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Objective: Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of CD5+CD19+ B cells in the bone marrow and peripheral blood. Recent studies indicated that expression of IL-10, AID and mir-155 which are regulated by STAT3 are increased in CLL patients. CD5+CD19+ regulator B (Breg) cells secrete IL-10 and suppress the immune system. While the CLL cells show similar immunophenotypic properties to Breg cells, they are also thought to be functionally similar. In this study, STAT3 and IL-10 levels of CLL patients were investigated. Methods: Peripheral blood samples obtained from patients (n:24) and healthy controls (n:14). Peripheral blood mononuclear cells were cultured for 48 hours in the presence and absence of CpG for IL-10 expression and cultured with and without PMA for STAT3 expression. IL-10 and STAT3 expression were analyzed with anti-CD5, anti-CD19, anti-CD38, anti-STAT3 and anti-IL-10 monoclonal antibodies by using flow cytometry. Results: Compared to healthy subjects, increased IL-10+, IL-10+CD19+, STAT3+CD19+ were obtained in lymphocyte population of patients. Increased IL-10 was showed CD19+ B cells of CLL patients. Our results showed that IL-10 levels had no significant difference between CD5+CD19+ cells, whereas STAT3 levels were found lower in patient compared to healthy controls. Conclusion: These results made us thought that the levels of IL-10 and STAT3 expression in CLL B cells is clearly different from normal B lymphocytes might have a role in the biology of CLL. It is believed that the presented data will contribute to the studies that scrutinize the similarity of CLL cells to Breg cells.

Objective: Acute lymphoblastic leukemia (ALL) is the most prevalent type of cancer in children. Minimal residual disease (MRD) is still the most important indicator of clinical results and relapse after chemotherapy. Multidrug resistance is the main obstacle to successful treatment. Multidrug resistance-related protein 1 (MRP1) may play a key role in throwing the chemical drug out of cells leading to therapy resistance. This study aims to detect MRP1 protein in the bone marrow cells of children with B-ALL and determine its value as a prognostic factor in comparison with other factors such as DNA index and MRD obtained by flow cytometric measurement. Methods: Bone marrow samples were obtained from children who are diagnosed as B-ALL (n=20) at day 0 (diagnosis) and 15 of therapy. Risk groups’ classification is based on discrimination of age and white cell count on day 0. The expressions of MRP1 levels and DNA index at diagnosis and MRD on the 15th day of treatment in the bone marrow were detected by using flow cytometry. The B-ALL blast cells were stained using anti-CD10, -CD19, -CD20, -CD34, -CD45 monoclonal antibodies. MRP1 content of cells was detected in an intracellular manner. Results: There was no statistically significant difference in MRP1 expression between risk groups and the other prognostic factor as Flow MRD and DNA index. Conclusion: The utilization of MRP1 as a predictive factor may not provide information on the B-ALL prognosis. Our results can help to better understand the nature of MRP1 in B-ALL patients.

Breast cancer is the most common type of cancer in females and the second most common cause of cancer mortality after lung cancer. Cancer stem cells represent a novel approach to target cancer and reduce cancer recurrence and metastasis. Many patients with breast cancer continue to smoke after receiving their diagnosis. Nicotine is a key factor in tobacco addiction and also changes some cellular functions, such as activation of mitogenic pathways, angiogenesis and cell proliferation. In the present study, the impact of nicotine was assessed in a population of MCF-7 human breast cancer cells. Cluster of differentiation (CD)44+CD24− cancer stem cell population of MCF-7 cells were evaluated using flow cytometry and scanning electron microscopy. Chemoresistance effects of nicotine were demonstrated in these cells. These findings demonstrated harmful effects of nicotine following metastasis of cancer, owing to the chemoresistance produced through uninterrupted smoking, which may impact the effectiveness of treatment.

Metformin is the most widely used anti-diabetic drug in the world. It reduces advanced glycation end product (AGEs)-induced ROS generation in high glucose condition. Protein glycation contributes to skin aging as it deteriorates the existing collagen by crosslinking. The progressive increase of AGE during aging not only causes oxidative damage to cellular macromolecules but also modulates the activation of transcription factors nuclear factor kappa-B(NF-kB). However, it is still unclear whether metformin can change collagen production and NF-kB activity induced by high glucose conditions in 3T3 fibroblast. The effects of metformin on proliferation, apoptosis, and collagen levels and NF-kB activity of in vitro cell aging model of 3T3 fibroblast cells in high glucose conditions. At first, we investigated the effects of 50 mM high glucose concentration, with or without metformin, on 3T3 fibroblast proliferation, by BrdU immunostaining for cell proliferation. Apoptotic levels were analyzed by flow cytometric assay. NF-kB(p65) activity was measured by transcription factor assay kit and collagen I and III levels by Collagen Estimation Assay through ELISA. We observed that metformin exposure leads to decreased apoptosis levels and increased proliferation of 3T3 fibroblast in high glucose media. We also determined that metformin exposure leads to increased production of collagen I–III and decreased activation of NF-kB(p65) activity. The data are consistent with the observation that metformin has a protective effect in this in vitro model of aging 3T3 fibroblasts under high glucose conditions inducing cell proliferation, collagen I and III production, protection from apoptosis, and reducing NF-kB(p65) activity.

Metformin has been successfully used as an anti-aging agent but exact molecular mechanisms of metformin in anti-aging remain unknown. Hyperglycemia during skin aging not only causes oxidative damage to cellular macromolecules, like dermal collagen, but also modulates the activation of transcription factor nuclear factor kappa B (NF-kB). We aimed to investigate in vitro effects of high glucose (HG) and metformin treatment on proliferation and apoptosis of human primary dermal fibroblasts (HDFs), and the expression of COL1A1 , COL3A1 , and RELA/p65 genes. Effects of normal glucose (5.5 mM) and HG concentration (50 mM HG) on HDFs, with two doses of metformin (50 μM and 500 μM), were investigated by immunostaining. Apoptotic levels were analyzed by flow cytometry. Expression of COL1A1 , COL3A1 , and RELA/p65 genes was measured by quantitative real-time PCR. The proliferation of HDFs was decreased significantly ( P  < 0.01) and expression of COL1A1 was downregulated by HG without metformin, whereas proliferation was elevated and expression was upregulated with 500 μM metformin + HG compared to 5.5 mM glucose ( P  < 0.05). The expression of COL3A1 and RELA /p65 were upregulated ( P  < 0.01 for COL3A1 ), and percentage of late apoptotic cells increased significantly by HG without metformin ( P  < 0.001) while it decreased in two concentrations of metformin dramatically compared with 5.5 mM glucose (P < 0.01 for expressions and < 0.001 for apoptosis). Metformin not only significantly downregulated RELA/p65 expression, but also inhibited the apoptosis of HDFs from aged human skin at toxic glucose concentrations which could be inversely mediated via COL1A1 and COL3A1 expression.

Bone marrow examination revealed 30% of blast-like cells, suspecting leukemia (Figure-1). The CD20 was shown as variable expressions from negative to bright consisting of a “J shaped trail pattern” on CD10/CD20 gating (Figure-2). [See PDF for image] Fig. 1 Complete blood counts, bone marrow smear, and flow cytometric analysis in a child with suspected leukemia (a) Changes in platelet counts (b) Changes in hemoglobin and neutrophil counts (c) Blast like cells as immature B lymphoid cells in different stages of hematogones (black arrows) in bone marrow smear (d) Variable expressions of CD10 Phycoerythrin-Cyanin 7 (PC7), CD19 Phycoerythrin-Texas Red-X (ECD), CD33 Phycoerythrin-Cyanin 5 (PC5), myeloperoxidase Phycoerythrin (PE), Cytoplasmic CD3 APC (Allophycocyanin)-Alexa Fluor 750 (A750) and Terminal deoxynucleotidyl transferase Fluorescein Isothiocyanate (FITC) in blastic cells populations using SSC/CD45 Krome Orange (KO) gating [See PDF for image] Fig. 2 Demonstrating hematogones [H] in total B lymphoid cells (a) using SSC/CD19 ECD gating (b) stage 1[H-1] -2[H-2] -3[H-3] hematogones using CD10 A750/ CD19 ECD gating (c) J shaped hematogon using CD A750/CD20 PC5 gating (d) stage 1[H-1] -2[H-2] -3[H-3] hematogones using CD10 A750/ CD45 KO gating (e) stage 1-2-3 hematogones using CD38 APC/ CD45 KO gating (f) stage 1[H-1] -2[H-2] -3[H-3] hematogones using CD10 A750/ CD34 PC7 gating by multiparameter 10-color flow cytometric analysis.

Purpose: Odontogenic cysts (OCs) are pathological lesions that include liquid or semi-liquid surrounded with epithelium. Radicular cysts' (RCs) and odontogenic keratocysts' (OKCs) are common odontogenic cysts of jaws, and may reach to a substantial size without symptoms for a long time. It is known that cytokines secreted during infection and inflammation regulate the immune response. This study aims to describe the relationship between bacteria as infection agents and the levels of interferon-gamma (IFN-γ) cytokine of innate and adaptive immune response. Materials and Methods: OC fluid samples with a history of infection were collected from a total of 39 OCs consisting 25 samples of odontogenic keratocysts (OKC) and 14 samples of radicular cysts (RC). Anaerobic bacteria detection was performed by a polymerase chain reaction (PCR) based on bacterial 16S rRNA genes. IFN-γ levels in OC fluids were determined using the luminex method. Results: No significant differences in IFN-γ levels and T cell type 1 cytokine responses were observed between the cystic fluid samples classified on the basis of age, gender, cyst-type, cyst-size, bacterial species and the number of bacterial species contained. The measured concentrations IFN-γ, which is a helper T cell type 1 cytokine were consistent with published data from experimental animal models, immunohistochemical studies, and molecular studies. Conclusion: Luminex method can detect the concentration of many different types of protein in a small sample volume and is suitable for determining the protein content of odontogenic cysts.

Masitinib mesylate, a selective tyrosine kinase inhibitor of the c-KIT receptor, is used for the treatment of mast cell tumours in dogs. Masitinib has previously been investigated in various cancers; however, its potential anticancer effect in canine mammary tumours (CMTs) is unknown. In the present paper, we investigated the antiproliferative effect of masitinib in CMT cells and its possible mechanisms of action. The effect of masitinib on the proliferation of CMT-U27 and CMT-U309 cells was assessed by MTT assay and DNA fragmentation. Flow cytometric analysis was used to measure the effect of masitinib on apoptosis and the cell cycle. Additionally, vascular endothelial growth factor levels (VEGF) were measured, and the proliferation marker Ki-67 was visualised in immunocytochemical stainings in CMT cells. Treatment with masitinib inhibited the proliferation of CMT cells in a concentration-dependent manner. Maximal apoptotic activity and DNA fragmentation were observed at approximately IC of masitinib in both cell lines. In addition, cell cycle distribution was altered and VEGF levels and Ki-67 proliferation indices were decreased in masitinib-treated cells in comparison with control cells. In this study, masitinib suppressed cell proliferation concomitantly induction of apoptosis and cell cycle arrest by decreasing VEGF levels and the Ki-67 proliferation index in CMT-U27 and CMT-U309 cells , suggesting its potential as a therapeutic tool in the clinical setting of mammary cancer treatment in dogs.

In subacute sclerosing panencephalitis (SSPE) the persistence of measles virus (MeV) may be related to the altered immune response. In this study, cytokine responses of lymphocytes and monocytes were evaluated in SSPE compared to controls with non-inflammatory (NICON) and inflammatory (ICON) diseases. Patients with SSPE (n = 120), 78 patients with ICON and 63 patients with NICON were included in this study. Phenotypes of peripheral blood mononuclear cells (PBMC) have been analyzed by flow cytometry. CD3 and CD28, and S . aureus Cowan strain I (SAC) stimulated and unstimulated cells were cultured and IL-2, IL-10, IFN-γ, IL-12p40, IL-12p70 and IL-23 were detected in supernatants by ELISA. MeV peptides were used for MeV-specific stimulation and IFN-γ secretion of PBMC was measured by ELISPOT. Spontaneous and stimulated secretions of IL-10 were lower in SSPE compared to both control groups. T cell stimulation induced lower IFN-γ production than ICON group, but higher IL-2 than NICON group in SSPE. Stimulated PBMC produced lower IL-12p70 in SSPE and had decreased CD46 on the cell surface, suggesting the interaction with the virus. IFN-γ responses against MeV peptides were not prominent and similar to NICON patients. The immune response did not reveal an inflammatory activity to eliminate the virus in SSPE patients. Even IL-10 production was diminished implicating that the response is self-limited in controlling the disease.