Origanum Sipyleum Methanol Extract in Combination with Ponatinib Shows Synergistic anti-Leukemic Activities on Chronic Myeloid Leukemia Cells.

Origanum sipyleum is used in folk medicine due to its anti-inflammatory, antimicrobial, and antioxidant properties. Ponatinib, an effective tyrosine kinase inhibitor in the treatment of chronic myeloid leukemia (CML), has severe side effects. Thus, we aimed to determine a novel herbal combination therapy that might not only increase the anti-leukemic efficacy but also reduce the dose of ponatinib in targeting CML cells. Origanum sipyleum was extracted with methanol (OSM), and secondary metabolites were determined by phytochemical screening tests. The cytotoxic effects of OSM on K562 cells were measured by WST-1 assay. Median-effect equation was used to analyze the combination of ponatinib and OSM (p-OSM). Apoptosis, proliferation, and cell-cycle were investigated by flow-cytometry. Cell-cycle-related gene expressions were evaluated by qRT-PCR. OSM that contains terpenoids, flavonoids, tannins, and anthracenes exhibited cytotoxic effects on K562 cells. The median-effect of p-OSM was found as synergistic; OSM reduced the ponatinib dose ∼5-fold. p-OSM elevated the apoptotic and anti-proliferative activity of ponatinib. Consistently, p-OSM blocked cell-cycle progression in G0/G1, S phases accompanied by regulations in TGFB2, ATR, PP2A, p18, CCND1, CCND2, and CCNA1 expressions. OSM enhanced the anti-leukemic activity of ponatinib synergistically via inducing apoptosis, suppressing proliferation, and cell-cycle. As a result, OSM might offer a potential strategy for treating patients with CML.

Immunologic changes during desensitization with cow's milk: How it differs from natural tolerance or nonallergic state?

BACKGROUND: Oral immunotherapy (OIT) is a novel allergen-specific treatment for food allergies. OBJECTIVE: To investigate the effect of OIT on blocking antibodies, T cell regulation, and cytokine response during immunoglobulin (Ig)E-mediated cow's milk allergy (CMA) treatment. METHODS: A total of 59 children with IgE-mediated CMA who were followed in pediatric allergy outpatient clinic and 18 healthy children were included. The children were evaluated in the following 4 groups: OIT group, elimination group (patients receiving dairy elimination diet), tolerance group (patients who developed tolerance), and healthy control group. Milk-specific IgE, IgG4, and IgA levels, cow's milk induration diameters in skin prick test, CD4 + CD25 + FoxP3 + Treg cell percentages, messenger RNA (mRNA) expressions, and interleukin (IL)-10, transforming growth factor-beta (TGF-ß), IL-2, IL-4, and IL-13 cytokine levels were compared between the groups. RESULTS: The mean age of the patients was 42.6 ± 39 (6-201) months, and 63.6% (n = 49) of the patients were girls. We observed an increase in total IgE levels (P = .02), a decrease in cow's milk sIgE (P = .08, NS), and an increase in cow's milk component (ß-lactoglobulin and casein) specific IgA (P < .05) and IgG4 (P < .001) levels at 2 months after the maintenance phase of OIT. In addition, the immune response after OIT treatment, which had a 100% clinical success rate, was notable for similar CD4 + CD25 + FoxP3 + cell percentages (P = .8), and increased IL-10 (P = .04) levels and increased but statistically nonsignificant TGF-ß levels (P = .17) compared with those before treatment. FoxP3 mRNA expression was similar to that of patients who developed natural tolerance. Pretreatment and post-treatment FoxP3 mRNA-FoxP3 flow cytometric expressions were positively correlated with TGF-ß concentrations in the OIT group. CONCLUSION: A successful immune response to OIT was found, possibly through the blockage of IgE-mediated allergen presentation by blocking antibodies, marked IL-10 cytokine response, and TGF-ß response. FoxP3 mRNA expression was similar to the natural tolerance mechanism, but more studies are needed.

[Investigation of the Anti-Leishmanial Effects of Prangos ferulacea and Ferula orientalis Extracts Collected from Sirnak Province Against Leishmania tropica Isolated in Turkey]. / Sirnak Ili ve Çevresinden Toplanan Prangos ferulacea ve Ferula orientalis Ekstrelerinin Türkiye'den Izole Edilmis Leishmania tropica'ya Karsi Anti-Leishmanial Etkilerinin Arastirilmasi.

Leishmaniasis is a vector-borne disease that is caused by the protozoa of Leishmania genus. Leishmaniasis is endemic in tropical, subtropical, and large areas of the Mediterranean basin, and covers a total of 98 countries worldwide. It is estimated, according to the World Health Organization (WHO) data, that approximately 350 million people are at risk in these areas, and approximately 12 million people are infected. Increased drug resistance has been documented lately, in the treatment of leishmaniasis which causes almost 1.2 million new cases annually. Thus, interest in plant-derived active substances has increased in recent years, and new anti-leishmanial agents are investigated with in vitro studies. The aim of the present study was to investigate the anti-leishmanial effects of Prangos ferulacea and Ferula orientalis plant extracts collected from the rural areas of Sirnak province against Leishmania tropica. The water, chloroform, and ethanol extracts of the roots, stems, and fruits of P.ferulaceae and F.orientalis plants were obtained, and the cytotoxic activity tests of the extracts were performed. L.tropica isolate obtained from the Parasite Bank in Manisa Celal Bayar University in Turkey (MHOM/TR/2012/CBCL-LT) was grown on NNN and RPMI 1640 broth medium. The cytotoxicity of each extract on the L.tropica isolate was evaluated with the XTT test. Amphotericin B (AmpB) was used as the positive control, and the IC50 values were determined. The lowest IC50 values of the plant extracts were found to be as follows: P.ferulaceae root chloroform extract 36 µg/ml and fruit chloroform extract 20 µg/ml, F.orientalis root ethanol extract 2.5 µg/ml, and fruit ethanol extract 48 µg/ml, stem chloroform extract 24 µg/ml, and fruit chloroform extract 3.1 µg/ml. It was also determined in our study that only P.ferulaceae root ethanol extract showed cytotoxic activity on the WI-38 fetal lung fibroblast cell line at 65.19 µg/ml at 72 hours. This is the first study that assessed the anti-leishmanial activities of P.ferulaceae and F.orientalis plants that grow in high altitude areas of our country. It was determined that P.ferulaceae root ethanol extract and fruit chloroform extract had the lowest IC50 values among the 18 plant extracts that we examined for their anti-leishmanial activities. The outcomes of this study will be useful in further studies for the determination of active compounds in P.ferulaceae and F.orientalis plant extracts.

Investigation of miRNA and cytokine expressions in latent tuberculosis infection and active tuberculosis.

BACKGROUND: In tuberculsosis (TB), miRNA has been used as a biomarker to distinguish between healthy individuals and TB patients. The aim of this study was to investigate (i) the association of the miRNA and cytokine expression levels, the course of tuberculosis infection, clinical forms and response to treatment, and (ii) the effects of genotypic features of bacteria on the course of tuberculosis and the relationship between miRNA and cytokine expressions and bacterial genotypes. METHODS: A total of 200 cases (100: culture positive active tuberculosis, 50: quantiferon positive latent tuberculosis infection and 50: quantiferon negative healthy controls) were included in the study. For the tuberculosis group at the time of admission and after treatment, for the latent tuberculosis infection and healthy control groups at the time of admission, miRNA and cytokine expressions were determined. Genotyping of M.tuberculosis isolates was performed by spoligotyping method. RESULTS: While, in the comparison of miRNA expressions between the pretreatment patient group and the healthy control group, there was a statistically significant decrease in the expression of miR-454-3p, miR-15a-5p, miR-590-5p, miR-381, and miR-449a in the Pulmonary TB group, there was no significant change in miRNA expression in extrapulmonary TB patients. When the cytokine expressions of the patient group and the healthy control group were compared before treatment, the expressions of all cytokines in the patient group decreased. However, the only cytokine that showed a significantly lower expression was IL12A in PTB patients. DISCUSSION: There is no significant relationship between the clinical course of the disease, cytokine and miRNA expression, and the genotype of the bacteria.

Temozolomide treatment combined with AZD3463 shows synergistic effect in glioblastoma cells.

Temozolomide (TMZ) is used in the standard therapy regimen for patients with glioblastoma (GBM). However, some GBM patients do not respond to TMZ therapy. The combining therapeutic agents in GBM treatment are attracting considerable interest due to TMZ resistance. This study aims to identify the combinatorial effect of TMZ and AZD3463 on the viability of the T98G GBM cells. The cytotoxic effects of compounds were determined by using WST-8 assay. Flow cytometry was used to determine apoptosis and cell cycle profiles after treatments. Real-time PCR was used to identify mRNA expression of genes in the PI3K/AKT signaling pathway after treatments. IC50 concentrations of TMZ and AZD3463 were found to be 1.54 mM and 529 nM after incubation for 48 h, respectively. The combination treatment showed a synergistic effect on reducing the viability of GBM cells. Each one of TMZ, AZD3463, and combination treatments induced apoptosis. Treatments, either alone or the combination of these agents, caused the cell cycle arrest in distinct phases. TMZ and AZD3463 treatments, either alone or in combination, downregulated mRNA expression of genes in the PI3K/AKT signaling pathway. The combination of TMZ with AZD3463 may increase the efficacy of single TMZ treatment in GBM cells due to decreased expression of genes in the PI3K/AKT signaling pathway that is responsible for drug resistance and intratumoral heterogeneity.

Effects of metformin and pioglitazone combination on apoptosis and AMPK/mTOR signaling pathway in human anaplastic thyroid cancer cells.

Anaplastic cancer constitutes 1% of thyroid cancers, and it is one of the most aggressive cancers. Treatment options are external radiation therapy and/or chemotherapy. The success rate with these treatment modalities is not satisfactory. We aimed to evaluate the effects of metformin (MET) and pioglitazone (PIO) combination on apoptosis and AMP-activated protein kinase/mammalian target of rapamycin (mTOR) signaling pathway in human anaplastic thyroid cancer cells. In this study, we evaluated the effects of MET and PIO individually and the combination of the two drugs on the cellular lines SW1736 and C643 ATC. Genes contained in the mTOR signaling pathway were examined using human mTOR Signalization RT2 Profiler PCR Array. In C643 and SW1736 cell lines, IC50 doses of MET and PIO were found out as 17.69 mM, 11.64 mM, 27.12 µM, and 23.17 µM. Also, the combination of MET and PIO was determined as an additive according to isobologram analyses. We have found the downregulation of the expression levels of oncogenic genes: AKT3, CHUK, CDC42, EIF4E, HIF1A, IKBKB, ILK, MTOR, PIK3CA, PIK3CG, PLD1, PRKCA, and RICTOR genes, in the MET and PIO combination-treated cells. In addition, expression levels of tumor suppressor genes, DDIT4, DDIT4L, EIF4EBP1, EIF4EBP2, FKBP1A, FKBP8, GSK3B, MYO1C, PTEN, ULK1, and ULK2, were found to have increased significantly. The MET + PIO combination was first applied to thyroid cancer cells, and significant reductions in the level of oncogenic genes were detected. The decreases, particularly, in AKT3, DEPTOR, EIF4E, ILK, MTOR, PIK3C, and PRKCA expressions indicate that progression can be prevented in thyroid cancer cells and these genes could be selected as therapeutic targets.

Fetal Gene Therapy Using a Single Injection of Recombinant AAV9 Rescued SMA Phenotype in Mice.

Symptoms of spinal muscular atrophy (SMA) disease typically begin in the late prenatal or the early postnatal period of life. The intrauterine (IU) correction of gene expression, fetal gene therapy, could offer effective gene therapy approach for early onset diseases. Hence, the overall goal of this study was to investigate the efficacy of human survival motor neuron (hSMN) gene expression after IU delivery in SMA mouse embryos. First, we found that IU-intracerebroventricular (i.c.v.) injection of adeno-associated virus serotype-9 (AAV9)-EGFP led to extensive expression of EGFP protein in different parts of the CNS with a great number of transduced neural stem cells. Then, to implement the fetal gene therapy, mouse fetuses received a single i.c.v. injection of a single-stranded (ss) or self-complementary (sc) AAV9-SMN vector that led to a lifespan of 93 (median of 63) or 171 (median 105) days for SMA mice. The muscle pathology and number of the motor neurons also improved in both study groups, with slightly better results coming from scAAV treatment. Consequently, fetal gene therapy may provide an alternative therapeutic approach for treating inherited diseases such as SMA that lead to prenatal death or lifelong irreversible damage.

[Diversity of Leishmania Strains Isolated from Cutaneous Leishmaniasis Patients in Turkey and its Reflection to Clinics in Mice Model]. / Türkiye'de Kutanöz Leysmanyazis Hastalarindan Elde Edilen Leishmania Izolatlarindaki Farkliliklar ve Bunlarin Fare Modeline Klinik Yansimasi.

Although asexual reproduction has been attributed to Leishmania species, genetic exchange has recently been demonstrated, which helped emerging of hybrid isolates. Situated on the crossroads between three continents, Leishmania hybrids may be present in Turkey. In Turkey, visceral leishmaniasis caused by Leishmania infantum is less common, while cutaneous leishmaniasis (CL) caused by Leishmania tropica and L.infantum could reach 2500 reported cases a year. Our aim was to investigate genetic variability of local Leishmania species and presence of hybrid Leishmania strains in Turkey. Twenty CL patients from Sanliurfa and Hatay, where only L.tropica and both L.tropica and L.infantum cause CL, respectively, were registered equally. All isolates were assessed with real-time polymerase chain reaction (Rt-PCR), isoenzyme analysis, gene sequencing, two-dimensional gel electrophoresis (2D-PAGE) and MALDI-TOF/TOFMS followed by in vivo analyses on mouse model. Identification of differentially expressed proteins was performed. These proteins were confirmed by sequence analysis. All isolates from Sanliurfa were found to be L.tropica which caused cutaneous infection in mice. However, one of 10 isolates from Hatay was found as Leishmania major which caused cutaneous infection. Five isolates were found as L.tropica with Rt-PCR and gene sequencing, one of which had one different protein from the reference L.tropica strain and caused cutaneous infection. Four of the five isolates had five different proteins compared to reference strain and caused both cutaneous and visceral infections. Remaining four isolates showed double melting curves in Rt-PCR, which were concordant with L.tropica and L.infantum. Their sequencing and isoenzyme analyses indicated them as L.infantum. They had six different proteins compared to reference L.infantum strain and caused cutaneous and visceral infections. It is concluded that the isolates with different proteins were hybrid Leishmania species. In the present study, outcomes of the proteomics, genomics, clinical manifestations and tissue tropism on animal models were evaluated together for the first time. In addition to L.tropica and L.infantum, L.major was identified as a causative agent for CL and hybrids of L.infantum/tropica were also shown to be present.

[Determination of Antimony Resistance Mechanism of Leishmania tropica Causing Cutaneous Leishmaniasis in Turkey]. / Türkiye'de Kutanöz Leysmanyazis Etkeni Leishmania tropica'da Antimon Direnç Mekanizmasinin Belirlenmesi.

World Health Organization reported that approximately one billion people are at risk in endemic areas, one million cases of cutaneous leishmaniasis (CL) and approximately 300,000 cases of visceral leishmaniasis (VL) were reported per year in the last five years. The number of deaths due to VL is reported to be approximately 20,000 per year. Approximately 2500 cases/year have been reported as CL, caused by Leishmania tropica and Leishmania infantum, in Turkey. The significant increase observed in many cities mainly in the provinces of Mediterranean and Aegean regions in cases and foci in recent years, suggests that there may be an increase in this infections in the following years as well. In Turkey, the causative agent of CL is L.tropica and meglumine antimoniate is used in the treatment of CL. We aimed to determine antimony resistance genes specific for L.tropica by comparing the gene and protein expressions of antimony-resistant and non-resistant L.tropica strains. L.tropica isolates obtained from 3 CL patients without antimonate resistance from Aegean, Mediterranean and Southeastern regions of Turkey were provided to transform into 3 resistant isolates against meglumine antimony in the laboratory conditions. Gene expression alterations by microarray method; protein profiles by two-dimensional gel electrophoresis (2D-PAGE) and relevant proteins by MALDI-TOF/TOF MS of these isolates were accomplished and compared. L.tropica isolates from 10 CL patients who did not respond to antimony therapy were analyzed for resistance to antimonial compounds and quantitative real-time polymerase chain reaction was performed to detect the expression of genes responsible for resistance development. Moreover, differences in protein expression levels in isolates with and without antimony resistance were determined by comparing protein profiles and identification of proteins with different expression levels was carried out. Enolase, elongation factor-2, heat shock protein 70, tripanthione reductase, protein kinase C and metallo-peptidase proteins have been shown to play roles in L.tropica isolates developing resistance to antimonial compounds and similar expression changes have also been demonstrated in naturally resistant isolates from patients. In conclusion, it was revealed that L.tropica strains in our country may gain resistance to meglumine antimoniate in a short time. It is foreseen that if the patients living in our country or entering the country are treated inadequately and incompletely, there may be new, resistant leishmaniasis foci that may increase the number of resistant strains and cases rapidly.

Epigenetic modifications in chronic myeloid leukemia cells through ruxolitinib treatment.

Chronic myeloid leukemia is a clonal malignancy of hematopoietic stem cell that is characterized by the occurrence of t(9;22)(q34;q11.2) translocation, named Philadelphia chromosome. Ruxolitinib is a powerful Janus tyrosine kinase 1 and 2 inhibitor that is used for myelofibrosis treatment. DNA-histone connection mediates a wide range of genes that code methylation, demethylation, acetylation, deacetylation, ubiquitination, and phosphorylation enzymes. Epigenetic modifications regulate chromatin compactness, which plays pivotal roles in critical biological processes including the transcriptional activity and cell proliferation as well as various pathological mechanisms, including CML. This study is aimed to determine the alterations of the expression levels of epigenetic modification-related genes after ruxolitinib treatment. Total RNA was isolated from K-562 cells treated with the IC50 value of ruxolitinib and untreated K-562 control cells. A reverse transcription procedure was performed for complementary DNA synthesis, and gene expressions were detected by real-time polymerase chain reaction compared with the untreated cells. Ruxolitinib treatment caused a significant alteration in the expression levels of epigenetic regulation-related genes in K-562 cells. Our novel results suggested that ruxolitinib has inhibitor effects on epigenetic modification-regulator genes.

The effect of ICRT-3 on Wnt signaling pathway in head and neck cancer.

The effect of Wnt pathway in head and neck cancer could not be elucidated, even though the aberrant Wnt signaling plays a key role in the development of many types of cancer. The inhibitor of ß-catenin responsive transcription (ICRT-3) blocks the Wnt signaling pathway by binding to ß-catenin, which is a coactivator of the Wnt signaling pathway and a promising agent for inhibiting aberrant signaling. In our study, we aimed to evaluate the effect of ICRT-3 on the cytotoxicity, apoptosis, cell cycle progression, migration, and gene expressions in head and neck cancer stem cell (HNCSC) and hypopharynx cancer. The effect of this compound on cytotoxicity and cell viability in FaDu and HNCSC line was assessed by using the water-soluble tetrazolium salt-1 method. The effect of ICRT-3 on apoptosis was detected by using Annexin V and caspase-3, caspase-9 kit, on cell cycle progression by cycle test plus DNA reagent kit, on gene expression by dual luciferase reporter assay, and on migration activity by wound healing assay in both cell lines. ICRT-3 was determined to have cytotoxic and apoptotic effect in both cell lines. In addition, it was also found that the administration of ICRT-3 caused cell cycle arrest and significant decrease in gene expression level and migration ability of the cells.

Investigation of the effect of telomerase inhibitor BIBR1532 on breast cancer and breast cancer stem cells.

It is emphasized that cancer stem cells (CSCs) forming the subpopulation of tumour cells are responsible for tumour growth, metastasis, and cancer drug resistance. Inadequate response to conventional therapy in breast cancer leads researchers to find new treatment methods and literature surveys that support CSC studies. A selective anticancer agent BIBR1532 inhibits the telomerase enzyme. Many of the chemotherapeutic drugs used in clinical trials have harmful effects, but the advantage of telomerase-based inhibitors is that they are less toxic to healthy tissues. The phosphoinositide 3-kinase (PI3K)/serine/threonine kinase (Akt)/mammalian target of rapamycin (mTOR) pathway is common in breast cancer, and the interaction between the mTOR pathway and human telomerase reverse transcriptase (hTERT) is essential for the survival of cancer cells. In our study, we treated MCF-7, breast cancer stem cell (BCSC) and normal breast epithelial cell MCF10A with the BIBR1532 inhibitor. The IC 50 doses for the 48th hour of BIBR1532 treatment were detected as 34.59 µM in MCF-7, 29.91 µM in BCSCs, and 29.07 µM in MCF10A. It has been observed that this agent induces apoptosis in the BCSC and MCF-7 cell lines. According to the results of cell cycle analysis, G 2 /M phase accumulation was observed in BCSC and MCF-7 cell lines. It has also been shown that BIBR1532 suppresses telomerase activity in BCSC and MCF-7. The effect of BIBR1532 on the mTOR signalling pathway has been investigated for the first time in this study. It is thought that the telomerase inhibitor may bring a new approach to the treatment and it may be useful in the treatment of CSCs.

First Report and In Silico Analysis of Leishmania virus (LRV2) identified in an autochthonous Leishmania major isolate in Turkey.

Leishmania virus (LRV) has previously been identified in different Leishmania species. Host-LRV interaction is associated with exacerbated clinical manifestations of cutaneous leishmaniasis (CL) and may cause poor therapeutic response. CL cases due to L. major with large skin lesions resistant to routine therapy were recently identified in Turkey. Here, we report the first autochthonous case of cutaneous leishmaniasis caused by LRV-positive Leishmania major, using conventional PCR targeting the viral capsid protein of LRV. The lesion of the case was 6 months old, relatively large (4 cm), and did not recover despite three consecutive intralesional applications of glucantime. Assessment of LRV's influence on prognosis and clinical outcomes of leishmaniasis, based on additional studies, is required.

Effects of telomerase inhibitor on epigenetic chromatin modification enzymes in malignancies.

Telomerase has a critical role in cell proliferation, tumor maintaining, and therapy resistance, which act by modifying many signaling pathways. 2-[(E)-3-Naphtalen-2-yl-but-2-enoylamino]-benzoic acid (BIBR1532) is one of the most studied telomerase inhibitors, and it targets telomerase components TERC and TERT. In this novel study, we aimed to investigate the epigenetic effects of BIBR1532 on both hematologic malignancies and solid tumors. K-562 human chronic myeloid leukemia cell line and U87MG glioblastoma cell line were compared with control groups without BIBR1532 treatment. Cytotoxic effects of BIBR1532 were determined by using WST-1 assay. Apoptotic effects of BIBR1532 were detected by using annexin V method. To assess expression changes in the human epigenetic chromatin modification enzyme genes, total RNA was isolated from K-562 and U87MG cells treated with BIBR1532 and untreated control cells. BIBR1532 induced 2.41-fold apoptotic cell death in U87MG cell lines compared with control groups. Apoptosis was slightly induced in K-562 cells with BIBR1532 treatment compared with control cells. We observed that BIBR1532 also regulates similar genes in both cell lines, and it is useful on epigenetic mechanisms. As a result, telomerase inhibitor BIBR1532 has a significant effect on both hematological malignancies and solid tumors.

Comparative analysis of human UCB and adipose tissue derived mesenchymal stem cells for their differentiation potential into brown and white adipocytes.

The differentiation potential of umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) into brown and white adipocytes in comparison to Adipose tissue derived MSCs (AD-MSCs) were investigated in order to characterize their potency for future cell therapies. MSCs were isolated from ten UCB samples and six liposuction materials. MSCs were differentiated into white and brown adipocytes after characterization by flow cytometry. Differentiated adipocytes were stained with Oil Red O and hematoxylin/eosin. The UCP1 protein levels in brown adipocytes were investigated by immunofluoresence and western blot analysis. Cells that expressed mesenchymal stem cells markers (CD34-, CD45-, CD90+ and CD105+) were successfully isolated from UCB and adipose tissue. Oil Red O staining demonstrated that white and brown adipocytes obtained from AD-MSCs showed 85 and 61% of red pixels, while it was 3 and 1.9%, respectively for white and brown adipocytes obtained from UCB-MSCs. Fluorescence microscopy analysis showed strong uncoupling protein 1 (UCP1) signaling in brown adipocytes, especially which were obtained from AD-MSCs. Quantification of UCP1 protein amount showed 4- and 10.64-fold increase in UCP1 contents of brown adipocytes derived from UCB-MSCs and AD-MSCs, respectively in comparison to undifferentiated MSCs (P < 0.004). UCB-MSCs showed only a little differentiation tendency into adipocytes means it is not an appropriate stem cell type to be differentiated into these cell types. In contrast, high differentiation efficiency of AD-MSCs into brown and white adipocytes make it appropriate stem cell type to use in future regenerative medicine of soft tissue disorders or fighting with obesity and its related disorders.

[Do the rodents have a role in transmission of cutaneous leishmaniasis in Turkey?] / Türkiye'de kutanöz leysmanyaziste kemiricilerin rolü var mi?

Leishmaniasis is a zoonotic/anthroponotic vector borne parasitic infection which is caused by Leishmania species and transmitted by sand flies (Phlebotomus spp.) The reservoirs of Leishmania species in nature are various wild and domestic carnivores, rodents and human. The aim of this study was to investigate whether the rodents in genera Meriones, Mesocricetus, Rattus and Mus which inhabit in the natural habitat of our country could be natural reservoirs of Leishmania tropica, Leishmania infantum, Leishmania major and Leishmania donovani for cutaneous Leishmaniasis (CL)., The rodents Mus musculus (Balb/C mouse), Mesocricetus auratus (hamster), Meriones unguiculatus (gerbil) and Rattus norvegicus (rat) which are part of the natural habitat in Turkey were used in the study. L.tropica, L.infantum, L.major and L.donovani promastigote isolates obtained from CL patients and cultured in enriched media were injected in the footpads of the animals intradermally using the density of 108 promastigote/ml. The scale of the lesions on the footpads of the animals were measured for 12 weeks. At the end of the experiment, the animals were sacrificed and "touch preparations" were prepared using footpad, liver, spleen and testicles of the sacrified animals and were examined using Giemsa stained slides following culturing in enriched NNN medium. Leishmania amastigotes were seen in the slides prepared from the footpads of the all experimental animals and all cultures were positive for promastigotes prepared from the same clinical material. But not all the experiment groups were positive for the liver, spleen and testicle preparations. According to these results it was concluded that while all rodents in the experiment groups were positive for CL, only a part of the experiment groups were positive for internal organ involvement. Accordingly, (a) All Leishmania strains caused both CL and internal organ involvement in M.unguiculatus and M.musculus, (b) only L.tropica caused CL and internal organ involvement in R.norvegicus, while other Leishmania strains only caused CL in this group, (c) in M.auratus only L.donovani caused CL while other strains caused both CL and internal organ involvement. In our study, it was determined that the rodents Meriones, Mesocricetus, Rattus and Mus genera which are part of our country's natural habitat could serve as natural reservoirs of L.tropica, L.infantum, L.major and L.donovani, thus having the potential for the spreading of Leishmaniasis in our country and important information were gathered concerning the clinical aspects of the infection caused by Leishmania species in their potential reservoir hosts.

[The comparison of microscopy and real time polymerase chain reaction methods for the diagnosis of Pneumocystis Jirovecii pneumonia: evaluation of clinical parameters]. / Pneumocystis jirovecii pnömonisi tanisinda mikroskopi ve gerçek zamanli polimeraz zincir reaksiyonu yöntemlerinin karsilastirilmasi: Klinik bulgular ile yorumlanmasi.

INTRODUCTION: Pneumocystis jirovecii pneumonia (PCP) causes serious infections, especially in patients with immunosuppressive diseases. In this study, it was aimed to evaluate the results of samples obtained from PCP suspected patients using two different methods together with clinical data. MATERIALS AND METHODS: Microscopy and real time polymerase chain reaction (real time PCR) methods were performed with bronchoalveolar lavage (BAL) samples sended to Ege University Medical Faculty Direct Parasitology Diagnostic Laboratory between March 2009 and June 2010. Demographic characteristics, clinical and laboratory data were also recorded retrospectively. The data were evaluated using the SPSS 16.0 program. RESULT: A total of 42 BAL samples collected from patients (24 males, mean age: 31.49 ± 26.14) were included. There were totally 16 P. jirovecii positives either one of the tests. Sixteen and three samples were detected positive by real time PCR and microscopy, respectively. Trimethoprim-sulfamethoxazole was prescribed in 11 PCP diagnosed cases and 6 of them died. CONCLUSIONS: Today, despite the growing opportunities in diagnosis and treatment, PCP pneumonia is associated with high mortality. Careful examination of clinical data and immune status of the patients are important. Multidisciplinary approach is required for early PCP diagnosis.

Ruxolitinib induces autophagy in chronic myeloid leukemia cells.

Ruxolitinib is the first agent used in myelofibrosis treatment with its potent JAK2 inhibitory effect. In this novel study, we aimed to discover the anti-leukemic effect of ruxolitinib in K-562 human chronic myeloid leukemia cell line compared to NCI-BL 2171 human healthy B lymphocyte cell line. Cytotoxic effect of ruxolitinib was determined by using WST-1 assay. IC50 values for K-562 and NCI-BL 2171 cell lines were defined as 20 and 23.6 µM at the 48th hour, respectively. Autophagic effects of ruxolitinib were detected by measuring LC3B-II protein formation. Ruxolitinib induced autophagic cell death in K-562 and NCI-BL 2171 cell lines 2.11- and 1.79-fold compared to control groups, respectively. To determine the autophagy-related gene expression changes, total RNA was isolated from K-562 and NCI-BL 2171 cells treated with ruxolitinib and untreated cells as control group. Reverse transcription procedure was performed for cDNA synthesis, and gene expressions were shown by RT-qPCR. Ruxolitinib treatment caused a notable decrease in expression of AKT, mTOR, and STAT autophagy inhibitor genes in K-562 cells, contrariwise control cell line. Ruxolitinib is a promising agent in chronic myeloid leukemia treatment by blocking JAK/STAT pathway known as downstream of BCR-ABL and triggering autophagy. This is the first study that reveals the relationship between ruxolitinib and autophagy induction.

miR-15a enhances the anticancer effects of cisplatin in the resistant non-small cell lung cancer cells.

Platinum-based chemotherapies have long been used as a standard treatment in non-small cell lung cancer. However, cisplatin resistance is a major problem that restricts the use of cisplatin. Deregulated cell death mechanisms including apoptosis and autophagy could be responsible for the development of cisplatin resistance and miRNAs are the key regulators of these mechanisms. We aimed to analyse the effects of selected miRNAs in the development of cisplatin resistance and found that hsa-miR-15a-3p was one of the most significantly downregulated miRNAs conferring resistance to cisplatin in Calu1 epidermoid lung carcinoma cells. Only hsa-miR-15a-3p mimic transfection did not affect cell proliferation or cell death, though decreased cell viability was found when combined with cisplatin. We found that induced expression of hsa-miR-15a-3p via mimic transfection sensitised cisplatin-resistant cells to apoptosis and autophagy. Our results demonstrated that the apoptosis- and autophagy-inducing effects of hsa-miR-15a-3p might be due to suppression of BCL2, which exhibits a major connection with cell death mechanisms. This study provides new insights into the mechanism of cisplatin resistance due to silencing of the tumour suppressor hsa-miR-15a-3p and its possible contribution to apoptosis, autophagy and cisplatin resistance, which are the devil's triangle in determining cancer cell fate.

Zoledronic acid induces apoptosis via stimulating the expressions of ERN1, TLR2, and IRF5 genes in glioma cells.

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor that affects older people. Although the current therapeutic approaches for GBM include surgical resection, radiotherapy, and chemotherapeutic agent temozolomide, the median survival of patients is 14.6 months because of its aggressiveness. Zoledronic acid (ZA) is a nitrogen-containing bisphosphonate that exhibited anticancer activity in different cancers. The purpose of this study was to assess the potential effect of ZA in distinct signal transduction pathways in U87-MG cells. In this study, experiments performed on U87-MG cell line (Human glioblastoma-astrocytoma, epithelial-like cell line) which is an in vitro model of human glioblastoma cells to examine the cytotoxic and apoptotic effects of ZA. IC50 dose of ZA, 25 µM, applied on U87-MG cells during 72 h. ApoDIRECT In Situ DNA Fragmentation Assay was used to investigate apoptosis of U87MG cells. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) (LightCycler480 System) was carried out for 48 gene expression like NF-κB, Toll-like receptors, cytokines, and inteferons. Our results indicated that ZA (IC50 dose) increased apoptosis 1.27-fold in U87MG cells according to control cells. According to qRT-PCR data, expression levels of the endoplasmic reticulum-nuclei-1 (ERN1), Toll-like receptor 2 (TLR2), and human IFN regulatory factor 5 (IRF5) tumor suppressor genes elevated 2.05-, 2.08-, and 2.3-fold by ZA, respectively, in U87MG cells. Our recent results indicated that ZA have a key role in GBM progression and might be considered as a potential agent in glioma treatment.