Determination of cell expansion and surface molecule expression on anti-CD3/28 expanded CD4+ T cells.

CD4+ T cell immunotherapy has potential for treatment in HIV-infected patients. A large number of expanded CD4+ T cells and confirmation of functional-related phenotypes are required for ensuring the successful outcomes of treatment. Freshly isolated CD4+ T cells from healthy donors were activated with anti-CD3/28-coated magnetic beads at different bead-to-cell ratios and cultured in the absence and presence of IL-2 supplementation for 3 weeks. Fold expansion, cell viability, growth kinetic and lymphocyte subset identities were determined. Data demonstrated that a 1:1 bead-to-cell ratio rendered the highest expansion of 1044-fold with 88% viability and 99.5% purity followed by the 2:1 and 0.5:1 ratios. No significant difference in proliferation and phenotypes was found between non-IL-2 and IL-2 supplementation groups. Several specific surface molecule expressions of the expanded cells including chemokine receptors, adhesion molecules, co-stimulatory molecules, activation molecules, maturation markers, cytokine receptors and other molecules were altered when compared to the unexpanded cells. This optimized expansion protocol using the 1:1 bead-to-cell ratio of anti-CD3/28-coated magnetic beads and culture condition without IL-2 supplementation provided the satisfactory yield with good reproducibility. Specific surface molecule expressions of the expanded cells presented potential roles in proliferation, differentiation, homeostasis, apoptosis and organ homing.

In vitro anti-lithogenic activity of lime powder regimen (LPR) and the effect of LPR on urinary risk factors for kidney stone formation in healthy volunteers.

Hypocitraturia, hypokaliuria, and increased oxidative stress are common lithogenic risk factors found in nephrolithiasis patients, especially in Thailand. We previously developed lime powder regimen (LPR), and demonstrated that LPR delivered citraturic, alkalinizing, and antioxidative effects in kidney stone patients. In this study, in vitro anti-lithogenic activity, in vivo acute toxicity, and crossover-designed phase 1 trial (in 13 healthy volunteers) of LPR were investigated. LPR inhibited the growth of calcium oxalate monohydrate (COM) crystals in dose-dependent manner, and inhibited the intracellular production of reactive oxygen species (ROS) in COM-treated HK-2 cells. LPR did not significantly alter viability of HK-2 cells. No acute toxicity was detected in mice orally fed with LPR (10 g/kg). No adverse effect and complaint of LPR ingestion (5 g/dose) were observed in the tested volunteers. Plasma citrate was elevated at 30 min after LPR load, which was higher than the water load control. Plasma potassium was significantly elevated at 30 min after LPR load and remained high for 2 h, and at 2 h, it was significantly higher than the water load. Urinary citrate was significantly increased at 1 h after LPR load and remained high for 2 h, and at 2 h, it was significantly higher than the water load. Urinary potassium was significantly increased at 1 h after LPR load and remained high for 3 h, and its levels at 1, 2, and 3 h were significantly higher than the water load. Urinary total antioxidant status was significantly increased at 2 h after LPR load. In conclusion, LPR had an inhibitory effect on COM growth and exerted as antioxidant to attenuate ROS production in the COM-treated renal tubular cells. LPR provided citraturic, kaliuric, and antioxidative responses in healthy individuals without any adverse events. This suggests that LPR is well tolerated and safe for daily consumption.