ArgD of Mycobacterium tuberculosis is a functional N-acetylornithine aminotransferase with moonlighting function as an effective immune modulator

Mycobacterium tuberculosis (M. tuberculosis) encodes an essential enzyme acetyl ornithine aminotransferase ArgD (Rv1655) of arginine biosynthetic pathway which plays crucial role in M. tuberculosis growth and survival. ArgD catalyzes the reversible conversion of N-acetylornithine and 2 oxoglutarate into glutamate-5-semialdehyde and L-glutamate. It also possesses succinyl diaminopimelate aminotransferase activity and can thus carry out the corresponding step in lysine biosynthesis. These essential roles played by ArgD in amino acid biosynthetic pathways highlight it as an important metabolic chokepoint thus an important drug target. We showed that M. tuberculosis ArgD rescues the growth of ΔargD E. coli grown in minimal media validating its functional importance. Phylogenetic analysis of M. tuberculosis ArgD showed homology with proteins in gram positive bacteria, pathogenic and non-pathogenic mycobacteria suggesting the essentiality of this protein. ArgD is a secretory protein that could be utilized by M. tuberculosis to modulate host innate immunity as its moonlighting function. In-silico analysis predicted it to be a highly antigenic protein. The recombinant ArgD protein when exposed to macrophage cells induced enhanced production of pro-inflammatory cytokines TNF, IL6 and IL12 in a dose dependent manner. ArgD also induced the increased production of innate immune effector molecule NOS2 and NO in macrophages. We also demonstrated ArgD mediated activation of the canonical NFkB pathway. Notably, we also show that ArgD is a specific TLR4 agonist involved in the activation of pro-inflammatory signaling for sustained production of effector cytokines. Intriguingly, ArgD protein treatment activated macrophages to acquire the M1 phenotype through the increased surface expression of MHCII and costimulatory molecules CD80 and CD86. ArgD induced robust B-cell response in immunized mice, validating its antigenicity potential as predicted by the in-silico analysis. These properties of M. tuberculosis ArgD signify its functional plasticity that could be exploited as a possible drug target to combat tuberculosis.     

Noscapine hydrochloride (benzyl-isoquinoline alkaloid) effectively prevents protein denaturation through reduction of IL-6, NF-kB,COX-2, Prostaglandin-E2 in rheumatic rats

Noscapine hydrochloride (benzyl-isoquinoline antitussive alkaloid) is an opium derivative and generally used as a cough suppressant. Numerous studies on noscapine hydrochloride have reported that it has potent anti-inflammatory activity. However, the mechanisms by which it exerts an anti-inflammatory function is not well understood. Protein denaturation is the primary step that leads to the organ destruction and permanent arthritic disability. The above-mentioned facts provided the ground to plan this study using different in-vitro and in-vivo approaches. RT-qPCR and ELISA assays were used to assess the inflammatory markers related to protein denaturation in complete adjuvant persuaded rheumatism in Sprague - Dawley rats. The results were collected as paw volume and body weight changes, arthritic scoring and serum antioxidant enzymes assays. These findings demonstrated that all doses of noscapine hydrochloride (10, 20 and 40 mg/kg) studied in this study, significantly (p < 0.001) decreased the protein denaturation by preventing the increase in levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), nuclear factor-kB (NF-kB), cyclooxygenase-2 (COX-2) and prostaglandin E2. Noscapine hydrochloride significantly reduced the paw volume (p < 0.001), arthritic scoring and reversed the body mass as compared to arthritic control diseased rats.     

Cytokine and LL-37 gene expression levels in Bartonella spp. seropositive and seronegative patients of a rheumatology clinic

PurposeThe variation in the immune response to Bartonella spp. infection in humans remains unclear. The present study compares the expression of selected interleukins, cytokines and cathelicidin (LL-37) in rheumatology clinic patients suffering from musculoskeletal symptoms with healthy blood donors. The patients had previously been tested for the presence of Bartonella henselae antibodies.MethodsGene expression of LL-37, interleukin (IL)-2, IL-4, IL-6, IL-12, interferon-(IFN)-γ, and tumor necrosis factor (TNF-α)-α was determined in blood samples using quantitative Polymerase Chain Reaction (qPCR). Statistical analysis was prepared with STATISTICA.ResultsStatistically significant differences in the mRNA levels of the tested cytokines (IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-12; p<0.0001) were observed between the healthy controls and patients; however, no difference was observed for LL37 mRNA (p ?= ?0.1974). No significant differences in mRNA expression were observed between IgG in anti-Bartonella seropositive and seronegative individuals (p>0.05). The only significant differences between the Bartonella spp. DNA positive and negative patients, indicated by PCR, were observed for TNF-α and IL-12 mRNA (p ?= ?0.0045 and p ?= ?0.0255, respectively).ConclusionA broadly similar immune response to the tested cytokines was observed among the participants irrespective of anti-Bartonella spp. IgG seropositivity. However, the Bartonella DNA-positive participants demonstrated significantly lower expression of IL-12 and TNF-α mRNA; this may indicate that these bacteria have a suppressive influence on the immune system.     

南星骨痛凝胶贴膏对兔膝骨关节炎关节形态及血清IL-1、TNF-α、MMP-13、TIMP-1表达的影响

目的观察南星骨痛凝胶贴膏对兔膝骨关节炎关节形态及血清IL-1、TNF-α、MMP-13、TIMP-1表达的影响,探讨南星骨痛凝胶贴膏治疗兔膝骨关节炎可能的作用机制。方法 18只健康6月龄新西兰兔随机分成3组:空白组(A组)、模型组(B组)、骨痛膏治疗组(C组). BC组用3%木瓜蛋白酶关节腔内注射造模后C组给予南星骨痛凝胶贴膏外敷,给药4周后,用Aloka Latheta LCT-200观察兔膝骨关节影像学改变、用ELISA法检测血清IL-1、TNF-α、MMP-13、TIMP-1含量。结果 C组兔膝关节病变程度较B组轻,C组兔血清中IL-1、TNF-α、MMP-13含量较B组降低(P<0. 05),TIMP-1含量较B组升高(P <0. 05)。结论南星骨痛凝胶贴膏能减轻兔膝关节骨关节炎病变程度,对关节软骨有保护作用,其作用机制可能与抑制炎症因子IL-1、TNF-α及MMP-13表达,增加TIMP-1表达有关。     

Anti-tumor and Phenotypic Regulation Effect of Matrine on Dendritic Cells through Regulating TLRs Pathway

Objective: To investigate the effects of matrine on antigen presentation of dendritic cells (DCs), and to explore the pharmacological mechanism of matrine on anti-tumor effect. Methods: Different concentrations (0, 1, 2, 4, 8 and 16 μ g/mL) of matrine were co-cultured with DCs, the harvested DCs were co-cultured with antigens of Lewis lung cancer (LLC) cells, and then DCs and T cells were co-cultured to produce DCs-activated killer (DAK) cells, which have significant tumor-killing activity. The expression of cytokines, mRNA and protein of toll-like receptors (TLRs) in DCs were detected by enzyme linked immunosobent assay, polymerase chain reaction and Western blot, respectively. And the killing effect of DAK were measured by MTT assay. Results: Matrine significantly increased the mRNA expression of TLR7, TLR8, myeloid differentiation factor 88 (MyD88), tumor necrosis factor receptor-associated factor 6 (TRAF-6) and Iκ B kinase (IKK), as well as the protein expression of TLR7 and TLR8, and up-regulated the levels of interleukin-12 (IL-12), IL-6 and tumor necrosis factor-α (TNF-α ), meanwhile, it also increased the expressions of MHC-Ⅱ , CD54, CD80 and CD86 in DCs. DCs-activated effector T cells had significant tumor-killing activity. When the concentration of matrine was more than 4 μ g/mL, all indices had significant difference (P<0.01 or P<0.05). Conclusion: Matrine plays an anti-tumor role by regulating TLRs signal transduction pathway, promoting the secretion of inflammatory cytokines and enhancing immune function.     

Iron overload in congenital haemolytic anaemias: role of hepcidin and cytokines and predictive value of ferritin and transferrin saturation

Iron overload (IO) is poorly investigated in the congenital haemolytic anaemias (CHAs), a heterogeneous group of rare inherited diseases encompassing abnormalities of the erythrocyte membrane and metabolism, and defects of the erythropoiesis. In this study we systematically evaluated routine iron parameters and cardiac and hepatic magnetic resonance imaging, together with erythropoietin, hepcidin, non-transferrin bound iron (NTBI), and cytokine serum levels in patients with different CHAs. We found that 40% of patients had a liver iron concentration (LIC) >4 mg Fe/g dry weight. Hepatic IO was associated with ferritin levels (P = 0·0025), transferrin saturation (TfSat, P = 0·002) and NTBI (P = 0·003). Moreover, ferritin >500 μg/l plus TfSat >60% was demonstrated as the best combination able to identify increased LIC, and TfSat alteration as more important in cases with discordant values. Possible confounding factors, such as transfusions, hepatic disease, metabolic syndrome and hereditary haemochromatosis-associated mutations, had negligible effects on IO. Erythropoietin and hepcidin levels were increased in CHAs compared with controls, correlating with LIC and ferritin, respectively. Regarding cytokines, γ-interferon (IFN-γ) was increased, and both interleukin 6 and IFN-γ levels positively correlated with ferritin and hepcidin levels. Overall, these findings suggest the existence of a vicious cycle between chronic haemolysis, inflammatory response and IO in CHAs.     

红藤虎杖复方免煎剂对急性痛风性关节炎模型小鼠细胞因子表达的影响

目的观察红藤虎杖复方免煎剂对急性痛风性关节炎模型小鼠细胞因子的表达,并探讨其可能作用机制。方法将KM雌雄性小鼠各36只,称重后随机分成空白组、模型组、阳性组和中药高、中、低剂量组,正常饲养1周后,连续灌胃5 d,第6天在小鼠踝关节注射尿酸钠诱导急性痛风性关节炎模型。测定足趾容积、HE染色观察滑膜组织病理形态,以及ELISA法检测IL-1β、TGF-β1、TNF-α、IL-1、IL-8、IL-1Ra在踝关节滑膜组织中的表达。结果与空白组比较,模型组右足趾容积明显增加;IL-1、IL-1β、IL-8、TNF-α水平均显著增高(P<0.01);IL-1Ra、TGF-β1较空白组增高(P<0.05)。与模型组比较,阳性组、中药高、中剂量组右足趾容积明显降低;IL-1、IL-1β、IL-8、TNF-α水平均显著性降低(P<0.01);TGF-β1、IL-1Ra水平均显著性增高(P<0.01)。与阳性组比较,中药高剂量组IL-1显著降低((P<0.01);中药高、中剂量组IL-1β、TNF-α较阳性组降低(P<0.05);中药高、中剂量组IL-8、IL-1Ra、TGF-β1与阳性组比较无显著性差异(P>0.05);HE染色观察显示高剂量组滑膜组织炎症浸润改善明显。结论红藤虎杖复方免煎剂能治疗小鼠急性痛风性关节炎,其作用机制可能与MSU诱导的促炎细胞因子及抗炎细胞因子的产生有关,且高剂量效佳。