cancer agent against lung carcinoma.

Keywords: angiogenesis, anti-cancer, marine drugs, matrix metalloproteinases

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Authors: Husham Bayazed

Abstract:

Immune responses following surgical trauma play a pivotal role in predicting postoperative outcomes from healing and recovery to postoperative complications. Postoperative complications, including infections and protracted recovery, occur in a significant number of about 300 million surgeries performed annually worldwide. Complications cause personal suffering along with a significant economic burden on the healthcare system in any community. The accurate prediction of postoperative complications and patient-targeted interventions for their prevention remain major clinical provocations. Recent Findings: Recent studies are focusing on immune dysregulation mechanisms that occur in response to surgical trauma as a key determinant of postoperative complications. Antecedent studies mainly were plunging into the detection of inflammatory plasma markers, which facilitate in providing important clues regarding their pathogenesis. However, recent Single-cell technologies, such as mass cytometry or single-cell RNA sequencing, have markedly enhanced our ability to understand the immunological basis of postoperative immunological trauma complications and to identify their prognostic biological signatures. Summary: The advent of proteomic technologies has significantly advanced our ability to predict the risk of postoperative complications. Multiomic modeling of patients' immune states holds promise for the discovery of preoperative predictive biomarkers and providing patients and surgeons with information to improve surgical outcomes. However, more studies are required to accurately predict the risk of postoperative complications in individual patients.

Keywords: immune dysregulation, postoperative complications, surgical trauma, flow cytometry

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Authors: Kumari Rekha, Mathur K Dhananjay, Maheshwari Deepanshu, Nautiyal Nidhi, Shubham Smriti, Laal Deepika, Sinha Swati, Kumar Anupam, Biswas Subhrajit, Shiv Kumar Sarin

Abstract:

Background: Mesenchymal stem cells are multipotent stem cells derived from mesoderm and are used for therapeutic purposes because of their self-renewal, homing capacity, Immunomodulatory capability, low immunogenicity and mitochondrial transfer signaling. MSCs have the ability to regulate the mechanism of both innate as well as adaptive immune responses through the modulation of cellular response and the secretion of inflammatory mediators. Different sources of MSC are UC MSC, BM MSC, Dental Pulp, and Adipose MSC. The most frequent source used is umbilical cord tissue due to its being easily available and free of limitations of collection procedures from respective hospitals. The immunosuppressive role of MSCs is particularly interesting for clinical use since it confers resistance to rejection by the host immune response. Methodology: In this study, T helper cells (TH4), Cytotoxic T cells (CD-8), immunoregulatory cells (CD25 +FOXP3+) are compared from isolated MSC from three different methods, UC Dissociation Kit (Miltenyi), Explant Culture and Collagenase Type-IV. To check the immunomodulatory property, these MSCs were seeded with PBMC(Coculture) in CD3 coated 24 well plates. Cd28 antibody was added in coculture for six days. The coculture was analyzed in FACS Verse flow cytometry. Results: From flow cytometry analysis of coculture, it found that All over T helper cells (CD4+) number p<0.0264 increases in (All Enzymes) MSC rather than explant MSC(p>0.0895) as compared to Collagenase(p>0.7889) in a coculture of Activated T cell and Mesenchymal Stem Cell. Similar T reg cells (CD25+, FOXP3+) expression p<0.0234increases in All Enzymes), decreases in Explant and Collagenase. Experiments have shown that MSCs can also directly prevent the cytotoxic activity of CD8 lymphocytes mainly by blocking their proliferation rather than by inhibiting the cytotoxic effect. And promoting the t-reg cells, which helps in the mediation of immune response in various diseases. Conclusion: MSC suppress Cytotoxic CD8 T cell and Enhance immunoregulatory T reg (CD4+, CD25+, FOXP3+) Cell expression. Thus, MSC maintains a proper balance(ratio) between CD4 T cells and Cytotoxic CD8 T cells.

Keywords: MSC, disease, T cell, T regulatory

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Authors: Dace Grauda, Inta Belogrudova, Alexei Katashev, Linda Lancere, Isaak Rashal

Abstract:

The goal of study was the elaboration of easy applicable flow cytometry method for detection of influence of abiotic stress factors on plants, which could be useful for detection of environmental stresses in urban areas. The lime tree Tillia vulgaris H. is a popular tree species used for urban landscaping in Europe and is one of the main species of street greenery in Riga, Latvia. Tree decline and low vitality has observed in the central part of Riga. For this reason lime trees were select as a model object for the investigation. During the period of end of June and beginning of July 12 samples from different urban environment locations as well as plant material from a greenhouse were collected. BD FACSJazz® cell sorter (BD Biosciences, USA) with flow cytometer function was used to test viability of plant cells. The method was based on changes of relative fluorescence intensity of cells in blue laser (488 nm) after influence of stress factors. SpheroTM rainbow calibration particles (3.0–3.4 μm, BD Biosciences, USA) in phosphate buffered saline (PBS) were used for calibration of flow cytometer. BD PharmingenTM PBS (BD Biosciences, USA) was used for flow cytometry assays. The mean fluorescence intensity information from the purified cell suspension samples was recorded. Preliminary, multiple gate sizes and shapes were tested to find one with the lowest CV. It was found that low CV can be obtained if only the densest part of plant cells forward scatter/side scatter profile is analysed because in this case plant cells are most similar in size and shape. The young pollen cells in one nucleus stage were found as the best for detection of influence of abiotic stress. For experiments only fresh plant material was used– the buds of Tillia vulgaris with diameter 2 mm. For the cell suspension (in vitro culture) establishment modified protocol of microspore culture was applied. The cells were suspended in the MS (Murashige and Skoog) medium. For imitation of dust of urban area SiO2 nanoparticles with concentration 0.001 g/ml were dissolved in distilled water. Into 10 ml of cell suspension 1 ml of SiO2 nanoparticles suspension was added, then cells were incubated in speed shaking regime for 1 and 3 hours. As a stress factor the irradiation of cells for 20 min by UV was used (Hamamatsu light source L9566-02A, L10852 lamp, A10014-50-0110), maximum relative intensity (100%) at 365 nm and at ~310 nm (75%). Before UV irradiation the suspension of cells were placed onto a thin layer on a filter paper disk (diameter 45 mm) in a Petri dish with solid MS media. Cells without treatment were used as a control. Experiments were performed at room temperature (23-25 °C). Using flow cytometer BS FACS Software cells plot was created to determine the densest part, which was later gated using oval-shaped gate. Gate included from 95 to 99% of all cells. To determine relative fluorescence of cells logarithmic fluorescence scale in arbitrary fluorescence units were used. 3x103 gated cells were analysed from the each sample. The significant differences were found among relative fluorescence of cells from different trees after treatment with SiO2 nanoparticles and UV irradiation in comparison with the control.

Keywords: flow cytometry, fluorescence, SiO2 nanoparticles, UV irradiation

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Authors: Nahid Eskandari, Marjan Ghagozolo, Ehsan Almasi

Abstract:

Introduction: Silymarin, as a polyphenolic flavonoid derived from milk thistle (Silybum marianum), is known to have antioxidant, immunomodulatory, antiproliferative, antifibrotic, and antiviral effects. The goal of this study was to determine immunosuppressive effect of Silymarin on proliferation and apoptosis of human T cells in comparison with Rapamycin and FK506. Methods: Peripheral Blood Mononuclear Cells (PBMCs) from healthy individuals were activated with Con A (5µg/ml) and then treated with Silymarin, Rapamycin and FK506 in various concentrations (0.001, 0.01, 0.1, 1, 10,100 and 200M) for 5 days. PBMCs were examined for proliferation using CFSE assay and the concentration that inhibited 50% of the cell proliferation (IC50) was determined for each treatment. For apoptosis assay using flow cytometry, PBMCs were activated with Con A and treated with IC50 dose of Silymarin, Rapamycin and FK506 for 5 days, then cell apoptosis was analysed by FITC-annexin V/PI staining and flow cytometry. The effects of Silymarin, Rapamycin and FK506 on the activation of PARP (poly ADP ribose polymerase) pathway in PBMCs stimulated with Con A and treated with IC50 dose of drugs for 5 days evaluated using the PathScan cleaved PARP sandwich ELISA kit. Results: This study showed that Silymarin had the ability to inhibit T cell proliferation in vitro. Moreover, our results indicated that 100 μM (P < 0.001) and 200 μM (P < 0.001) of Silymarin has more inhibitory effect on T cells proliferation than FK506 and Rapamycin. Our data showed that the effective doses (IC50) of Silymarin, FK506 and Rapamycin were 3×10-5 µM, 10-8 µM and 10-6 µM respectively. Data showed that the inhibitory effect of Silymarin, FK506 and Rapamycin on T cell proliferation was not due to cytotoxicity and none of these drugs at IC50 concentration had not affected the level of cleaved PARP. Conclusion: Silymarin could be a good candidate for immunosuppressive therapy for certain medical conditions with superior efficacy and lesser toxicity in comparison with other immunosuppressive drugs.

Keywords: silymarin, immunosuppressive effect, rapamycin, immunology

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Authors: Melanie Ostermann, Eivind Birkeland, Ying Xue, Alexander Sauter, Mihaela R. Cimpan

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Background: New reliable and robust techniques to assess biological effects of nanomaterials (NMs) in vitro are needed to speed up safety analysis and to identify key physicochemical parameters of NMs, which are responsible for their acute cytotoxicity. The central aim of this study was to validate and evaluate the applicability and reliability of an impedance-based flow cytometry (IFC) technique for the high-throughput screening of NMs. Methods: Eight inorganic NMs from the European Commission Joint Research Centre Repository were used: NM-302 and NM-300k (Ag: 200 nm rods and 16.7 nm spheres, respectively), NM-200 and NM- 203 (SiO₂: 18.3 nm and 24.7 nm amorphous, respectively), NM-100 and NM-101 (TiO₂: 100 nm and 6 nm anatase, respectively), and NM-110 and NM-111 (ZnO: 147 nm and 141 nm, respectively). The aim was to assess the biological effects of these materials on human monoblastoid (U937) cells. Dispersions of NMs were prepared as described in the NANOGENOTOX dispersion protocol and cells were exposed to NMs at relevant concentrations (2, 10, 20, 50, and 100 µg/mL) for 24 hrs. The change in electrical impedance was measured at 0.5, 2, 6, and 12 MHz using the IFC AmphaZ30 (Amphasys AG, Switzerland). A traditional toxicity assay, Trypan Blue Dye Exclusion assay, and dark-field microscopy were used to validate the IFC method. Results: Spherical Ag particles (NM-300K) showed the highest toxic effect on U937 cells followed by ZnO (NM-111 ≥ NM-110) particles. Silica particles were moderate to non-toxic at all used concentrations under these conditions. A higher toxic effect was seen with smaller sized TiO2 particles (NM-101) compared to their larger analogues (NM-100). No interferences between the IFC and the used NMs were seen. Uptake and internalization of NMs were observed after 24 hours exposure, confirming actual NM-cell interactions. Conclusion: Results collected with the IFC demonstrate the applicability of this method for rapid nanotoxicity assessment, which proved to be less prone to nano-related interference issues compared to some traditional toxicity assays. Furthermore, this label-free and novel technique shows good potential for up-scaling in directions of an automated high-throughput screening and for future NM toxicity assessment. This work was supported by the EC FP7 NANoREG (Grant Agreement NMP4-LA-2013-310584), the Research Council of Norway, project NorNANoREG (239199/O70), the EuroNanoMed II 'GEMN' project (246672), and the UH-Nett Vest project.

Keywords: cytotoxicity, high-throughput, impedance, nanomaterials

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Authors: Reza Fardid, Aliyeh Alipour

Abstract:

Introduction: Coronary heart disease (CHD) is the most common and deadliest diseases. A coronary angiography is an important tool for the diagnosis and treatment of this disease. Because angiography is performed by exposure to ionizing radiation, it can lead to harmful effects. Ionizing radiation induces double-stranded breaks in DNA, which is a potentially life-threatening injury. The purpose of the present study is an investigation of the phosphorylation of histone H2AX in the location of the double-stranded break in Peripheral blood lymphocytes as an indication of Biological effects of radiation on angiography patients. Materials and Methods: This method is based on measurement of the phosphorylation of histone (gamma-H2AX, gH2AX) level on serine 139 after formation of DNA double-strand break. 5 cc of blood from 24 patients with angiography were sampled before and after irradiation. Blood lymphocytes were removed, fixed and were stained with specific ϒH2AX antibodies. Finally, ϒH2AX signal as an indicator of the double-strand break was measured with Flow Cytometry Technique. Results and discussion: In all patients, an increase was observed in the number of breaks in double-stranded DNA after irradiation (20.15 ± 14.18) compared to before exposure (1.52 ± 0.34). Also, the mean of DNA double-strand break was showed a linear correlation with DAP. However, although induction of DNA double-strand breaks associated with radiation dose in patients, the effect of individual factors such as radiosensitivity and regenerative capacity should not be ignored. If in future we can measure DNA damage response in every patient angiography and it will be used as a biomarker patient dose, will look very impressive on the public health level. Conclusion: Using flow cytometry readings which are done automatically, it is possible to detect ϒH2AX in the number of blood cells. Therefore, the use of this technique could play a significant role in monitoring patients.

Keywords: coronary angiography, DSB of DNA, ϒH2AX, ionizing radiation

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Authors: Bader Alruwaili, Surajit Ray

Abstract:

Finite mixtures are a flexible and powerful tool that can be used for univariate and multivariate distributions, and a wide range of research analysis has been conducted based on the multivariate normal mixture and multivariate of a t-mixture. Determining the number of modes is an important activity that, in turn, allows one to determine the number of homogeneous groups in a population. Our work currently being carried out relates to the study of the modality of the skew normal distribution in the univariate and multivariate cases. For the skew normal distribution, the aims are associated with studying the modality of the skew normal distribution and providing the ridgeline, the ridgeline elevation function, the $\Pi$ function, and the curvature function, and this will be conducive to an exploration of the number and location of mode when mixing the two components of skew normal distribution. The subsequent objective is to apply these results to the application of real world data sets, such as flow cytometry data.

Keywords: mode, modality, multivariate skew normal, finite mixture, number of mode

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Authors: Mashitoh Abd Rahman, Najihah Mohd Hashim, Faiqah Ramli, Syam Mohan, Noraziah Nordin, Hamed Karimian, Hapipah Mohd Ali

Abstract:

Ovarian cancer is the most lethal gynecological malignancies. HME5, a prenylflavanoid has been isolated from local medicinal plant. This compound has been reported to possess a broad spectrum of biological activities including anticancer property. However, the potential of HME5 as an antiproliferative and cytotoxic agent on an ovarian cancer cells has not yet been investigated. In this present study, we examined the antiproliferative and cytotoxic effect of HME5 on Caov-3 (Human Ovarian Adenocarcinoma) cell line by using 3-[4,5-dimethylthizol-2-y]-2,5-diphenyltetrazolium bromide (MTT) assay, Acridine orange and propidium Iodide (AOPi) and cell cycle analysis study. HME5 has shown to inhibit Caov-3 in a time-dependent manner with the IC50 values of 5µg/ml, 2µg/ml and 1µg/ml after 24h, 48h and 72h treatment, respectively. Morphological study from AOPi analysis showed that HME5 induced apoptosis after 24 and 48h post-treatment. Nevertheless, HME5 exhibited cell cycle arrest at G1 phase as indicated in flow cytometry cell cycle profiling. In conclusion, HME5 inhibited proliferation of Caov-3 through induction of apoptosis and cell cycle arrest at G1 phase.

Keywords: apoptosis, prenylflavanoid, ovarian cancer, HME5

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Authors: Soumya S., Manju Nidagodu Jayakumar, Vellore Kannan Gopinath

Abstract:

Dental pulp inflammation resulting from dental caries often leads to a pathologic condition known as irreversible pulpitis and the currently managed by root canal treatment. Extirpation of the entire pulp tissue is done during this procedure, and the canal space is filled with synthetic materials. Recent studies in the stem cell biology state that some portion of the irreversibly inflamed pulp tissue could be viable with progenitor cells, having the properties similar to that of Mesenchymal stem cells. Hence, we aim to isolate Dental Pulp Stem Cells (DPSCs) from patients diagnosed with severe irreversible pulpitis and characterize the cells for the MSC specific markers. The pulp tissue was collected from the dental clinic and subjected to collagenase/dispase digestion. The isolated cells were expanded in culture, and the phenotypic characterization was done using flow cytometry. MSC specific markers such as CD-90, CD-73, and CD-105 were analysed along with negative markers such as CD-14 and CD-45. The isolated cells expressed positive expression for CD markers with CD90 and CD105 ( > 95%) and CD73 (19%). The cells did not express the negative markers CD-14 and CD-45. The commercially available DPSCs from vital extracted teeth, preferably molar/wisdom teeth with large pulp cavity or incomplete root growth in young patients (aged 15-30 years) showed more than 90% expression for all the CD markers such as CD-90, 73 and 105, whereas negative for CD-14 and CD-45. The DPSCs isolated from inflamed pulp tissue showed a less expression for CD-73 compared to the commercially available DPSCs whereas, as the other two markers were found to show similar percentage of positive expression. This could be attributed to the fact that the pulp population is very heterogeneous and we used the pooled tissue from different patients. Hence the phenotypic characterization and comparison with the commercially available DPSCs proved that the inflamed pulp tissue is a good source of MSC like cells which can be utilized further for regenerative application.

Keywords: collagenase/dispase, dental pulp stem cells, flow cytometry, irreversible pulpitis

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Authors: G. Cunningham, E. Cantor, X. Wu, F. Shen, G. Jiang, S. Philips, C. Bales, Y. Xiao, T. R. Cummins, J. C. Fehrenbacher, B. P. Schneider

Abstract:

Background: Taxane-induced peripheral neuropathy (TIPN) is the most devastating survivorship issue for patients receiving therapy. Dose reductions due to TIPN in the curative setting lead to inferior outcomes for African American patients, as prior research has shown that this group is more susceptible to developing severe neuropathy. The mechanistic underpinnings of TIPN, however, have not been entirely elucidated. While it would be appealing to use primary tissue to study the development of TIPN, procuring nerves from patients is not realistically feasible, as nerve biopsies are painful and may result in permanent damage. Therefore, our laboratory has investigated paclitaxel-induced neuronal morphological and molecular changes using an ex vivo model of human-induced pluripotent stem cell (iPSC)-derived neurons. Methods: iPSCs are undifferentiated and endlessly dividing cells that can be generated from a patient’s somatic cells, such as peripheral blood mononuclear cells (PBMCs). We successfully reprogrammed PBMCs into iPSCs using the Erythroid Progenitor Reprograming Kit (STEMCell Technologiesᵀᴹ); pluripotency was verified by flow cytometry analysis. iPSCs were then induced into neurons using a differentiation protocol that bypasses the neural progenitor stage and uses selected small-molecule modulators of key signaling pathways (SMAD, Notch, FGFR1 inhibition, and Wnt activation). Results: Flow cytometry analysis revealed expression of core pluripotency transcription factors Nanog, Oct3/4 and Sox2 in iPSCs overlaps with commercially purchased pluripotent cell line UCSD064i-20-2. Trilineage differentiation of iPSCs was confirmed with immunofluorescent imaging with germ-layer-specific markers; Sox17 and ExoA2 for ectoderm, Nestin, and Pax6 for mesoderm, and Ncam and Brachyury for endoderm. Sensory neuron markers, β-III tubulin, and Peripherin were applied to stain the cells for the maturity of iPSC-derived neurons. Patch-clamp electrophysiology and calcitonin gene-related peptide (CGRP) release data supported the functionality of the induced neurons and provided insight into the timing for which downstream assays could be performed (week 4 post-induction). We have also performed a cell viability assay and fluorescence-activated cell sorting (FACS) using four cell-surface markers (CD184, CD44, CD15, and CD24) to select a neuronal population. At least 70% of the cells were viable in the isolated neuron population. Conclusion: We have found that these iPSC-derived neurons recapitulate mature neuronal phenotypes and demonstrate functionality. Thus, this represents a patient-derived ex vivo neuronal model to investigate the molecular mechanisms of clinical TIPN.

Keywords: chemotherapy, iPSC-derived neurons, peripheral neuropathy, taxane, paclitaxel

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Authors: K. Santacruz-Gomez, E. Silva-Campa, S. Álvarez-García, V. Mata-Haro, D. Soto-Puebla, M. Pedroza-Montero

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Blood gamma irradiation is the only available method to prevent transfusion-associated graft versus host disease (TA-GVHD). However, when blood is irradiated, determine blood shelf time is crucial. Non-irradiated blood has a self-time from 21 to 35 days when is preserved with an anticoagulated solution and stored at 4°C. During their storage, red blood cells (RBC) undergo a series of biochemical, biomechanical and molecular changes involving what is known as storage lesion (SL). SL include loss of structural integrity of RBC, a decrease of 2,3-diphosphatidylglyceric acid levels, and an increase of both ion potassium concentration and hemoglobin (Hb). On the other hand, Atomic force Microscopy (AFM) represents a versatile tool for a nano-scale high-resolution topographic analysis in biological systems. In order to evaluate SL in irradiated and non-irradiated blood, RBC topography and morphometric parameters were obtained from an AFM XE-BIO system. Cell viability was followed using flow cytometry. Our results showed that early markers as nanoscale roughness, allow us to evaluate blood quality since another perspective.

Keywords: AFM, blood γ-irradiation, roughness, storage lesion

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Authors: Michelle C. S. Azevedo, Priscila M. Colavite, Carolina F. Francisconi, Ana P. Trombone, Gustavo P. Garlet

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VIP (vasoactive intestinal peptide) know as a potential protective factor in the view of its marked immunosuppressive properties. In this work, we investigated a possible association of VIP with the clinical status of experimental periapical granulomas and the association with expression markers in the lesions potentially associated with periapical lesions pathogenesis. C57BL/6WT mice were treated or not with recombinant VIP. Animals with active/progressive (N=40), inactive/stable (N=70) periapical granulomas and controls (N=50) were anesthetized and the right mandibular first molar was surgically opened, allowing exposure of dental pulp. Endodontic pathogenic bacterial strains were inoculated: Porphyromonas gingivalis, Prevotella nigrescens, Actinomyces viscosus, and Fusobacterium nucleatum subsp. polymorphum. The cavity was not sealed after bacterial inoculation. During lesion development, animals were treated or not with recombinant VIP 3 days post infection. Animals were killed after 3, 7, 14, and 21 days of infection and the jaws were dissected. The extraction of total RNA from periodontal tissues was performed and the integrity of samples was checked. qPCR reaction using TaqMan chemistry with inventoried primers were performed in ViiA7 equipment. The results, depicted as the relative levels of gene expression, were calculated in reference to GAPDH and β-actin expression. Periodontal tissues from upper molars were vested and incubated supplemented RPMI, followed by processing with 0.05% DNase. Cell viability and couting were determined by Neubauer chamber analysis. For flow cytometry analysis, after cell counting the cells were stained with the optimal dilution of each antibody; (PE)-conjugated and (FITC)-conjugated antibodies against CD4, CD25, FOXP3, IL-4, IL-17 and IFN-γ antibodies, as well their respective isotype controls. Cells were analyzed by FACScan and CellQuest software. Results are presented as the number of cells in the periodontal tissues or the number of positive cells for each marker in the CD4+FOXp3+, CD4+IL-4+, CD4+IFNg+ and CD4+IL-17+ subpopulations. The levels mRNA were measured by qPCR. The VIP expression was predominated in inactive lesions, as well part of the clusters of cytokine/Th markers identified as protective factors and a negative correlation between VIP expression and lesion evolution was observed. A quantitative analysis of IL1β, IL17, TNF, IFN, MMP2, RANKL, OPG, IL10, TGFβ, CTLA4, COL5A1, CTGF, CXCL11, FGF7, ITGA4, ITGA5, SERP1 and VTN expression was measured in experimental periapical lesions treated with VIP 7 and 14 days after lesion induction and healthy animals. After 7 days, all targets presented a significate increase in comparison to untreated animals. About migration kinetics, profile of chemokine receptors expression of TCD4+ subsets and phenotypic analysis of Tregs, Th1, Th2 and Th17 cells during the course of experimental periodontal disease evaluated by flow cytometry and depicted as the number of positive cells for each marker. CD4+IFNg+ and CD4+FOXp3+ cells migration were significate increased 7 days post VIP treatment. CD4+IL17+ cells migration were significate increased 7 and 14 days post VIP treatment, CD4+IL4+ cells migration were significate increased 14 and 21 days post VIP treatment compared to the control group. In conclusion, our experimental data support VIP involvement in determining the inactivity of periapical lesions. Financial support: FAPESP #2015/25618-2.

Keywords: chronic inflammation, cytokines, osteolytic lesions, VIP (Vasoactive Intestinal Peptide)

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