Prognostic role of natural killer cells in pediatric mixed cellularity and nodular sclerosing Hodgkin's disease

OBJECTIVE: To study natural killer cells' spontaneous cytotoxic capacity against tumor cells and their prognostic significance in classical Hodgkin's disease. STUDY DESIGN: Thirty-eight pediatric mixed cellularity and nodular sclerosing Hodgkin's disease patients were included in the study. Immunohistochemical staining was performed for natural killer cells in the background using the monoclonal antibody CD57 in serial sections of B5-formalin-fixed, paraffin-embedded tissue blocks. CD57-positive cells were counted with an immersion objective among 5,000 cells on representative areas of the tumors. The degree of natural killer cells was classified as low (<150 cells) or high (> or = 150 cells). Multivariate regression analysis was performed to determine the differences between patients with and without relapse. RESULT: The mean of CD57-positive cell numberfor all the cases was 173.42 +/- 117.34 (range, 20-500). CD57-positive cells were high in 21 cases and low in 17. The mean of CD57-positive cell numbers was 191.85 +/- 115.33 in the disease-free group and 84.44 +/- 57.90 in the relapsing group. Log rank analysis showed statistical significance between event-free survival and number of CD57-positive cells (P = .0207). CONCLUSION: In multivariate analysis, CD57 expression proved to be a prognostic factor independent from otherfactors. As a result, CD57 expression by background natural killer cells may be used as a prognostic parameter in mixed cellularity and nodular sclerosing Hodgkin's disease.     

Polyaniline grafted polyacylonitrile conductive composite fibers for reversible immobilization of enzymes: Stability and catalytic properties of invertase

Polyacylonitrile fibers (PAN) surfaces were modified with chemical polymerization of conductive polyaniline (PANI) in the presence of potassium dichromate as an oxidizing agent. The effect of aniline concentration on the grafting efficiency and on the electrical surface resistance of PAN/PANI composite fibers was investigated. The surface resistance of the conductive composite fibers in this work was found to be between 8.0 and 0.5 kΩ/cm. As the amount of grafted PANI increased on the PAN fibers the electrical resistance of composite fibers decreased. The PAN/PANI composite fibers were characterized by SEM and FTIR studies. Composite PAN/PANI fibers were used for reversible immobilization of invertase. The immobilization efficiency and the activity of the immobilized invertase (from 1.0 mg/mL invertase solution at pH 5.5) were increased with increasing PANI contents of the composite fibers. The maximum amount of immobilized enzyme onto composite fibers containing 2.0% PANI was about 76.6 mg/g. The optimum pH for the free enzyme was observed at 5.0. On the other hand, immobilized invertase yielded a broad optimum pH profile between pH 5.0 and 7.0. Immobilized invertase exhibited 83% of its original activity even after two months storage at 4 °C while the free enzyme showed only 7% of its initial activity.     

CD4+CD25hiFOXP3+ Regulatory T Cells and Cytokine Responses in Human Schistosomiasis before and after Treatment with Praziquantel

Background

Chronic schistosomiasis is associated with T cell hypo-responsiveness and immunoregulatory mechanisms, including induction of regulatory T cells (Tregs). However, little is known about Treg functional capacity during human Schistosoma haematobium infection.

Methodology

CD4⁺CD25^hiFOXP3⁺ cells were characterized by flow cytometry and their function assessed by analysing total and Treg-depleted PBMC responses to schistosomal adult worm antigen (AWA), soluable egg antigen (SEA) and Bacillus Calmette-Guérin (BCG) in S. haematobium-infected Gabonese children before and 6 weeks after anthelmintic treatment. Cytokines responses (IFN-γ, IL-5, IL-10, IL-13, IL-17 and TNF) were integrated using Principal Component Analysis (PCA). Proliferation was measured by CFSE.

Principal Findings

S. haematobium infection was associated with increased Treg frequencies, which decreased post-treatment. Cytokine responses clustered into two principal components reflecting regulatory and Th2-polarized (PC1) and pro-inflammatory and Th1-polarized (PC2) cytokine responses; both components increased post-treatment. Treg depletion resulted in increased PC1 and PC2 at both time-points. Proliferation on the other hand, showed no significant difference from pre- to post-treatment. Treg depletion resulted mostly in increased proliferative responses at the pre-treatment time-point only.

Conclusions

Schistosoma-associated CD4⁺CD25^hiFOXP3⁺Tregs exert a suppressive effect on both proliferation and cytokine production. Although Treg frequency decreases after praziquantel treatment, their suppressive capacity remains unaltered when considering cytokine production whereas their influence on proliferation weakens with treatment.     

Comparing ethylene glycol with glycerol and with or without dithiothreitol and sucrose for cryopreservation of bull semen in egg-yolk containing extenders

There are few studies performed for investigating the roles of different ratio and cryoprotectants with dithiothreitol or sucrose on sperm motility characteristics and antioxidant capacities of post-thawed bull spermatozoa. The objectives of this study were to compare glycerol (G) and ethylene glycol (EG) at different concentrations as cryoprotectants and dithiothreitol (D) or sucrose (S) (with/without) as antioxidants in Tris extender for cryopreservation of bull semen. Twenty-four ejaculates obtained from three bulls were included in the study. Each ejaculate was split into four equal aliquots and diluted using both of the Tris extenders with glycerol (5% or 7%) or ethylene glycol (3% or 5%). After that, each extenders were split into three equal aliquots and diluted using both of the dithiothreitol 5 mM or sucrose 25 mM, and control (without additives) was cooled to 4 °C and frozen in 0.25-ml French straws. when compared to control, different doses cryoprotectants and antioxidants addition no significantly increased the percentages of post-thaw sperm progressive and motitilities, acrosome abnormality and plasma membrane integrity (P > 0.05). However, EG3 + S yielded the greatest percentages of the total abnormality (P < 0.05). As regard to antioxidant activities G7 and EG5 led to lowest MDA activity with or without D or S but, these results were not supported to the GPx activity (P < 0.01). The sperm motion characteristics such as VAP, VCL, ALH and BCF gave significantly different results (P < 0.05). When compared the DNA integrity, different doses cryoprotectants without antioxidants addition significantly increased the percentages of the tail intensity and tail moment (P < 0.05). There were no significant differences observed in non-return rates among all treatment groups (P > 0.05).     

Value of human amniotic epithelial cells in tissue engineering for cornea

Human amniotic epithelial cells (hAECs) are potentially one of the key players in tissue engineering due to their easy availability. The aim of the present study was to develop an optimal isolation and transportation technique, as well as to determine the immunophenotype and epithelial gene expression of hAECs. Amnion was mechanically peeled off from the chorion and digested with trypsin-ethylenediaminetetraacetic acid. The isolated hAECs were cultured in medium containing 10 ng/mL epidermal growth factor until P4. The epithelial gene expression, cell surface antigen and protein expression of hAECs were analyzed by quantitative polymerase chain reaction, flow cytometry and immunocytochemistry. hAECs were also cultured in adipogenic, osteogenic and neurogenic induction media. The best cell yield of hAECs was seen in the digestion of 15 pieces of amnion (2 × 2 cm) and isolated 30 min after digestion with trypsin. F12:Dulbecco's modified eagle medium was the best medium for short term storage at 4 °C. hAECs expressed CD9, CD44, CD73 and CD90, and negligibly expressed CD31, CD34, CD45 and CD117. After serial passage, CK3, CK19 and involucrin gene expressions were upregulated, while p63, CK1 and CK14 gene expressions were downregulated. Sustained gene expressions of integrin β1 and CK18 were observed. At initial culture, these cells might have stem-like properties. However, they differentiated after serial passage. Nonetheless, hAECs have epithelial stem cell characteristics and have the potential to differentiate into corneal epithelial cells. Further investigations are still needed to elucidate the mechanism of differentiation involved and to optimize the culture condition for long term in vitro culture.     

Effects of Selenium Supplementation on Antioxidant Defense and Glucose Homeostasis in Experimental Diabetes Mellitus

The objective of this study was to investigate the effects of different forms of Se supplementation on the antioxidant defense and glucose homeostasis in experimental diabetes. Sodium selenate (SS) or selenomethionine (SM) were administered (2 μmol Se kg⁻¹ day⁻¹) via orogastric route to streptozotocine (STZ)-induced diabetic rats in addition to basal diet for 12 weeks. Glucose levels in whole blood, glutathione peroxidase (GSH-Px) activity in erythrocytes, Se and fructosamine levels in plasma were evaluated monthly. Plasma Se levels increased significantly in all diabetic groups compared to basal measurements, being more prominent in SM group [p(SM₃/SM₀) = 0.018]. The increase in GSH-Px activities was significant at the end of the second month in SS [p(SS₂/SS₀) = 0.028], whereas at the end of the third month in SM the value was lower [p(SM₃/SM₀) = 0.018] and the unsupplemented diabetic control (DC) groups, p(DC₃/DC₀) = 0.012. Glucose increased significantly only in DC group. Fructosamine increased gradually in all diabetic groups, being significant in DC and SS groups. At the end of the third month, highest fructosamine levels were observed in SS group, which were significantly higher than the SM group [p(SM/SS) = 0.010]. In conclusion, Se augmented the antioxidant defense by increasing GSH-Px activity and this effect was more prominent when Se was supplemented as SM, which exerted positive effects also on glucose homeostasis.     

Comparison of five methods for determination of total plasma protein concentration

Quantitation of exact total protein content is often a key step and is common to many applications in general biochemistry research and routine clinical laboratory practice. Before embarking on any type of protein analysis, particularly comparative techniques, it is important to accurately quantitate the amount of protein in the sample. In order to assess the quality of total protein estimation results, five methods were tested and were applied to the same pooled plasma sample. For this aim, Bradford (Coomassie Brilliant Blue), Lowry (Folin-Ciocalteau), Biüret, Pesce and Strande (Ponceau-S/TCA), and modified method of Schaffner-Weismann (Amido Black 10B) were used. The last two methods employ simultaneous precipitation of proteins with the acid containing dye solutions followed by dissolution of precipitate in a NaOH solution. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in literature, accuracy and reproducibility/coefficient of variation. All of the methods tested show a CV % < 6. Besides pooled plasma, a known concentration of human serum albumin was also analyzed and discussed by means of standardization of plasma total protein content.     

Persistence of hyperbaric oxygen-induced oxidative effects after exposure in rat brain cortex tissue

Hyperbaric oxygen (HBO) causes oxidative stress in several organs and tissues. Due to its high rate of blood flow and oxygen consumption, the brain is one of the most sensitive organs to this effect. Many studies have reported oxidative effects of HBO, but there is no comprehensive data about how long this effect persists. The aim of this study was to elucidate the duration of HBO-induced oxidative/antioxidant action. Male Sprague-Dawley rats were divided into 5 groups. Except for the controls, the animals were subjected to 100% oxygen for 2 h at 3 atm and differed from each other by the time to dissection after exposure that began at 30, 60, 90, or 120 min. Thiobarbituric acid-reactive substances (TBARS), as well as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity was determined in brain cortex tissue. Additionally, nitrite-nitrate (NO(x)) concentrations were measured. All measured parameters were found to be significantly increased 30 min after exposure. SOD and GSH-Px levels persisted at significantly high levels for 60 min. In conclusion, the oxidative effect of HBO was shown to persist only for 1 h. Further studies should be performed to elucidate the possible molecular interactions during this period.