Contribution of Multiparameter Flow Cytometry Immunophenotyping to the Diagnostic Screening and Classification of Pediatric Cancer

Pediatric cancer is a relatively rare and heterogeneous group of hematological and non-hematological malignancies which require multiple procedures for its diagnostic screening and classification. Until now, flow cytometry (FC) has not been systematically applied to the diagnostic work-up of such malignancies, particularly for solid tumors. Here we evaluated a FC panel of markers for the diagnostic screening of pediatric cancer and further classification of pediatric solid tumors. The proposed strategy aims at the differential diagnosis between tumoral vs. reactive samples, and hematological vs. non-hematological malignancies, and the subclassification of solid tumors. In total, 52 samples from 40 patients suspicious of containing tumor cells were analyzed by FC in parallel to conventional diagnostic procedures. The overall concordance rate between both approaches was of 96% (50/52 diagnostic samples), with 100% agreement for all reactive/inflammatory and non-infiltrated samples as well as for those corresponding to solid tumors (n = 35), with only two false negative cases diagnosed with Hodgkin lymphoma and anaplastic lymphoma, respectively. Moreover, clear discrimination between samples infiltrated by hematopoietic vs. non-hematopoietic tumor cells was systematically achieved. Distinct subtypes of solid tumors showed different protein expression profiles, allowing for the differential diagnosis of neuroblastoma (CD56^hi/GD2⁺/CD81^hi), primitive neuroectodermal tumors (CD271^hi/CD99⁺), Wilms tumors (>1 cell population), rhabdomyosarcoma (_nuMYOD1⁺/_numyogenin⁺), carcinomas (CD45⁻/EpCAM⁺), germ cell tumors (CD56⁺/CD45⁻/NG2⁺/CD10⁺) and eventually also hemangiopericytomas (CD45⁻/CD34⁺). In summary, our results show that multiparameter FC provides fast and useful complementary data to routine histopathology for the diagnostic screening and classification of pediatric cancer.     

Cardiomyocyte marker expression in a human lymphocyte cell line using mouse cardiomyocyte extract

Cell transplantation shows potential for the treatment of cardiac diseases. Embryonic stem cells, cord blood and mesenchymal stem cells have been suggested as sources for transplantation therapy. Because of some technical limitations with the use of stem cells, transdifferentiation of fully differentiated cells is a potentially useful alternative. We investigated whether human peripheral blood cells could transdifferentiate into cardiomyocyte. Transdifferentiation was induced in a human B lymphocyte cell line (Raji). Cardiomyocyte extract was prepared from adult mouse cardiomyocytes. The cells were treated with 5-aza-2-deoxycytidine and trichostatin A, permeabilized with streptolysin O, and exposed to the mouse cardiomyocyte extract. They were cultured for 10 days, 3 weeks and 4 weeks. Cardiomyocyte markers were detected with immunohistochemistry and flow cytometry. Immunocytochemistry revealed that some cells expressed myosin heavy chain, α-actinin and cardiac troponin T after 3 and 4 weeks. Flow cytometry confirmed these data. In cells exposed to trichostatin A and 5-aza-2-deoxycytidine and permeabilized in the presence of the cardiomyocyte extract, troponin T expression was seen in 3.53% of the cells and 3.11% of them expressed α-actinin. After exposure to the cardiomyocyte extract, some permeabilized cells adhered to the plate loosely; however, the morphology did not change significantly, and they continued to show a rounded shape after 4 weeks. Our treated lymphocytes expressed cardiomyocyte markers. Our results suggest that lymphocytes may be useful in future research as a source of cells for reprogramming procedures.     

Modified low density lipoproteins decrease the activity and expression of lysosomal acid lipase in human endothelial and smooth muscle cells

Lysosomal acid lipase (LAL), the only lysosomal enzyme involved in the hydrolysis of LDL-cholesteryl esters, is a key regulator of cellular cholesterol and fatty acid homeostasis and its deficiency contributes to the pathophysiology of various diseases. In this study, we questioned whether oxidized or glycated LDL, a common occurrence in atherosclerosis and diabetes, affect the activity and expression of LAL in vascular endothelial cells (EC) and smooth muscle cells (SMC). LAL activity and expression were assayed in cultured human EC and SMC exposed to oxidized LDL (oxLDL), (±)9-hydroxyoctadecadienoic acid-cholesteryl ester (HODE), glycated LDL (gLDL), or native LDL (nLDL) as control, in the presence or absence of LXR or PPAR-gamma agonists. We found that LAL activity and expression were significantly down regulated by oxLDL and HODE in EC, and by gLDL in SMC. The LXR agonist T0901317 reversed the decreased LAL expression in modified LDL- or HODE-exposed EC (P < 0.001) and in gLDL-exposed SMC, whereas PPAR-gamma agonist rosiglitazone induced a low effect only in EC. In conclusion, modified LDL down regulates LAL expression in human EC and SMC by a process involving the LXR signaling pathway. This is the first demonstration that modified LDL modulate LAL expression, in a cell specific manner.     

Gluten screening of several dietary supplements by immunochromatographic assay

Celiac disease (CD) is a chronic intestinal disorder of public health concern caused by gluten ingestion in sensitive individuals. Gluten is a protein found not only in gluten-containing food but also as normal component of drugs and dietary supplements. Detection of gluten in dietary supplements is a very important task required for establishing their gluten status, which is highly important for the safety of products consumed by CD and gluten-sensitive patients. In this paper, we investigated the presence of gluten in twenty one common dietary supplements from the national market using the immunochromatographic assay. This visual assay proved to be an efficient rapid tool for gluten screening as an alternative to the ELISA techniques. The results have shown the presence of gluten in 23.8% of the investigated samples (vitamins, minerals, plant extracts, probiotics supplements, lactoferrin, propolis supplements). The results provide information which may contribute to the completion of the existing lists of gluten-free pharmaceuticals. It is known that for CD patients obtaining accurate information about the gluten content of a particular item is a difficult and time-consuming process.     

Multi objective land allocation (MOLA) for zoning Ghamishloo Wildlife Sanctuary in Iran

Protected area zoning is an approach towards decreasing conflict between the possible uses of land and providing an opportunity for policy making. GIS data processing and spatial analysis along with decision analysis techniques, were used in this study to define zones for Ghamishloo Wildlife Sanctuary according to I.U.C.N. category IV in Isfahan Province of Iran. We used multi-criteria evaluation and multi-objective land allocation for zoning the sanctuary, which covers an area of about 866 km². First, we prepared a land use map of the area using classification of the IRS 6 (AWiFS) data of May 2005. For zoning this region, nine major criteria including wildlife habitat, vegetation cover, soil, distance to historical places, water resources, road, scenic beauties in the landscape, and also to residential areas, and to the core zone were considered. We used the analytical hierarchy process to derive weights of the criteria and then applied a weighted linear combination technique to combine the factors. The degree of suitability was defined by applying Fuzzy membership function. The wildlife sanctuary was divided into four zones including conservation, recreation, rehabilitation, and cultural zones, consisting of 69%, 21%, 9.5% and 0.5% of the area, respectively. Finally, multi-objective land allocation (MOLA) function was used for allocation of the sanctuary's land area to the zones which produced reasonable results.     

The Effect of Iron–Vitamin C Co-supplementation on Biomarkers of Oxidative Stress in Iron-Deficient Female Youth

There is no study that assessed the effect of co-supplementation of iron and vitamin C on biomarkers of oxidative stress in non-anemic iron-deficient females. We investigated the effects of iron vs. iron?+?vitamin C co-supplementation on biomarkers of oxidative stress in iron-deficient girls. In a double-blind randomized controlled clinical trial, performed among 60 non-anemic iron-deficient girls, participants were randomly assigned to receive either 50 mg/day elemental iron supplements or 50 mg/day elemental iron?+?500 mg/day ascorbic acid for 12 weeks. Fasting blood samples were taken at baseline, weeks 6 and 12 for assessment of biomarkers of oxidative stress. Compared with the baseline levels, both iron and iron?+?vitamin C supplementation resulted in a significant reduction in serum malondialdehyde (MDA) levels (P _time?<?0.001) and remarkable elevation in serum total antioxidant capacity (TAC; P _time?<?0.001) and vitamin C levels (P _time?=?0.001); however, comparing the two groups we failed to find an additional effect of iron?+?vitamin C supplementation to that of iron alone on serum TAC and MDA levels (P _group was not statistically significant). Iron?+?vitamin C supplementation influenced serum vitamin C levels much more than that by iron alone (P _group?<?0.01). We also found a significant interaction term between time and group about serum vitamin C levels while this interaction was not significant about serum TAC and MDA levels. In conclusion, we found that iron supplementation with/without vitamin C improve biomarkers of oxidative stress among non-anemic iron-deficient females and may strengthen the antioxidant defense system by decreasing reactive oxygen species. Co-supplementation of iron?+?vitamin C has no further effect on oxidative stress compared with iron alone.     

Autophagy modulates transforming growth factor beta 1 induced epithelial to mesenchymal transition in non-small cell lung cancer cells

Lung cancer is considered one of the most frequent causes of cancer-related death worldwide and Non-Small Cell Lung Cancer (NSCLC) accounts for 80% of all lung cancer cases. Autophagy is a cellular process responsible for the recycling of damaged organelles and protein aggregates. Transforming growth factor beta-1 (TGFβ₁) is involved in Epithelial to Mesenchymal Transition (EMT) and autophagy induction in different cancer models and plays an important role in the pathogenesis of NSCLC. It is not clear how autophagy can regulate EMT in NSCLC cells. In the present study, we have investigated the regulatory role of autophagy in EMT induction in NSCLC and show that TGFβ₁ can simultaneously induce both autophagy and EMT in the NSCL lines A549 and H1975. Upon chemical inhibition of autophagy using Bafilomycin-A1, the expression of the mesenchymal marker vimentin and N-cadherin was reduced. Immunoblotting and immunocytochemistry (ICC) showed that the mesenchymal marker vimentin was significantly downregulated upon TGFβ₁ treatment in ATG7 knockdown cells when compared to corresponding cells treated with scramble shRNA (negative control), while E-cadherin was unchanged. Furthermore, autophagy inhibition (Bafilomycin A1 and ATG7 knockdown) decreased two important mesenchymal functions, migration and contraction, of NSCLC cells upon TGFβ₁ treatment. This study identified a crucial role of autophagy as a potential positive regulator of TGFβ₁-induced EMT in NSCLC cells and identifies inhibitors of autophagy as promising new drugs in antagonizing the role of EMT inducers, like TGFβ₁, in the clinical progression of NSCLC.     

Mobile blood collection sites and their roles in providing safe and adequate supply: A six-year experience

Background

The determination of the role of mobile sites, as compared with fixed sites, in providing safe blood supply will help with the planning of future programs.

Materials and Methods

This retrospective study was carried out at the Khuzestan Blood Transfusion Organization from 2007 to 2012. Samples of the blood collected at mobile sites and fixed sites were compared. Comparisons took into consideration noticeable trends as well as the prevalence of major TTIs including HIV, HBV and HCV.

Results

The total number of blood donations from 2007 to 2012 was 621117 out of which 89590 (14.43%) were collected from mobile sites. The overall blood donation index was estimated at 23.8 per 1000 population. The prevalence of HIV, HBV and HCV in mobile site donations was 5.31, 320.34 and 117.4, and in fixed sites was 5.31, 214.72 and 104.83 per 100000 donations respectively. HBV prevalence in mobile sites was significantly higher than in fixed sites (p = 0.014).

Conclusion

The blood donation index in Khuzestan province is much better when compared with areas of similar socioeconomic status as well as neighboring countries. The allotment of blood units collected by mobile teams is lower than that of national reports. In addition, the prevalence of TTIs in mobile site blood donations was higher than at fixed sites.

     

Production of p-hydroxycinnamic acid from glucose in Saccharomyces cerevisiae and Escherichia coli by expression of heterologous genes from plants and fungi

Biological production of p-hydroxycinnamic acid (pHCA) from glucose can be achieved via deamination of the aromatic amino acids l-tyrosine or l-phenylalanine. Deamination of l-phenylalanine produces trans-cinnamic acid (CA) which is further hydroxylated in the para position to produce pHCA. However, when tyrosine is used as the substrate, trans-pHCA is produced in one step. This reaction is accomplished by phenylalanine ammonia-lyase (PAL)/tyrosine ammonia-lyase (TAL). Various bacteria and eukaryotic microorganisms were screened for their ability to produce a PAL/TAL enzyme with high TAL activity. Cell-free extracts of the yeast Rhodotorula glutinis possessed the highest level of TAL activity (0.0143U/mg protein) and the lowest PAL/TAL ratio (1.68) amongst species examined. The gene for this enzyme was cloned and expressed in Escherichia coli and the kinetics of the purified PAL/TAL determined. The recombinant PAL/TAL possessed characteristics similar to those of the wild-type enzyme. Functional expression of R. glutinis PAL/TAL enzyme in Saccharomyces cerevisiae cells containing the plant C4H P-450 and P-450 reductase enzymes from Helianthus tuberosus allowed conversion of glucose to pHCA. Addition of l-phenylalanine to these cultures increased pHCA production confirming its production via the PAL route. When R. glutinis PAL/TAL was synthesized in an E. colil-phenylalanine producing strain (ATCC 31882) and grown on glucose, pHCA was formed in the absence of the Cytochrome P-450 and the P-450 reductase enzymes underlining its production via the TAL route without CA intermediacy.