Gammadelta T-lymphocyte cytotoxic activity against Mycobacterium bovis analyzed by flow cytometry

Human exposure to bovine serum albumin (BSA) is very common and occurs through dietary and medicinal routes. Although great effort has been made to reduce exposure to BSA in pharmaceuticals to eliminate the threat of bovine spongiform encephalopathy, less attention has been given to assessing the human immune response after exposure to BSA. A sensitive quantitative radioimmunoassay was therefore developed to measure anti-BSA IgG antibodies in healthy subjects and in cancer patients participating in a randomized, placebo controlled clinical trial where they were exposed to BSA as an intrathoracic surgical sealant during pneumonectomy. Anti-BSA antibodies were detected in 55% of 60 healthy blood donors and 51% of 83 patients before lung cancer resection. The median antibody levels were the same in both cohorts; 0.086 μg/mL (range 0.016–19.5 μg/mL) for health blood donors and 0.062 μg/mL (range 0.009–44 μg/mL) for cancer patients. Six months after exposure of the cancer patients to BSA, the percentage of patients with anti-BSA antibody rose to 96% and the median antibody level rose to 19 μg/mL (range 0.009–258 μg/mL). Placebo-treated cancer patients showed no significant increase in the percentage of patients with anti-BSA antibody (41%) or the median antibody level (0.047 μg/mL; range 0.008–1.58) over 6 months. Western blot analysis confirmed the presence of anti-BSA antibody. Elevated levels of anti-BSA antibody were not associated with any detectable clinical events in either the healthy blood donors or the cancer patients.     

Properties of HTLV-I transformed CD8 T-cells in response to HIV-1 infection

HIV-1 infection studies of primary CD8⁺ T-cells are hampered by difficulty in obtaining a significant number of targets for infection and low levels of productive infection. Further, there exists a paucity of CD8-expressing T-cell lines to address questions pertaining to the study of CD8⁺ T-cells in the context of HIV-1 infection. In this study, a set of CD8⁺ T-cell clones were originated through HTLV-I transformation in vitro, and the properties of these cells were examined. The clones were susceptible to T-cell tropic strains of the virus and exhibited HIV-1 production 20-fold greater than primary CD4⁺ T-cells. Productive infection resulted in a decrease in expression of CD8 and CXCR4 molecules on the surface of the CD8⁺ T-cell clones and antibodies to these molecules abrogated viral binding and replication. These transformed cells provide an important tool in the study of CD8⁺ T-cells and may provide important insights into the mechanism(s) behind HIV-1 induced CD8⁺ T-cell dysfunction.     

磺化聚醚砜对光敏剂-亚甲蓝的吸附去除研究Ⅱ.磺化聚醚砜对血浆中亚甲蓝的吸附去除研究

研究了磺化聚醚砜吸附柱对血浆中亚甲蓝的吸附效果,并检测了流经吸附柱的牛血清白蛋白随时间变化的规律以及过柱前后血浆中主要生化指标的变化情况.结果:磺化聚醚砜对亚甲蓝的吸附效果明显优于聚醚砜对亚甲蓝的吸附效果;其对白蛋白的吸附较小;对血浆中主要生化指标的影响可以忽略不计.     

Serpin-1 splicing isoform J inhibits the proSpätzle-activating proteinase HP8 to regulate expression of antimicrobial hemolymph proteins in Manduca sexta

The innate immune system of insects include the Toll pathway, which is mediated by an extracellular serine proteinase cascade. In the tobacco hornworm, Manduca sexta, hemolymph proteinase 8 (HP8) promotes the synthesis of antimicrobial proteins by cleaving proSpätzle, the putative ligand of M. sexta Toll. HP8 has been observed to form a complex in hemolymph with M. sexta serpin-1, which has multiple alternative splicing isoforms. To investigate the regulation of HP8 and its processing of proSpätzle, we characterized the interaction of recombinant HP8 with serpin-1 isoform J (serpin-1J). Recombinant serpin-1J formed an SDS-stable complex with HP8 in vitro. The association rate constant of serpin-1J and HP8 was 1.3 × 10⁴ M⁻¹ s⁻¹, with a stoichiometry of inhibition of 5.4. Serpin-1J inhibited the cleavage of proSpätzle by HP8. Injection of serpin-1J into M. sexta larvae resulted in decreased bacteria-induced antimicrobial activity in hemolymph and reduced expression of cecropin, attacin and hemolin mRNA in fat body. Altogether, these results suggest that serpin-1J functions to inhibit HP8 and thereby modulates the concentration of active Spätzle to regulate the Toll pathway response in M. sexta.     

Preparation and properties of cryogel based on poly(hydroxypropyl methacrylate)

A novel supermacroporous poly(hydroxypropyl methacrylate) (p(HPMA)) cryogel was synthesized by cryogelation method at –16 °C. In this synthesis process, HPMA was used as a monomer, and N,N′-methylenebisacrylamide (MBAAm) was used as cross-linker; the reaction was carried out in the presence of redox initiator pair N,N,N′,N′-tetramethylene diamine (TEMED) and ammonium persulfate (APS). The effect of monomer concentration, cross-linker content, cooling rate, and dioxane co-solvent were determined with respect to the pore structure, mechanical behavior, swelling degree, and porosity of cryogel. The ESEM images indicate that the pore wall structure of cryogels was rough; moreover, small holes were present in the pore walls of cryogels. The result of compression test indicates that cryogels can be compressed by at least 80% without any breakdown. The result of swelling kinetics indicates that cryogels attain swelling equilibrium in 10 s. Furthermore, p(HPMA)-Cu²⁺ cryogel was prepared by loading Cu²⁺ ions on functionalized poly(hydroxypropyl methacrylate)-iminodiacetic acid (p(HPMA)-IDA) cryogel. We investigated the adsorption of bovine serum albumin (BSA) on cryogels. The results indicate that compared to Freundlich isotherm, Langmuir isotherm could more suitably describe the adsorption process of BSA on cryogels. Meanwhile, the adsorption capacity of p(HPMA)-Cu²⁺ cryogel was significantly greater than that of p(HPMA) cryogel. The maximum adsorption capacity of BSA on p(HPMA)-Cu²⁺ cryogel, which was treated with 1 M Cu²⁺ ions, was as high as 196.87 mg/g cryogel (equivalent to 20.48 mg/mL cryogel) at 25 °C and pH = 7.8; therefore, the maximum adsorption capacity of BSA on p(HPMA)-Cu²⁺ cryogel was 4.35 times higher than that of p(HPMA) cryogel. Thus, the adsorption capacity of cryogels was strongly influenced by Cu²⁺ concentration, moreover, temperature changes clearly affected the adsorption capacity of p(HPMA)-Cu²⁺cryogel. The adsorption capacity at 25 °C was twice as that at 15 °C. By calculating Gibbs free energy change (?G) of adsorption, we found that the adsorption process was spontaneous; moreover, adsorption process occurred better at higher temperature.     

The effects of systemic treatment with aminobisphosphonates and statins on circulating Vγ9Vδ2-T cells in patients with advanced cancer

Aminobisphosphonates (NBP) are used for treatment of metastatic bone disease. Frequently, patients undergoing NBP-treatment experience side-effects, known as acute phase response (APR), resulting from cytokine production by Vγ9Vδ2-T cells. As opposed to NBP, statins reduce intracellular phosphoantigen levels and prevent NBP-induced Vγ9Vδ2-T cell activation in vitro. We conducted a pilot study in patients with (bone-)metastasized malignancies receiving NBP-treatment and evaluated the phenotype and function of circulating Vγ9Vδ2-T cells in vivo and the effects of statins on Vγ9Vδ2-T cell responses and the associated APR. We observed reduced expression of perforin, granzyme B and HLA-DR on Vγ9Vδ2-T cells in patients treated with NBP and statins. However, statins could not prevent NBP-induced changes in circulating Vγ9Vδ2-T cell numbers or production of IFNγ and TNFα. Consistent with this, simvastatin could not prevent the occurrence of APR upon NBP-infusion. These observations call for the exploration of alternative strategies to prevent collateral APR upon NBP treatment.     

Human apolipoprotein A-I Gly26Arg stimulation of inflammatory responses via NF-kB activation: Potential roles in amyloidosis?

The cascade of molecular events leading to Human apolipoprotein A–I (apoA–I) amyloidosis is not completely understood, not even the pathways that determine clinical manifestations associated to systemic protein deposition in organs such as liver, kidney and heart. About twenty natural variants of apoA–I were described as inducing amyloidosis, but the mechanisms driving their aggregation and deposition are still unclear. We previously identified that the mutant Gly26Arg but not Lys107-0 induced the release of cytokines and reactive oxygen species from cultured RAW 264.7 murine macrophages, suggesting that part of the pathogenic pathway could elicit of an inflammatory signal. In this work we gained deep insight into this mechanism and determined that Gly26Arg induced a specific pro-inflammatory cascade involving activation of NF-κB and its translocation into the nucleus. These findings suggest that some but not all apoA–I natural variants might promote a pro-oxidant microenvironment which could in turn result in oxidative processing of the variants into a misfolded conformation.     

Biosynthesis of collagen I,II, RUNX2 and lubricin at different time points of chondrogenic differentiation in a 3D in vitro model of human mesenchymal stem cells derived from adipose tissue

The first aim of the study was to identify the most appropriate time for differentiation of adipose tissue derived mesenchymal stem cells (MSCs) to chondrocytes, through the self-assembly process. For this purpose, the expression of some chondrocyte markers, such as collagen type I, collagen type II, RUNX2 and lubricin was investigated at different times (7, 14, 21 and 28 days) of chondrogenic differentiation of MSCs, by using immunohistochemistry and Western blot analysis. The second aim of the study was to demonstrate that the expression of lubricin, such as the expression of collagen type II, could be a possible biomarker for the detection of chondrocytes well-being and viability in the natural self-assembling constructs, called ‘cell pellets’. Histology (hematoxylin and eosin) and histochemistry (alcian blue staining) methods were used to assess the chondrogenic differentiation of MSCs. The results showed that after 21 days the differentiated chondrocytes, when compared with MSCs cultured without chondrogenic medium (CD44, CD90 and CD105 positive; CD45, CD14 and CD34 negative), were able to produce significant quantities of collagen type I, collagen type II, and lubricin, suggesting hyaline cartilage formation. During the differentiation phase, the cells showed a reduced expression of RUNX2, a protein expressed by osteoblasts. Our studies demonstrated that 21 days is the optimum time for the implantation of chondrocytes differentiated from adipose tissue-derived MSCs. This information could be useful for the future development of cell-based repair therapies for degenerative diseases of articular cartilage.     

Expression of PDZ-binding kinase/T-LAK cell-originated protein kinase (PBK/TOPK) in human urinary bladder transitional cell carcinoma

The objective of this study was to evaluate the expression pattern of PDZ-binding kinase/T-LAK cell-originated protein kinase (PBK/TOPK) and its clinical significance in human bladder cancer (BC).