The effect of PRP and hyperosmolarity simultaneous use on expression profile alteration of miRNAs associated with cartilage differentiation in human adipose tissue-derived mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSC) are a type of multipotent stem cell whose differentiation into cartilage cells has been considered in recent years. Platelet-rich plasma (PRP) may impair cartilage differentiation due to its richness in growth factors and hyperosmolarity due to its proximity to the required cartilage environment. OBJECTIVES: The main purpose of this study was to treat human adipose tissue-derived MSCs concurrently with PRP and hyperosmolarity to investigate the expression profile of micro-RNA (miRNA) involved in the cartilage process differentiation. We examined the effect of PRP and the increase in osmolarity on the expression of miR-27, miR-101, miR-140, miR-145, miR-146, and miR-199. METHODS: Mesenchymal stem cells were extracted from human adipose tissue and differentiated into chondrocytes and the effect of baseline cultures (diff), PRP (prp), hyperosmolarity (os), base plus hyperosmolarity (diff + os), PRP plus hyperosmolarity (prp + os) next to the control group were studied in cartilage differentiation using specific stains such as Alcian blue, hematoxylin and eosin, and collagen type 2 and 10 immunohistochemistry. In addition, the expression of miR-27, miR-140, miR-199, miR-146, miR-101, and miR-145 was evaluated using real-time PCR. CONCLUSION: Human adipose tissue-derived MSCs with the ability to differentiate into adipocytes and osteocytes showed the properties of chondrocytes in all differentiation groups. Alkaline phosphatase (ALP) enzyme activity and calcium deposition were lower in the diff + os group than in other groups. Therefore, the diff + os group may be a more suitable environment for cartilage differentiation. Furthermore, 5% PRP concentration and hyperosmolarity showed a positive effect on miR-140, miR-199, miR-27, and, miR-146 and a negative effect on miR-101 and miR-145 on cartilage differentiation.

Assessment of trace elements in serum of acute lymphoblastic and myeloid leukemia patients.

AIM: Trace elements play a key role in human metabolism. The aim of the present study was to measure essential trace elements in the serum of patients with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). MATERIALS AND METHODS: 32 patients with ALL and 16 patients with AML were studied. The control group consisted of 36 subjects. Serum levels of the trace elements selenium, copper and zinc were measured by spectrophotometry. RESULTS: The mean of copper concentrations in the groups of patients with AML and ALL was significantly higher than in the control group (p < 0.0001), whereas serum levels of selenium and zinc were significantly lower in AML patients (p < 0.0001). Also in ALL patients the levels of selenium and zinc were significantly decreased compared with the control group (p < 0.0001; p = 0.0003). CONCLUSION: Our results indicate that the levels of zinc and selenium are significantly decreased and copper levels are significantly increased in the serum of patients with acute leukemia (AML, ALL).

Induction Effects of Bacteroides fragilis Derived Outer Membrane Vesicles on Toll Like Receptor 2, Toll Like Receptor 4 Genes Expression and Cytokines Concentration in Human Intestinal Epithelial Cells.

OBJECTIVE: Gastrointestinal (GI) tract, like other mucosal surface, is colonized with a microbial population known as gut microbiota. Outer membrane vesicles (OMVs) which are produced by gram negative bacteria could be sensed by Toll like receptors (TLRs). The interaction between gut microbiota and TLRs affects homeostasis and immune responses. In this study, we evaluated TLR2, TLR4 genes expression and cytokines concentration in Caco-2 cell line treated with Bacteroides fragilis (B. fragilis) and its OMVs. MATERIALS AND METHODS: In this experimental study, OMVs were extracted using sequential centrifugation and their physicochemical properties were evaluated as part of quality control assessment. Caco-2 cells were treated with B. fragilis and its OMVs (180 and 350 µg/ml). Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed to assess TLR2 and TLR4 mRNA expression levels. Pro-inflammatory (IFNᵧ) and anti-inflammatory (IL- 4 and IL-10) cytokines were evaluated by ELISA. RESULTS: B. fragilis significantly decreased TLR2 and slightly increased TLR4 mRNA levels in Caco-2 cell line. The TLR2 mRNA level was slightly increased at 180 and 350 µg/ml of OMVs. Conversely, the TLR4 mRNA level was decreased at 180 µg/ml of OMVs, while it was significantly increased at 350 µg/ml of OMVs. Furthermore, B. fragilis and its OMVs significantly increased and decreased IFNᵧ concentration, respectively. Anti-inflammatory cytokines were increased by B. fragilis and its OMVs. CONCLUSION: B. fragilis and its OMVs have pivotal role in the cross talk between gut microbiota and the host especially in the modulation of the immune system. Based on the last studies on immunomodulatory effect of B. fragilis derived OMVs on immune cells and our results, we postulate that B. fragilis derived OMVs could be possible candidates for the reduction of immune responses.

One missense mutation in exon 2 of the PAX5 gene in Iran.

The PAX5 gene, which encodes the B-cell specific activator protein, is one of the most important factors in determination of B-cell development. This gene is the main target of somatic mutations in acute B lymphoblastic leukemia (B-ALL). For example, point mutations, deletions, as well as other gene rearrangements may lead to several forms of B-cell malignancy. In this study, we obtained 50 blood samples from patients diagnosed with ALL, and screened for PAX5 mutations using sequencing in exons 1, 2 and 3. We found a heterozygous germline variant, c.113G>A (p.Arg38His), which affects the paired domain of PAX5. It seems that this mutation is pathogenic, but is recessive. Our findings suggest that this mutation in a single allele of the PAX5 gene is not sufficient to cause disease, and it is possible that other alleles are also involved in the onset of B-ALL.

Correlation between Plasma Interleukin-3, the α/ß Globin Ratio, and Globin mRNA Stability.

Background. Globin chain synthesis (GCS) analysis is used in the diagnosis of thalassemia. However, the wide reference range limits its use as a decisive diagnostic tool. It has been shown that α and ß globin mRNA increase through stimulation of cells by interleukin-3 (IL-3). Therefore, this study investigates the relationship between plasma IL-3 and the ß/α globin ratio. Methods. Blood samples were collected from 32 healthy participants on two occasions one month apart. GCS analysis, real-time PCR, and ELISA tests were conducted to determine the ß/α globin ratio, globin mRNA expression and stability rate, and IL-3 levels. Results. On the basis of IL-3 levels, the participants were divided in two groups. One group included participants who showed a significant increase in IL-3 as indicated by a significant rise in mean values of α, ß, and γ globin mRNA, α and ß globin, RBC, and hemoglobin. The other group included participants who showed no difference in IL-3 levels with no significant variations in the above-mentioned parameters. Conclusion. The results of this study indicate that IL-3 has an equivalent positive effect on α and ß globin chain synthesis. Therefore, IL-3 levels do not explain the wide reference range of the α/ß globin ratio.

Assessment of Thiopurine-based drugs according to Thiopurine S-methyltransferase genotype in patients with Acute Lymphoblastic Leukemia.

For the past half century, thiopurines have earned themselves a reputation as effective anti-cancer and immunosuppressive drugs. Thiopurine S-methyltransferase (TPMT) is involved in the metabolism of all thiopurines and is one of the main enzymes that inactivates mercaptopurine. 6-MP is now used as a combination therapies for maintenance therapy of children with acute lymphocytic leukemia (ALL). In all patients receiving mercaptopurine, there is a risk of bone marrow suppression. TPMT activity is inherited as a monogenic, co-dominant trait. More than 25 variants are known. Genetic testing is available for several TPMT variant alleles. Most commonly TPMT*2, *3A, and *3C are tested for, which account for >90% of inactivating alleles. Differences in DNA that alter the expression or function of proteins that are targeted by drugs can contribute significantly to variation in the responses of individuals.Genotyping may become part of routine investigations to help clinicians tailor drug treatment effectively. This success is mainly due to the development of combination therapies and stratification of patients according to risk of treatment failure and relapse, rather than the discovery of new drugs. The aim of this study was to investigate the effect of genotype or methyltransferase enzyme activity before starting therapy in children with ALL. This can prevent the side effect of thiopurine drugs. In fact, the common polymorphism of this enzyme in population could be a prognostic factor in relation to drug use and treatment of patients with ALL.

[Association of DNA polymorphisms of the growth hormone and prolactin genes with milk productivity in Yaroslavl and black-and-white cattle].

Polymorphisms of the prolactin (bPRL) and growth hormone (bGH) genes were studied comparatively in the Russian and German Black-and-White and Yaroslavl cattle breeds. Two polymorphisms were studied for each gene. In the case of the bPRL gene, the polymorphism of the 5'-untranslated region was examined by microsatellite analysis and the RsaI polymorphism of exon 3, by RFLP analysis. In the case of the bGH gene, the MspI polymorphism of intron III and the AluI polymorphism of exon 5 were assessed by RFLP analysis. Differences in allele and genotype frequencies were observed both between and within breeds. The heterozygosity at the RsaI marker was low (9.4%) in the Russian Black-and-White breed; that at the microsatellite of the bPRL gene was low (3.2-24%) in all breeds examined. Homozygotes BB at the bPRL gene, which had not been reported earlier for European cattle breeds, were detected in the German Black-and-White and Yaroslavl breeds (at frequencies 0.16 and 0.13, respectively). The frequency of allele MspI(-) of the bGH gene in the Yaroslavl breed was extremely low (0.02), comparable only with that of the Holstein cattle (0.02). The heterozygosity at the AluI polymorphism was higher than at the MspI polymorphism of the bGH gene and reached 55% in the Yaroslavl breed. Genotype BB of the RsaI polymorphism of the bPRL gene tended to show a negative association with the fat content in milk. The genotypes of the AluI polymorphism of the bGH gene were associated with the fat content in milk in the Yaroslavl (F = 4.56, P = 0.013) and German Black-and-White (F = 4.1, P = 0.014) breeds: the highest fat content in milk was observed in the subsample of cows with heterozygous genotype VL.

Effects of granulocyte-macrophage colony stimulating factor (GM-CSF) in vivo on cytokine production and proliferation by spleen cells.

GM-CSF is a potent stimulator of haematopoietic cells as well as some functions of granulocytes and macrophages. GM-CSF has many clinical uses; however, little is known about the effects of GM-CSF treatment in vivo on the responses of tissue lymphocytes in terms of secretion of Th-1 and Th-2 cytokines. We investigated this issue by measuring the responses of spleen cells from mice 24 h after treatment i.p. with saline or rmGM-CSF. GM-CSF at 16.7-50.0 microg/kg significantly increased (P < 0.01) spleen cellularity 2-2.5-fold and enhanced proliferative responses of non-stimulated (no mitogen) as well as concanavalin A (Con A)-stimulated spleen cells. Secretion of IFN-gamma by Con A (2.5 microg/ml)-stimulated spleen cells was significantly (P < 0.01) increased from 1.8 microg/ml by control spleen cells to 5.2 microg/ml by GM-CSF spleen cells. IL-10 production was greater (0.25 microg/ml, P < 0.05) by Con A-stimulated spleen cells from GM-CSF-treated mice compared to control spleen cells (0.06 microg/ml). By contrast, there were no significant differences in IL-4 production by Con A-stimulated spleen cells from the different groups. These results show that GM-CSF treatment increases spleen cellularity and primes lymphocytes for enhanced responses. The enhanced production of Th-1 cytokines by primed lymphocytes may partially explain the beneficial role of in vivo administration of GM-CSF in several clinical situations.

National university hospital discharge survey in the Islamic Republic of Iran.

We aimed to determine the rate of hospital discharge, average length of stay and bed occupancy rate in different hospital wards around the country. The survey consisted of health care service activities from 452 university-related hospitals in the country with a total of 59,348 beds. Because of missing data, the use of 56,315 of these beds was analysed. The countrywide discharge rate was 68.32 patients/1000 population per year with an average length of stay of 3.60 days and a bed occupancy rate of 57.44%. The data could be used to design a framework for prediction of inpatient health care facilities needed in the future.

National University Hospital discharge survey in the Islamic Republic of Iran

We aimed to determine the rate of hospital discharge, average length of stay and bed occupancy rate in different hospital wards around the country. The survey consisted of health care service activities from 452 university-related hospitals in the country with a total of 59 348 beds. Because of missing data, the use of 56 315 of these beds was analysed. The countrywide discharge rate was 68.32 patients/1000 population per year with an average length of stay of 3.60 days and a bed occupancy rate of 57.44%. The data could be used to design a framework for prediction of inpatient health care facilities needed in the future

The acute toxicity of phenol and unionized ammonia, separately and together, to the ephemeropteran Baetis rhodani (Pictet).

The acute toxicity of phenol and ammonia, singly and in combination, to larvae of the ephemeropteran, Baetis rhodani, was examined using a computerized continuous flow system in the laboratory. The 24 h LC50 values (with 95% confidence intervals) were calculated to be 29.9 (17.3-51.5) mg phenol litre(-1) and 8.2 (2.0-33.0) mg un-ionized ammonia litre(-1). When phenol and ammonia were together in low concentration (< 20 mg litre(-1) and < 3 mg litre(-1) [un-ionized], respectively), they expressed their toxicity additively, but at higher concentrations they behaved in a greater-than-additive manner. When the 24 h LC50 values were used to predict the toxicity of a coking plant effluent, containing principally ammonia and phenol, it was found that the B. rhodani larvae died quicker than expected indicating the presence of other toxic chemicals. This exemplifies the value of using direct toxicity assessments to detect the presence of unknown toxicants.

Polyunsaturated fatty acids in cultured cardiomyocytes: effect on physiology and beta-adrenoceptor function.

This study was carried out to investigate the influence of the membrane fatty acid composition on the basal electrical and contractile activities and the response to beta-adrenergic stimulation of rat cardiac muscle cells in culture. Cells were grown for 3 days in a conventional serum culture medium and then incubated for 24 h in synthetic media containing either n-6 or n-3 as the sole source of polyunsaturated fatty acids (PUFA). The n-6/n-3 ratio in the phospholipids was 0.9 in the n-3 cells and 13.1 in the n-6 cells compared with 6.3 in controls cells. Such modifications did not alter action potentials and the main parameters related to contraction, although shortening was slightly accelerated in the n-6 cells. On the other hand, the positive chronotropic effect induced by isoproterenol was more pronounced (P less than 0.01) in n-3 cells than in n-6 cells. In addition, isoproterenol caused a decrease in contraction duration and in shortening and relaxation time that was less pronounced in n-6 than in control cells (P less than 0.01, P less than 0.01 and P less than 0.05, respectively). These results suggest that the PUFA balance in the phospholipids may contribute to modulate the cardiac adrenergic receptor system but not the membrane properties related to electro-mechanical functions.

Oxygen and substrate deprivation on isolated rat cardiac myocytes: temporal relationship between electromechanical and biochemical consequences.

The effects of hypoxia and reoxygenation on action potentials (AP), contractions, and certain biochemical parameters were studied in isolated rat ventricular myocytes in monolayer culture in the presence and absence of glucose. Substrate deprivation alone had no influence on the basal properties. In the presence of glucose, a 4-h hypoxic treatment caused only a moderate decrease in AP amplitude and rate. In substrate-free conditions, hypoxia induced a gradual decline in plateau potential level and in AP duration and rate, followed by rhythm abnormalities and a failure of the electromechanical coupling. Spontaneous AP generation then ceased, and the resting potential decreased with increased duration of hypoxia. These alterations were associated with a decrease in ATP content, an increase in the lactate production, and a leakage of about 50% of the total cellular lactate dehydrogenase (LDH). Cells reoxygenated after 150 min hypoxia recovered near-normal function, while the ATP depletion ceased and the rate of lactate and LDH loss was diminished. Conversely, cells reoxygenated after 4 h hypoxia exhibited a further decrease of the residual resting polarization and no change in the decline of intracellular ATP and in the efflux of cytosolic lactate and LDH. The results of this study indicate that (1) the sequence and the extent of functional alterations are dependent on the duration of hypoxia in the absence of exogenous substrate and (2) ATP depletion and the amount of lactate and LDH released during hypoxia are related to the shift from reversibly to irreversibly damaged cells.

Modification of the n-6/n-3 fatty acid ratio in the phospholipids of rat ventricular myocytes in culture by the use of synthetic media: functional and biochemical consequences in normoxic and hypoxic conditions.

The respective roles of exogenous polyunsaturated fatty acids on the lipid composition, physiological properties and enzyme release was investigated on isolated cardiac muscle cells in normoxia and hypoxia. Rat neonatal ventricular myocytes were grown for 5 days in conventional serum-supplemented medium. Cells were then incubated for 24 h in fully chemically-defined media featuring a balanced fatty acid composition containing either linoleic acid (18:2 n-6) or linolenic acid (18:3 n-3) as sole polyunsaturated fatty acid source. Transmembrane potentials were monitored with microelectrodes and contractions with a photoelectric device. The radio of n-6 to n-3 phospholipid fatty acids increased from 6.3 in control cells to 20.2 in cells exposed to n-6 fatty acids (SM6) and decreased to 1.4 in those exposed to n-3 fatty acids (SM3). These modifications had no influence on the electrical and mechanical activities and on automaticity in normoxic conditions. The action potential depression under hypoxia was less severe in SM6 cells, whereas there was a better electrophysiological recovery upon reoxygenation in SM3 cells. However, the loss of lactate dehydrogenase during sustained hypoxic treatment was not affected by changes in phospholipid fatty acid pattern. These results suggest that the effect of the polyunsaturated fatty acid balance depends on the cellular function under study and on the environmental conditions.

Selective effects of ethanol on opiate receptor subtypes in brain.

Large concentrations of ethanol in vitro decreased ligand binding to mu and delta opiate receptors in the frontal cortex of the C57BL mouse, but did not alter binding to kappa opiate receptors. Mu and delta receptors were equally sensitive to the inhibitory effect of ethanol. Since the effects of ethanol were significant only in large concentrations, ethanol may alter opiate binding through its membrane lipid-perturbing actions, and the selectivity of the effects of ethanol may reflect differences in the microenvironments of the opiate receptor subtypes in membranes. After chronic ingestion of ethanol by mice, in vivo, there was a selective decrease in the number of mu receptors in the frontal cortex. This change may result from indirect effects of ethanol on the opiate receptor and may contribute to specific central effects of ethanol.

Comparison of typical and atypical benzodiazepines on the central and peripheral benzodiazepine receptors.

The affinities of typical and atypical benzodiazepines (BZs) for 3H-flunitrazepam (FLU) and 3H-Ro 5-4864, "peripheral" BZ, binding sites in the central nervous system (CNS) were compared. Each dissociation constant (Kd) value of the 3H-FLU binding sites in the cerebellum, cortex, hippocampus and spinal cord showed the similar results, although the binding maximum (Bmax) for the cortex and cerebellum yielded the largest value and the smallest Bmax appeared in the spinal cord. In contrast, Bmax for 3H-Ro 5-4864 binding sites were about three-fold higher in the spinal cord than in the cerebellum. Among the drugs tested, brotizolam demonstrated the highest affinity for 3H-FLU binding sites in the four regions. Typical and atypical BZs did not normally show different affinities for the two BZ receptor subtypes, except for CL 218,872 and Ro 5-4864. A series of compounds including Ro 5-4864, PK 11195, diazepam and brotizolam had high affinity for the 3H-Ro 5-4864 binding sites in the cerebellum and spinal cord. Other BZs did not show affinity for "peripheral" BZ binding sites in the CNS. In conclusion, brotizolam (or atypical BZ) had the binding affinity to both "central" and "peripheral" BZ receptors, like diazepam (a typical BZ).

The neonatal development of benzodiazepine receptor subtypes in the rat cerebral cortex and cerebellum.

The development of benzodiazepine receptor subtypes in the rat cerebral cortex and cerebellum was studied. The proportions of Type I (CL218872 sensitive binding sites) and Type II (CL218872 insensitive binding sites) in both regions of the neonatal brain were quite different from those of adult. In one day old rat cortex and cerebellum, about 95% of the benzodiazepine receptor were Type II receptor, while these receptors comprised about 50% and 15% of the total in the adult cortex and cerebellum, respectively. From 21 days onward, these ratios in both brain regions did not significantly change through adult age (over 55 days old). The proportion of receptor subtypes in the 21 day cortex (50% type I, 50% type II) was approximately equivalent to that seen in the cerebellum at the 9th day of age. The 3H-flunitrazepam specific binding sites in both brain regions increased markedly after birth, although Type II receptor binding sites did not show any notable gain. Type II receptor specific binding sites in the cortex were not significantly different between neonatal and adult, while these sites in the cerebellum to decrease after birth. These results suggest that Type I and Type II receptors seemed to develop independently during their embryogenesis.