Optimization of methods for peripheral blood mononuclear cells isolation and expansion of human gamma delta T cells

Human Vg9/Vδ2 T cells (γδ T cells) are immune surveillance cells both in innate and adaptive immunity and are a possible target for anticancer therapies, which can induce immune responses in a variety of cancers. Small non-peptide antigens such as zoledronate can do activation and expansion of T cells in vitro. It is evident that for adoptive cancer therapies, large numbers of functional cells are needed into cancer patients. Hence, optimization of methods needs to be carried out for the efficient expansion of these T cells. Standardization of peripheral blood mononuclear cells (PBMCs) isolation was devised. Cytokines (interleukin 2 (IL-2) and interleukin 15 (IL-15)) and zoledronate were also standardized for different concentrations. It was found that an increased number of PBMCs were recovered when washing was done at 1100 revolution per minute (rpm). Significantly high expansion fold was (2524 ± 787 expansion fold) achieved when stimulation of PBMCs was done with 1 µM of zoledronate and both cytokines IL-2 and IL-15 supported the expansion and survival of cells at the concentrations of 100 IU/ml and 10 ng/ml respectively. 14-day cultures showed highly pure (91.6 ± 5.1%) and live (96.5 ± 2.5%) expanded γδ T cells. This study aimed to standardize an easy to manipulate technique for the expansion of γδ T cells, giving a higher yield.     

Sub-clinical mastitis and associated risk factors on lactating cows in the Savannah Region of Nigeria

ABSTRACT: BACKGROUND: Sub-clinical mastitis limits milk production and represents an important barrier to profitable livestock economics worldwide. Milk production from cows in Nigeria is not at optimum levels in view of many factors including sub-clinical mastitis. RESULTS: The overall herd-level prevalence rate for SCM was 85.33% (256/300 heads of cows) while the quarter-level prevalence rate of SCM was 43.25% (519/1,200 quarters). The prevalence of SCM was 50.67%, 43.67%, 39.67% and 39.13% for the left fore-quarter, right hind-quarter, left hind-quarter and right fore-quarter, respectively. The Rahaji breed had the highest prevalence of SCM with 65.91% (29/44), Sokoto while the White Fulani breed had the least with 32.39% (57/176). A total of 32.33% (97/300) had only one mammary quarter affected, 30.33% (91/300) had two quarters affected, 16.00% (48/300) had three quarters affected while 6.67% (20/300) had all the four quarters affected. A total of 53.00% had SCM in multiple quarters (159/300). The risk of SCM decreased significantly among young lactating cows compared to older animals (OR = 0.283; P < 0.001; 95%CI = 0.155; 0.516). The Rahaji breed had significantly higher risk compared with the White Fulani breed (OR = 8.205; P = 0.013; 95%CI = 1.557; 43.226). Improved sanitation (washing hands before milking) will decrease the risk of SCM (OR = 0.173; P = 0.003; 95%CI = 0.054; 0.554). CONCLUSION: SCM is prevalent among lactating cows in the Nigerian Savannah; and this is associated with both animal characteristics (age, breed and individual milk quarters) and milking practices (hand washing).Good knowledge of the environment and careful management of the identified risk factors with improved sanitation should assist farm managers and veterinarians in implementing preventative programmes to reduce the incidence of SCM.     

Selective Divalent Cobalt Ions Detection Using Ag2O3-ZnO Nanocones by ICP-OES Method for Environmental Remediation

Here, we have synthesized Ag₂O₃-ZnO nanocones (NCs) by a wet-chemical route using reducing agents at low temperature. The structural, optical and morphological properties of Ag₂O₃-ZnO NCs were investigated by several conventional techniques such as powder XRD, XPS, FESEM, XEDS, FTIR and UV/vis. spectroscopy. The analytical parameters of prepared NCs were also calculated for a selective detection of divalent cobalt [Co(II)] prior to its determination by inductively coupled plasma-optical emission spectrometry (ICP-OES). The selectivity of NCs toward various metal ions, including Cd(II), Co(II), Cr(III), Cu(II), Fe(III), Ni(II), and Zn(II) was studied. Results of the selectivity study demonstrated that Ag₂O₃-ZnO NC phase was the most selective towards Co(II) ion. The uptake capacity for Co(II) ion was experimentally calculated to be ∼76.69 mgg⁻¹. Moreover, adsorption isotherm data provided that the adsorption process was mainly monolayer on homogeneous adsorbent surfaces of Ag₂O₃-ZnO NCs. Kinetic study revealed that the adsorption of Co(II) on Ag₂O₃-ZnO NCs phase followed the pseudo-second-order kinetic model. In addition, thermodynamic results provided that the adsorption mechanism of Co(II) ions on Ag₂O₃-ZnO NCs was a spontaneous process and thermodynamically favorable. Finally, the proposed method was validated by applying it to real environmental water samples with reasonable results.     

Binding of Net-Fla,a Netropsin-Flavin Hybrid Molecule,to DNA: Molecular Mechanics and Dynamics Studies in vacuo and in Water Solution

Abstract

We have studied the binding of the hybrid netropsin-flavin (Net-Fla) molecule onto four sequences containing four A.T base pairs. Molecular mechanics minimizations in vacuo show numerous minimal conformations separated by one base pair. 400 ps molecular dynamics simulations in vacuo have been performed using the lowest minima as the starting conformations. During these simulations, the flavin moiety of the drug makes two hydrogen bonds with an amino group of a neighboring guanine. A 200 ps molecular dynamics simulation in explicit water solution suggests that the binding of Net-Fla upon the DNA substrate is enhanced by water bridges. A water molecule bridging the amidinium of Net-Fla to the N3 atom of an adenine seems to be stuck in the dmg-DNA complex during the whole simulation. The fluctuations of the DNA helical parameters and of the torsion angles of the sugar-phosphate backbone are very similar in the simulations in vacuo and in water. The time auto-correlation functions for the DNA helical parameters decrease rapidly in the picosecond range in vacuo. The same functions computed from the water solution molecular dynamics simulations seem to have two modes: the rapid mode is similar to the behavior in vacuo, and is followed by a slower mode in the 10 ps range.     

The activated hepatic glucocorticoid-receptor complex: A simple one-step method for its separation from nonactivated complex

Protamine sulfate was found to precipitate completely the nonactivated [³H]-dexamethasone-receptor complex of rat liver. This observation was then used as the basis of a method to separate activated from nonactivated complex. Thus, addition of 10 mg/ml of protamine sulfate to the rat hepatic cytosol [³H]dexamethasone-receptor complex, incubated at 0–4°C for 2 hr, resulted in the complete precipitation of [³H]dexamethasone-receptor complex. The remaining supernatant obtained on centrifugation at 800g was unable to bind either to nuclei or to DNA-cellulose. An increase in temperature to 25°C or the addition of 10 mm CaCl₂ to the cytosol resulted in the appearance of activated [³H]dexamethasone-receptor complex in the supernatant obtained by addition of protamine sulfate. This was determined by characteristic binding to nuclei or DNA cellulose and by pI. Protamine sulfate could not affect the separation of activated [³H]dexamethasone-receptor complex at salt concentrations above 100 mm NaCl. This procedure therefore had to be carried out under conditions of relatively low ionic strength. Finally, a one-step rapid method is described for the separation of activated [³H]dexamethasone-receptor complex from nonactivated receptor complex. The homogeneous population of activated complex thus obtained should have considerable applicability in studies of the mechanisms of in vitro glucocorticoid-receptor activation.