increased genotoxic risk in lymphocytes from phototherapy-treated hyperbilirubinemic neonates

Background: Phototherapy is believed to be a safe method for the management of hyperbilirubinemia. However, there are some controversial issues regarding the genotoxic effects of phototherapy on DNA. The aim of this study was to investigate morphologically both phototherapy-induced DNA double-strand breaks [DSBs] and apoptosis in lymphocytes derived from jaundiced and non-jaundiced neonates

Methods: Newborns were divided into three groups, including phototherapy-treated [PT, n=30] jaundiced newborns with total serum bilirubin [TSB] levels >15 mg/dl, non-treated jaundiced newborns [C+, n=27], as positive, as well as healthy negative [C-, n=30] controls with TSB levels ranging from 10 and 15 mg/dl and less than 5 mg/dl, respectively. Lymphocytes were isolated from whole blood samples by Ficoll-isopaque density gradient centrifugation and then assessed for DNA damage and apoptosis before and 24 hours after incubation at 37 degree C in 5% CO2 using the neutral comet assay

Results: DSB levels were significantly much higher in the PT group compared to the controls before incubation but decreased remarkably after the incubation period. As expected, no statistical differences were found between the two control groups before and after incubations. The frequency of apoptotic cells showed no significant differences among all the three groups before incubation; however, it was significantly increased in the PT group after incubation

Conclusion: It seems that phototherapy in jaundiced infants is able not only to induce apoptosis in newborn lymphocytes but also to affect indirectly DNA integrity

L-carnitine Effectively Induces hTERT Gene Expression of Human Adipose Tissue-derived Mesenchymal Stem Cells Obtained from the Aged Subjects

BACKGROUND AND OBJECTIVES: Human mesenchymal stem cells (hMSCs) are attractive candidates for cell therapy and regenerative medicine due to their multipotency and ready availability, but their application can be complicated by the factors such as age of the donors and senescence-associated growth arrest during culture conditions. The latter most likely reflects the fact that aging of hMSCs is associated with a rise in intracellular reactive oxygen species, loss of telomerase activity, decrease in human telomerase reverse transcriptase (hTERT) expression and finally eroded telomere ends. Over-expression of telomerase in hMSCs leads to telomere elongation and may help to maintain replicative life-span of these cells. The aim of this study was to evaluate of the effect of L-carnitine (LC) as an antioxidant on the telomerase gene expression and telomere length in aged adipose tissue-derived hMSCs. METHODS: For this purpose, cells were isolated from healthy aged volunteers and their viabilities were assessed by MTT assay. Quantitative gene expression of hTERT and absolute telomere length measurement were also performed by real- time PCR in the absence and presence of different doses of LC (0.1, 0.2 and 0.4 mM). RESULTS: The results indicated that LC could significantly increase the hTERT gene expression and telomere length, especially in dose of 0.2 mM of LC and in 48 h treatment for the aged adipose tissue-derived hMSCs samples. CONCLUSION: It seems that LC would be a good candidate to improve the lifespan of the aged adipose tissue-derived hMSCs due to over-expression of telomerase and lengthening of the telomeres.

Induction of bone marrow stromal cells into cholinergic-like cells by nerve growth factor

Bone marrow stromal cells [BMSC] are used as a source for cell therapy in different model for neurological disorder such as stroke and spinal cord injury. However, the transdifferentiation of BMSC into cholinergic phenotype requires more investigation. BMSC were isolated from adult rats, pre-induced with [beta-mercaptoethanol [BME] and followed by nerve growth factor [NGF] induction. Neurofilaments of 68 kDa, 160 kDa and 200 kDa [NF-200, NF-160 and NF-68, respectively] immuno-staining were used for evaluating the transdifferentiation of BMSC into neuronal phenotype. The percentage of neurofilaments immuno-reactive cells was applied in order to evaluate the results at the pre-induction and the induction stages. Also, NeuroD and Oct-4 expressions, using RT-PCR, were used in assessing the progression of BMSC into neuronal lineage. Choline acetyltransferase immuno-reactive cells were used for estimating the percentage of cholinergic neuronal phenotype. Immuno-staining with anti-microtubule-associated protein-2 [MAP-2] and anti-synapsin-I antibodies was done in order to evaluate cell tendency for synaptogenesis. The yield of cholinergic neurons with BME as pre-inducer and NGF as inducer was 80%. Also, NF-200, NF-160, NF-68, MAP-2 and synapsin-I were detected in the transdifferentiated cells. RT-PCR showed the expression of NeuroD, while Oct-4 was not detected. BME as pre-inducer and NGF as inducer for BMSC transdifferentiation into cholinergic phenotype are potential sources in traumatic injury therapy in the central nervous system

Enhanced cytochrome C oxidase activity in WBC of COPD patients

Chronic obstructive pulmonary disease [COPD] is characterized by decreased expiratory flow rates, increased pulmonary resistance and hyperinflation. Cytochrome C Oxidase [COX] as a key oxidative enzyme modulates oxygen uptake and catalyzes the oxidation of reduced cytochrome C by molecular oxygen. In vitro studies indicate that the activity of COX can be directly regulated by the presence of molecular oxygen. Thus, a better understanding of the role of COX in patients with COPD can provide an important link between the availability of oxygen to tissues and the regulation of oxygen uptake and energy production in these patients. We studied 42 COPD patients [36 males, 6 females] with clinically stable conditions and 50 [42 males, 8 females] healthy sedentary volunteers of similar age. Whole blood was collected by venipuncture in sodium citrate tubes and WBCs were separated by Ficoll according to standard protocol and lysed with microtube pestle homogenizer. The homogenates were centrifuged and the supernatants were used as a cell extract for COX activity determination. Aliquots of this were assayed for total protein content and COX activity. Analysis of COX activity was performed using COX assay kit. Absolute specific COX activity was normalized for total protein. Relative activities were determined by dividing absolute specific COX activity on absolute specific citrate synthase activity. Mitochondrial COX activity and specific activity [absolute and relative] significantly increased in WBCs of patients with COPD in comparison with control samples [p< 0.05] These results indicated that the activity of COX was increased in WBCs of patients with COPD but whether this is a primary or secondary change relevant to hypoxic condition in these patients is not clear and needs further investigation.

Alteration in antioxidant capacity in patients with chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease [COPD] is a major public health problem that needs greater attention. Variability in the susceptibility to develop COPD is related to both genetic and environmental factors. Oxidative stress and inflammation are the major hallmarks of COPD and antioxidant status can be used as a biomarker to assess the risk of chronic diseases. We used the FRAP [ferric reducing ability of plasma] assay as a simple and powerful test for determination of the total antioxidant capacity of plasma of patients and normal subjects. The patients were selected by cross-sectional method. The mean average age +/- SD of normal subjects and patients was 56 +/- 4 and 60 +/- 2 years respectively. The spectrophotometeric method was used for this assay. The means of the FRAP assays in the patients were higher [about twice] than those of normal subjects. The differences were significant [p < 0.01]. The high levels of antioxidant capacity in the patient group indicated that the antioxidant defense system had been activated due to the oxidative stress and hypoxic condition. A though, FRAP assay can probably be used for demarcation of severity and risk of developing COPD, clinical follow-up and further investigation are required for the assessment of this hypothesis

Determination of alpha1-antitrypsin phenotypes and genotypes in Iranian patients

Alpha 1-antitrypsin [AAT] or alpha 1-protease inhibitor [PI] is the principal inhibitor of proteolytic enzyme in serum. Its phenotypic variability has been reported to be associated with liver, lung diseases and rheumatoid arthritis in humans. There is much documentation about high risk phenotypes of PI in some regions of the world, however, there are no reliable reports on these phenotypes and genotypes and their related diseases in Iranian population. The aim of this study was to determine PI phenotypes and genotypes in Iranian patients suffering from PI deficiency. For this purpose, whole blood samples from 307 patients suspected of diseases related to PI deficiency, and 156 healthy persons were examined. PI phenotypes and genotypes were determined by isoelectric focusing [IEF] and polymerase chain reactionrestriction fragment length polymorphism [PCR-RFLP], respectively. Allele frequencies from patients and normal subjects were compared. For reliability, a family study of the patients was also carried out. The PI phenotype frequencies of all six possible combinations of M, S and Z haplotypes in patients were: MM, 77.20%; MS, 6.18%; MZ, 7.17%; SS, 3.91%; ZZ, 4.56%; SZ, 0.98% and in normal subjects were: MM, 78.20%; MS, 5.76%; MZ, 15.38%; SS, 0.64%; 0% for ZZ and SZ. Analysis of data showed that there was a significant difference between patients [with liver, lung diseases and rheumatoid arthritis] and control subjects [p< 0.05]. In Conclusion, the allelic frequencies of S and Z in the patient group were 7.49% and 8.63%, while in the normal subjects were 5.13% and 4.17%, respectively. This is the first report of the prevalence of high risk alleles [Z and S] in patients suspected of PI deficiency and related diseases in Iran