Short-term effects of the 21-aminosteroid lazaroid tirilazad mesylate (PNU-74006F) and the pyrrolopyrimidine lazaroid PNU-101033E on energy metabolism of human peripheral blood mononuclear cells

Two groups of antioxidant compounds, the 21-aminosteroids and the pyrrolopyrimidines, have been found to act as neuroprotective drugs against lipid peroxidation in the injured CNS. Like glucocorticoids at high doses they are assumed to produce their effects at least in part by direct membrane stabilizing effects. In order to prove this hypothesis, we have investigated in this study the effects of these drugs on the energy metabolism of activated human peripheral blood mononuclear cells (PBMC) since these cells have been shown to serve as a suitable test system for substances affecting processes of ATP turnover. We compared the in vitro effects of (i) the 21-aminosteroid lazaroid tirilazad, (ii) the pyrrolopyrimidine lazaroid PNU-101033E and (iii) the glucocorticoid methylprednisolone on mitogen-induced respiration rate and ATP-consumption. We show that tirilazad inhibits concanavalin A-stimulated respiration rate and sodium cycling across the plasma membrane. The effect of methylprednisolone is similar indicating corresponding cellular mechanisms. However, unlike methylprednisolone, tirilazad produced no significant effect on calcium cycling across the plasma membrane. PNU-101033E in our test system caused cytotoxic effects on PBMC that did not allow us to quantify cellular actions on energy metabolism. Our results underline the view that tirilazad, first, is mimicking the high-dose immunosuppressive pharmacology of glucocorticoids such as methylprednisolone and, second, is likely to produce its therapeutic effects by direct physicochemical interactions with cellular membranes.     

The Design and Characterization of Oligospecific Antibodies for Simultaneous Targeting of Multiple Disease Mediators

Monoclonal antibodies are traditionally used to block the function of a specific target in a given disease. However, some diseases are the consequence of multiple components or pathways and not the result of a single mediator; thus, blocking at a single point may not optimally control disease. Antibodies that simultaneously block the functions of two or more disease-associated targets are now being developed. Herein, we describe the design, expression, and characterization of several oligospecific antibody formats that are capable of binding simultaneously to two or three different antigens. These constructs were generated by genetically linking single-chain Fv fragments to the N-terminus of the antibody heavy and light chains and to the C-terminus of the antibody C_H3 domain. The oligospecific antibodies were expressed in mammalian cells, purified to homogeneity, and characterized for binding to antigens, Fcγ receptors, FcRn, and C1q. In addition, the oligospecific antibodies were assayed for effector function, protease susceptibility, thermal stability, and size distribution. We demonstrate that these oligospecific antibody formats maintain high expression level, thermostability, and protease resistance. The in vivo half-life, antibody-dependent cellular cytotoxicity function, and binding ability to Fcγ receptors and C1q of the test oligospecific antibodies remain similar to the corresponding properties of their parental IgG antibodies. The excellent expression, biophysical stability, and potential manufacturing feasibility of these multispecific antibody formats suggest that they will provide a scaffold template for the construction of similar molecules to target multiple antigens in complex diseases.     

Further evidence for the contribution of the vascular endothelial growth factor gene in coronary artery disease susceptibility

Coronary artery disease (CAD) receives intensive attentions in the research of cardiovascular diseases, due to its high incidence and severe impact on the quality of life vascular endothelial growth factor (VEGF), a potent angiogenic and vascular permeability factor, has been strongly implicated in the pathogenesis of CAD. Genetic markers in different regions of the VEGF gene have a plausible role in modulating the risk of CAD. To identify the markers contributing to the genetic susceptibility to CAD, we examined the potential association between CAD and 10 single nucleotide polymorphisms (SNPs, rs699947, rs1570360, rs2010963, rs833068, rs3024997, rs3025000, rs3025010, rs3025020, rs3025030, rs3025039) of the VEGF gene using the MassARRAY system. Participants included 242 CAD patients and 253 healthy controls from a Chinese Han Population (He'nan Province, China). The allelic or genotypic frequencies of the rs699947 (5′ untranslated regions, 5′UTR) and rs2010963 (5′UTR) polymorphisms in the CAD patients were significantly different from those in the healthy controls. The CAD patients had significantly higher frequency of the rs699947 A allele (χ² = 11.141, P = 0.001, OR = 1.665, 95% CI = 1.232–2.250) and rs2010963 C allele (χ² = 13.593, P = 0.0002, OR = 1.611, 95% CI = 1.249–2.077). Strong linkage disequilibrium was observed in the rs699947–rs1570360–rs2010963 haplotype block (D’ > 0.9). Significantly more C–G–C haplotypes (P = 0.040) and significantly fewer C–G–G haplotypes (P = 0.0004) were found in the CAD patients. The possible association of rs699947 and rs2010963 with CAD risks warrant confirmation in independent case–control studies and may be informative for future investigations on the pathogenesis of CAD.     

The role of phagocytosis in IL-8 production by human monocytes in response to lipoproteins on Staphylococcus aureus

Cytokine responses to microbes are triggered by pattern recognition receptors, such as Toll-like receptors (TLRs), which sense pathogen-associated molecular patterns. Cell wall-associated triacylated lipoproteins in Staphylococcus aureus are known to be native TLR2 ligands that mediate host inflammatory responses against S. aureus. However, the mechanism by which these lipidated lipoproteins, which are buried under the thick S. aureus cell wall, work to stimulate TLR2 remains unclear. Heat-killed wild type S. aureus cells activated human monocytic THP-1 cells to produce proinflammatory cytokines, including interleukin (IL)-8, whereas the lipoprotein lipidation-deficient lgt mutant induced less than an eighth of the amount of IL-8 induced by the wild type. IL-8 induction in response to heat-killed S. aureus cells in THP-1 cells was not inhibited by a blocking antibody against cell surface TLR2, suggesting that intracellular TLR2 might be involved in the induction of IL-8 by S. aureus lipoprotein. The relationship between phagocytosis and IL-8 production in THP-1 cells was analyzed on a single-cell level by flow cytometry using fluorescein-labeled S. aureus cells and phycoerythrin-labeled anti-IL-8 antibody. Production of intracellular IL-8 was correlated with phagocytosis of S. aureus cells in THP-1 cells and in human peripheral blood mononuclear cells. Opsonization of S. aureus cells enhanced both the phagocytosis of S. aureus cells and the production of intracellular IL-8 in THP-1 cells. These results suggest that lipidated lipoproteins on S. aureus cells stimulate human monocytes after phagocytosis.     

Tetraspanins regulate the protrusive activities of cell membrane

Tetraspanins have gained increased attention due to their functional versatility. But the universal cellular mechanism that governs such versatility remains unknown. Herein we present the evidence that tetraspanins CD81 and CD82 regulate the formation and/or development of cell membrane protrusions. We analyzed the ultrastructure of the cells in which a tetraspanin is either overexpressed or ablated using transmission electron microscopy. The numbers of microvilli on the cell surface were counted, and the radii of microvillar tips and the lengths of microvilli were measured. We found that tetraspanin CD81 promotes the microvillus formation and/or extension while tetraspanin CD82 inhibits these events. In addition, CD81 enhances the outward bending of the plasma membrane while CD82 inhibits it. We also found that CD81 and CD82 proteins are localized at microvilli using immunofluorescence. CD82 regulates microvillus morphogenesis likely by altering the plasma membrane curvature and/or the cortical actin cytoskeletal organization. We predict that membrane protrusions embody a common morphological phenotype and cellular mechanism for, at least some if not all, tetraspanins. The differential effects of tetraspanins on microvilli likely lead to the functional diversification of tetraspanins and appear to correlate with their functional propensity.     

Induction of immunostimulatory cytokine genes expression in human PBMCs by a novel semiquinone glucoside derivative (SQGD) isolated from a Bacillus sp. INM-1

In the present study, a semiquinone glucoside derivative (SQGD) isolated from a radioresistant bacterium Bacillus sp. INM-1 was evaluated for its immunostimulatory activities. Human peripheral blood mononuclear cells (PBMCs) were stimulated by different doses (30–90 μg/ml) of SQGD for different time (3–12 h) intervals at 37 °C, and IL-12p40, IL-23p19, IL-10, RelA and c-Jun gene expression analysis was carried out by qRT-PCR method. SQGD dose dependent cytokines protein expression kinetic analysis was carried out using western blotting. As the results of SQGD (30 μg/ml) stimulation for 3 h at 37 °C, significant induction in IL-12p40, IL-23p19 and RelA gene expression was observed in PBMCs compared to unstimulated control cells. However, no such induction in IL-10 and c-Jun gene expression was observed. Time dependent protein expression study indicated significant increase in IL-12p40, IL-12p35, IL-23p19 and RelA protein expression at 3–6 h, which was found decrease at 12 h upon SQGD treatment. In contrast, IL-10 protein expression was found to enhance significantly at 12 h after SQGD treatment to the PBMCs. SQGD dose dependent study showed approximately similar level of induction in IL-12p40, IL-12p35, IL-23p19 and RelA proteins expression at all tested concentration (30–90 μg/ml) compared to control. However, no significant change in the IL-10 and c-Jun protein expression was observed at any SQGD concentration. SQGD treatment (0.25 mg/kg b wt.) was also found to enhance anti-keyhole Limpet Hemocynin (KLH) IgM antibodies significantly in the mice immunized by KLH.Thus, SQGD fraction stimulates cellular immunity by inducing immunostimulatory cytokines and humoral immunity by enhancing IgM antibodies and could be a promising immunostimulant. Further studies related to molecular mechanisms offering immunostimulation is underway, will certainly helpful to unravel its mode of action in the biological system.     

Droplet-digital PCR assay to detect Merkel cell polyomavirus sequences in chorionic villi from spontaneous abortion affected females

Droplet-digital polymerase chain reaction (ddPCR) technique was set up to detect/quantify Merkel cell polyomavirus (MCPyV) DNA in clinical specimens, including chorionic villi and peripheral blood mononuclear cells (PBMCs) from spontaneous abortion (SA)-affected females. This ddPCR assay showed high accuracy, sensitivity, and specificity in detecting MCPyV DNA cloned in a recombinant plasmid vector, the control. ddPCR was extended to MCPyV DNA to investigate/quantify its sequences in clinical samples. Overall, 400 samples were analyzed, that is, 100 chorionic villi and 100 PBMCs, from SA females (n = 100), the cases, and 100 chorionic villi and 100 PBMCs from females who underwent voluntary pregnancy interruption (VI, n = 100), the control. MCPyV DNA was detected in 4/100 (4%) and 5/100 (5%) of SA and VI chorionic villi, respectively. The mean viral DNA load was 1.99 ( ± 0.94 standard mean deviation [SD]) copy/10⁴ cells in SA and 3.02 ( ± 1.86 [SD]) copy/10⁴ cells in VI. In PBMCs, MCPyV DNA was revealed in 9/100 (9%) and 14/100 (14%) of SA and VI, with a mean of 2.09 ( ± 1.17 [SD]) copy/10⁴ cells and 4.09 ( ± 4.26 [SD]) copy/10⁴ cells in SA and VI, respectively. MCPyV gene expression analysis by quantitative PCR for the large T antigen (LT) and viral capsid protein 1 (VP1) showed their mRNAs in 2/4 (50%) SA- and 2/5 (40%) VI-MCPyV-positive samples. MCPyV DNA was detected/quantified using the ddPCR technique, in chorionic villi and PBMCs from SA and VI. In our experimental conditions, ddPCR provided a powerful tool to detect/quantify MCPyV DNA sequences in clinical samples.