Colcemid treatment of myeloma prior to cell fusion increases the yield of hybridomas between myeloma and splenocyte

Effect of Colcemid treatment of myeloma (X63-Ag8-6.5.3.) prior to fusion with mouse spleen cell was studied in terms of hybridoma formation. Spleen cells from BALB/c mice immunized with various soluble antigens were fused with the myeloma cells by using polyethylene glycol solution. Colcemid treatment of myeloma cells prior to fusion increased the average number of hybridoma colonies per well by 26-570%. The yield of hybridomas producing antigen-specific antibodies was also higher with the Colcemid treatment. The results suggest that most of the proliferative hybridomas are formed by fusion of cells in the M-phase of the cell cycle.     

Carbohydrate carbonyl mobility--the key process in the formation of alpha-dicarbonyl intermediates

Covalently cross-linked proteins are among the major modifications caused by the advanced Maillard reaction. In the present study, the formation pathway of the dideoxyosone N6-(2,3-dihydroxy-5,6-dioxohexyl)-L-lysine is shown. To elucidate the formation of this glucose-derived dideoxyosone D-lactose (O-beta-D-galp-(1-->4)-D-glcp) and D-glucose-6-phosphate were incubated with lysine in the presence of the trapping reagent o-phenylenediamine (OPD). Synthesis and unequivocal structural characterization were reported for the quinoxalines of the dideoxyosones N6-(5,6-dihydroxy-2,3-dioxohexyl)-L-lysine and N6-(2,3-dihydroxy-4,5-dioxohexyl)-L-lysine, respectively. Additionally, dicarbonyl compounds derived from D-erythrose, D-glycero-D-mannoheptose, and D-gluco-L-talooctose were synthesized and structurally characterized.     

Leishmania infantum: prime boost vaccination with C-terminal extension of cysteine proteinase type I displays both type 1 and 2 immune signatures in BALB/c mice

The aims of current study are to describe the immunogenicity and protective efficacy of prime boost vaccine using C-terminal extension (CTE) of cysteine proteinase type I of Leishmania infantum in BALB/c mice. Group I as vaccinated group primed with 100 microg of pcDNA-CTE and 3 weeks later boosted with combination of 30 microg rCTE, 50 microg of CpG and Montanide 720. Groups II and III were served as control groups. Although, this vaccination regimen did not protect mice against the infectious challenge but it was highly immunogenic. IgG2a has been raised strongly against rCTE in contrast to IgG1 and remains high at every time point under study. By analysis of CTE synthetic peptides (CTE100) before challenge, both IgG1 and IgG2a were produced and for all overlapping synthetic peptides (CTE 1-8) IgG1 raised significantly. This statue is changed at 7 weeks after challenge and only CTE2 and CTE3 have shown to induce considerable amount of IgG1. In all groups, the level of IL-5 started to increase with high concentration shortly passing only 3 weeks after infectious challenge. In compare with two control groups, the vaccinated group produced significantly higher level of IL-5 at 7 weeks post-infection. The parasite burden of all groups is similar at 4 weeks post-challenge in both liver and spleen. In contrast, at 8 weeks post challenge, the spleen of the vaccinated group showed significantly higher level of parasite load in compare with two control groups. This study demonstrated that immunization with CTE display both type 1 and 2 immune signatures in experimental murine model of L. infantum infection.     

Synthetic adjuvants for vaccine formulations: evaluation of new phytol derivatives in induction and persistence of specific immune response

Terpenoids are ubiquitous natural compounds that have been shown to improve vaccine efficacy as adjuvants. To gain an understanding of the structural features important for adjuvanticity, we studied compounds derived from a diterpene phytol and assessed their efficacy. In a previous report, we showed that phytol and one of its derivatives, PHIS-01 (a phytol-derived immunostimulant, phytanol), are excellent adjuvants. To determine the effects of varying the polar terminus of PHIS-01, we designed amine and mannose-terminated phytol derivatives (PHIS-02 and PHIS-03, respectively). We studied their relative efficacy as emulsions with soluble proteins, ovalbumin, and a hapten-protein conjugate phthalate-KLH. Immunological parameters evaluated consisted of specific antibody responses in terms of titers, specificities and isotype profiles, T cell involvement and cytokine production. Our results indicate that these new isoprenoids were safe adjuvants with the ability to significantly augment immunogen-specific IgG1 and IgG2a antibody responses. Moreover, there was no adverse phthalate cross-reactive anti-DNA response. Interestingly, PHIS-01 and PHIS-03 influenced differentially T-helper polarization. We also observed that these compounds modulated the immune response through apoptotic/necrotic effects on target tumor cells using murine lymphomas. Finally, unlike squalene and several other terpenoids reported to date, these phytol derivatives did not appear arthritogenic in murine models.     

In vitro and in vivo anti-inflammatory properties of imine resveratrol analogues

Resveratrol is a natural polyphenol found mainly on red grapes and in red wine, pointed as an important anti-inflammatory/immunomodulatory molecule. However, its bioavailability problems have limited its use encouraging the search for new alternatives agents. Thus, in this study, we synthetize 12 resveratrol analogues (6 imines, 1 thioimine and 5 hydrazones) and investigated its cytotoxicity, antioxidant activity and in vitro anti-inflammatory/immunomodulatory properties. The most promising compounds were also evaluated in vivo. The results showed that imines presented less cytotoxicity, were more effective than resveratrol on DPPH scavenger and exhibited an anti-inflammatory profile. Among them, the imines with a radical in the para position, on the ring B, not engaged in an intramolecular hydrogen-interaction, showed more prominent anti-inflammatory activity modulating, in vivo, the edema formation, the inflammatory infiltration and cytokine levels. An immunomodulatory activity also was observed in these molecules. Thus, our results suggest that imines with these characteristics presents potential to control inflammatory disorders.     

Genetic variants of TNFα, IL10, IL1β, CTLA4 and TGFβ1 modulate the indices of alcohol-induced liver injury in East Indian population

Alcohol induced liver disease or alcoholic liver disease (ALD), a complex trait, encompasses a gamut of pathophysiological alterations in the liver due to continuous exposure to a toxic amount of alcohol (more than 80g per day). Of all chronic heavy drinkers, only 15-20% develops hepatitis or cirrhosis concomitantly or in succession. Several studies revealed that inter-individual as well as inter-ethnic genetic variation is one of the major factors that predispose to ALD. The role of genetic factors in ALD has long been sought for in ethnically distinct population groups. ALD is fast emerging as an important cause of chronic liver disease in India; even in populations such as "Bengalis" who were "culturally immune" earlier. While the genetic involvement in the pathogenesis of ALD is being sought for in different races, the complex pathophysiology of ALD as well as the knowledge of population level diversity of the relevant alcohol metabolizing and inflammatory pathways mandates the need for well designed studies of genetic factors in ethnically distinct population groups. An array of cytokines plays a critical role as mediators of injury, inflammation, fibrosis and cirrhosis in ALD. We, therefore, studied the association of polymorphisms in five relevant cytokine genes with "clinically significant" ALD in an ethnic "Bengali" population in Eastern India. Compared with "alcoholic" controls without liver disease (n=110), TNFα -238AA genotype, IL1β -511CC genotype, TGFβ1 -509CC genotype and IL10 -592AA genotype were significantly overrepresented in ALD patients (n=181; OR=2.4 and 95% CI 1.2-5.5, P(genotype)=0.042, P(allelic)=0.008; OR=2.7 and 95% CI 1.2-5.9, P(genotype)=0.018, P(allelic)=0.023; OR=4.7 and 95% CI 1.7-13.1, P(genotype)=0.003, P(allelic)=0.014; and OR=2.2 and 95% CI 1.1-4.8, P(genotype)=0.04, P(allelic)=0.039 respectively). Moreover a cumulative genetic risk analysis revealed a significant trend for developing ALD with an increase in the number of risk alleles on IL10 and TGFβ1 loci among alcoholics. The risk genotype of IL1β and TGFβ1 also influences the total bilirubin, albumin and alanine aminotransferase levels among alcoholic "Bengalis". The present study is the first case-control study from Eastern India that comprehensively identified polymorphic markers in TNFα, IL10, IL1β and TGFβ1 genes to be associated with ALD in the Bengali population, accentuating the significance of genetic factors in clinical expressions of ALD.     

Superoxide Disrnutase Does Protect the Cdtured Rat Cardiac Myocytes Against Hypoxia/Reoxygenation Injury

The effect of superoxide dismutase (SOD) on membrane integrity and fluidity of the cultured neonatal rat cardiac myocytes in vitro was investigated under the condition of hypoxia and hypoxia/reoxygenation. Lactate dehydrogenase (LDH) concentration was used as the biochemical indicator for the loss of cell membrane integrity. Fluorescence polarization (FP), average microviscosity (N) and anisotropy (Ast), which are inversely proportional to the fluidity of cell membrane, were assayed. Cells were respectively exposed to hypoxia or hypoxia/reoxygenation for different periods of time in the absence or presence of SOD at various concentrations. Hypoxia alone or hypoxia/ reoxygenation brought injury to the cultured myocytes. This was demonstrated by changes in LDH and membrane fluidity. In the former LDH concentration gradually increased in a time-dependent manner and the values of FP, N and Asf were significantly increased. The changes in membrane integrity and fluidity induced by hypoxia or hypoxia/reoxygenation could be prevented by adding SOD to the culture medium. The results provide a direct evidence that SOD (740 u.ml^-1, the effective dose) was effective in protecting cultured myocytes against the injury as well as an indirect evidence of free radical generation. Based on the results obtained from this study and the establishment of concept of optimally effective dose by Bernier and Omar et al, it was suggested that some previous reports, in which no evidence was found both in protective effect of SOD and in free radical generation by using only one dose in hypoxia/reoxygenation model, should be interpreted with caution.     

尿微量白蛋白ELISA测定法最适条件的探讨

用自制兔抗人白蛋白抗体;酶标抗原和进口NUNC板, 进行了两种显色剂邻苯二胺(OPD)及四甲基联苯胺(TMB)测定尿微量白蛋白的比较,底物(H₂O₂)浓度对测定的影响,碳酸钠和戊二醛两种包被方法的比较,以及抗体和抗原浓度的选择等影响因素进行了实验探讨,并确定了本实验的最佳分析条件.