In vivo anti-inflammatory effects of the M20 IL-1 Inhibitor: II. Effects on serum reactants

We have previously described an IL-1 Inhibitor derived from the M20 myelomoncytic cell line. This line also secretes several molecules of IL-1. We have shown that this factor is specific to IL-1in vitro, as well asin vivo. In vitro IL-1 induced proliferative responses of mouse thymocytes, human T cells and fibroblasts and IL-1 stimulated PGE₂ secretion from fibroblasts, were all inhibited by the M20 IL-1 Inhibitor.In vivo, the IL-1 Inhibitor reduced parameters of acute inflammation such as fever, leukocytosis and local inflammation. This study describes additional effects of the M20 IL-1 Inhibitor on inflammatory serum reactants. Levels of corticosterone and fibrinogen were increased by injection of IL-1, and decreased by the IL-1 Inhibitor. IL-1 reduced zinc and iron plasma levels and elevated copper plasma levels. The M20 IL-1 Inhibitor reversed these changes in a dose dependent manner. Similar effects produced by IL-6 and TNF were unaffected by the M20 IL-1 Inhibitor. Our results indicate that the M20 IL-1 Inhibitor acts specifically on IL-1 induced responsesin vivo. Therefore we conclude that this IL-1 Inhibitor has a great potential as an anti-inflammatory agent.Abbreviations IL-1 Interleukin 1 - IL-6 Interleukin 6 - IL-1ra Interleukin 1 receptor antagonist - TNF tumor necrosis factor     

The M20 IL-1 inhibitor prevents onset of adjuvant arthritis

Cytokines, specifically IL-1 and TNF, have been implicated as important mediators of joint destruction in rheumatoid arthritis (RA). Elevated levels of IL-1 in the joint fluid of patients with RA have been reported, as well as the presence of IL-1 inhibitory activity. We have reported the characterization of an inhibitor derived from a myelomonocytic cell line cloned in our laboratory which is specific for IL-1. This IL-1 inhibitor is protein in nature which specifically inhibits activityin vitro andin vivo. Previous studies showed that the inhibitor reduced acute inflammatory reactions associated with IL-1 (fever, leukocytosis, local foot pad swelling, lymph node enlargement and acute phase reactants). Thus it was of interest to study whether the M20 IL-1 inhibitor could modify adjuvant-induced chronic inflammation in rats, which is often used as a model for human RA. Administration of complete Freund's adjuvant (CFA) into Lewis rats, resulted in a severe adjuvant arthritis (AA) which reached peak severity after 14 days. Daily administration of IL-1 inhibitor, beginning after injection of CFA, abolished the appearance of AA. The parameters investigated were: joint swelling (the increase in diameter of joints), peri-articular erythema, limping of the rats and histological examination. The effect of the M20 IL-1 inhibitor was shown to be dose dependent and the IL-1 inhibitor alone had no adverse effects. These results indicate that the M20 IL-1 inhibitor may have a role in the treatment of AA and may be used to reduce pathological processes in joint inflammation.     

Comparison of in vitro and in vivo cell cycle parameters of lymphoid cells in Pig spleens

Summary Parameters of the cell cycle of lymphoid cells were estimated by analyzing percent labeled mitoses curves after a ³H-thymidine flash. Either anaesthetized pigs were labeled and multiple biopsies taken from the spleen in vivo or isolated perfused pig spleens were labeled in vitro. The data from in vivo and in vitro experiments were very similar.The mean values for cell cycle parameters were: 20.2 to 20.5 hours for the generation time, about 0.5 to 1 hour for G₂, about 1.2 to 1.3 hours for M; about 17 to 16.5 hours for S and about 1.5 to 1.7 hours for G₁. The mean grain count halving time of labeled mitoses was in accordance with the measured generation time. The isolated perfused spleen seems to give results equal to in vivo data and could, therefore, be employed as a model for studying cell cycle parameters not only in animal but also in human lymphoid tissue.The expert technical assistance of Mrs. A. Fischer is gratefully acknowledged. This study was supported by the Deutsche Forschungsgemeinschaft, SFB 112.     

具核梭杆菌脂多糖诱导THP-1细胞M2极化及对低浓度白介素6产生的作用

【背景】在结直肠肿瘤等多种肿瘤中普遍存在的具核梭杆菌(Fusobacterium nucleatum)与结直肠肿瘤发生、预后不良、复发及化疗耐药等密切相关。其引发炎症、对肿瘤微环境中巨噬细胞等免疫细胞作用与机制尚待阐明。【目的】对比分析F.nucleatum脂多糖(Lipopolysaccharide,LPS)与嗜黏蛋白阿克曼氏菌(Akkermansia muciniphila)、大肠杆菌(Escherichia coli)的LPS诱导单核细胞极化、炎性细胞因子表达等活性差异,探讨F.nucleatum在诱发慢性炎症、致癌等过程中的作用与机制。【方法】分别用A.muciniphila、E.coli、F.nucleatum LPS或联合干扰素γ(Interferon-γ,IFN-γ)处理后,观察THP-1、THP-1 M0细胞的细胞形态变化,然后检测M0 (CD11B)、M1 (CD40、CD86)和M2 (CD163、CD206)巨噬细胞标志基因、TLR3、TLR4、IL-6、IL-10等基因转录水平,以及IL-6、IL-10、C反应蛋白(C Reactive Protein,CRP)...     

In vivo efficacy of praziquantel against Echinoparyphium aconiatum (Trematoda: Echinostomatidae) parasitizing the great pond snails Lymnaea stagnalis (Gastropoda: Lymnaeidae)

The present study had a practical goal. I aimed to determine whether praziquantel could reduce the production of Echinoparyphium aconiatum (Trematoda: Echinostomatidae) cercariae in infected snails Lymnaea stagnalis (Gastropoda: Lymnaeidae) without killing the hosts. Praziquantel is a broad-spectrum antihelminth agent. It caused a total cessation of cercaria shedding when the praziquantel concentration in the treatment bath was 10 mg/L and the treatment time was 30 h or longer. A next research step which has to be taken before giving detailed recommendations about using praziquantel for ceasing production of E. aconiatum cercariae in parasitized snails is to follow the survivorship and performance of treated snails after a praziquantel exposure for longer than in this medium-term (3 days) experiment.     

In Vivo Gene Electroporation of Glial Cell Line-Derived Neurotrophic Factor (GDNF) into Skeletal Muscle of SOD1 Mutant Mice

Motor neurons degenerate with intracellular vacuolar change and eventually disappear in spinal cords of SOD1 mutant mice, resembling human amyotrophic lateral sclerosis (ALS). The GDNF gene was electroporatically transferred into the leg muscles of SOD1 mutant mice and expressed in muscle cells. This gene therapy with GDNF delayed the deterioration of motor performance, being retrogradely transported into spinal motor neurons. However, the number of the motor neurons and survival of the mutant mice were not improved by GDNF treatment. These results indicate that in vivo gene electroporation of GDNF into muscles could be an appropriate therapeutic approach to ameliorate an early dysfunction of motor neurons in SOD1 mutant mice, but further improvement is needed to use this gene transfer as an effective treatment of ALS.     

In vivo effects of vanadium on GLUT4 translocation in cardiac tissue of STZ-diabetic rats

The effect of vanadium treatment on insulin-stimulated glucose transporter type 4 (GLUT4) translocation was studied in cardiac tissue of streptozotocin (STZ)-induced diabetic rats by determining the subcellular distribution of GLUT4. Four groups of rats were examined: control and diabetic, with or without bis(maltolato)oxovanadium(IV) (BMOV, an organic form of vanadium) treatment for 8 weeks. The effect of vanadium on insulin-induced GLUT4 translocation was studied at 5 min as the early insulin response and at 15 min after insulin injection as the maximal insulin response.At 5 min after insulin injection, plasma membrane GLUT4 level in the diabetic-treated group was not different from the control groups and was significantly higher than that of the insulin-stimulated diabetic group, indicating an enhancement of insulin response on GLUT4 translocation brought about by vanadium treatment. In contrast to that at 5 min after insulin injection, no significant difference in the plasma membrane GLUT4 level was observed between the diabetic and the diabetic-treated groups at 15 min after insulin injection. GLUT4 mobilization from the intracellular pool in response to insulin was also investigated at 15 min after insulin injection. Basal intracellular GLUT4 content was significantly higher in the diabetic-treated group when compared to the diabetic group under the same condition. However, the increased basal intracellular GLUT4 in the diabetic-treated group did not result in more insulin-mediated GLUT4 translocation at 15 min after insulin injection. In conclusion, the finding that plasma membrane GLUT4 in the diabetic-treated group is significantly higher than that of the diabetic group at 5 min but not at 15 min post-insulin injection indicates that vanadium treatment enhances insulin-mediated GLUT4 translocation in cardiac tissue by enhancing its early response.     

House dust mite, Dermatophagoides pteronissinus increases expression of MCP-1, IL-6, and IL-8 in human monocytic THP-1 cells

The house dust mite (Dermatophagoides pteronissinus) plays an important role in the pathogenesis of allergic diseases, including atopic dermatitis, and asthma. Monocyte chemotactic protein 1 (MCP-1/CCL2)/IL-6/IL-8 (CXCL8) plays a pivotal role in mediating the infiltration of various cells into the skin of atopic dermatitis and psoriasis. The aim of this study was to investigate the effect of D. pteronissinus extract (DpE) on expression of MCP-1/IL-6/IL-8 mRNA and protein and the signal transduction in the human monocytic cell line, THP-1. The mRNA and protein expression of MCP-1/CCL2, IL-6, and IL-8 were elevated by DpE in a time and dose-dependent manner in THP-1 cells. The increased expression of MCP-1, IL-6, and IL-8 was not affected by aprotinin (serine protease inhibitor) or E64 (cysteine protease inhibitor). We found that MCP-1 and IL-6 expression due to DpE was related to Src, protein kinase C δ (PKC δ), extracellular-signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) and IL-8 expression was involved in Src family tyrosine kinase, PKC δ, ERK. DpE increased the phosphorylation of ERK and p38 MAPK after 5 min and peaked at 30 min. The activation was significantly blocked by PP2, an inhibitor of Src family tyrosine kinase and rottlerin, an inhibitor of PKC δ (p < 0.01). DpE increases MCP-1, IL-6, and IL-8 expression and transduces its signal via Src family tyrosine kinase, PKC, and ERK in a protease-independent manner. This finding may contribute to the elucidation of the pathogenic mechanism triggered by DpE .     

Identification of Brucella melitensis 16M genes required for bacterial survival in the caprine host

Brucella species are gram-negative bacteria which belong to alpha-Proteobacteria family. These organisms are zoonotic pathogens that induce abortion and sterility in domestic mammals and chronic infections in humans known as Malta fever. The virulence of Brucella is dependent upon its ability to enter and colonize the cells in which it multiplies. The genetic basis of this aspect is poorly understood. Signature-tagged mutagenesis (STM) was used to identify potential Brucella virulence factors. PCR amplification has been used in place of DNA hybridization to identify the STM-generated attenuated mutants. A library of 288 Brucella melitensis 16M tagged mini-Tn5 Km2 mutants, in 24 pools, was screened for its ability to colonize spleen, lymph nodes and liver of goats at three weeks post-i.v. infection. This comparative screening identified 7 mutants (approximately 5%) which were not recovered from the output pool in goats. Some genes were known virulence genes involved in biosynthesis of LPS (lpsA gene) or in intracellular survival (the virB operon). Other mutants included ones which had a disrupted gene homologous to flgF, a gene coding for the basal-body rod of the flagellar apparatus, and another with a disruption in a gene homologous to ppk which is involved in the biosynthesis of inorganic polyphosphate (PolyP) from ATP. Other genes identified encoded factors involved in DNA metabolism and oxidoreduction metabolism. Using STM and the caprine host for screening, potential virulence determinants in B. melitensis have been identified.     

In vitro inhibitory effects of Daphne oleoides ssp. oleoides on inflammatory cytokines and activity-guided isolation of active constituents

Aerial parts of Daphne oleoides Schreber ssp. oleoides (Thymelaeaceae) are used to treat rheumatoid arthritis and lumbago in Turkish folk medicine. In order to evaluate folkloric utilization, in vitro inhibitory effects of the ethyl acetate extract and fractions obtained from this extract on interleukin 1 (IL-1alpha, IL-1beta) and tumour necrosis factor (TNF-alpha) biosynthesis were studied. Through chemical isolation techniques and activity-guided fractionation process, seventeen compounds were isolated and their structures were elucidated (numbered 1-17). Diterpenoids genkwadaphnin (3) and 1,2-dehydrodaphnetoxin (6) and a coumarin derivative daphnetin (9) showed potent inhibitory activity and were found to be the main active ingredients. Furthermore, gnidilatin (4), gnidilatin-20 palmitate (5), genkwadaphnin-20-palmitate (7) and gnidicin-20-palmitate (8), having diterpenoid structure, and eudesmine (12), wikstromol (13) and matairesinol (14), having lignan structure, were determined to possess moderate inhibitory activity and may have a contributory role in the effect of the remedy.     

Site-directed mutagenesis of the yeast PRP20/1SRM1 gene reveals distinct activity domains in the protein product

Prp20/Srm1, a homolog of the mammalian protein RCC1 in Saccharomyces cerevisiae, binds to double-stranded DNA (dsDNA) through a multicomponent complex in vitro. This dsDNA-binding capability of the Prp20 complex has been shown to be cell-cycle dependent; affinity for dsDNA is lost during DNA replication. By analyzing a number of temperature sensitive (ts) prp20 alleles produced in vivo and in vitro, as well as site-directed mutations in highly conserved positions in the imperfect repeats that make up the protein, we have determined a relationship between the residues at these positions, cell viability, and the dsDNA-binding abilities of the Prp20 complex. These data reveal that the essential residues for Prp20 function are located mainly in the second and the third repeats at the amino-terminus and the last two repeats, the seventh and eighth, at the carboxyl-terminus of Prp20. Carboxyl-terminal mutations in Prp20 differ from amino-terminal mutations in showing loss of dsDNA binding: their conditional lethal phenotype and the loss of dsDNA binding affinity are both suppressible by overproduction of Gsp1, a GTP-binding constituent of the Prp20 complex, homologous to the mammalian protein TC4/Ran. Although wild-type Prp20 does not bind to dsDNA on its own, two mutations in conserved residues were found that caused the isolated protein to bind dsDNA. These data imply that, in situ, the other components of the Prp20 complex regulate the conformation of Prp20 and thus its affinity for dsDNA. Gsp1 not only influences the dsDNA-binding ability of Prp20 but it also regulates other essential function(s) of the Prp20 complex. Overproduction of Gsp1 also suppresses the lethality of two conditional mutations in the penultimate carboxyl-terminal repeat of Prp20, even though these mutations do not eliminate the dsDNA binding activity of the Prp20 complex. Other site-directed mutants reveal that internal and carboxyl-terminal regions of Prp20 that lack homology to RCC1 are dispensable for dsDNA binding and growth.     

Inhibitor effects on the accumulation and efflux of nickel ions in plasmid pMOL28-harboring strains of Alcaligenes eutrophus

The accumulation and efflux of ⁶³Ni²⁺ ions were studied using two strains of the strictly respiratory bacterium Alcaligenes eutrophus, the wild type strain N9A and its transconjugant N9A-M243 which harbors plasmid pMOL28.1 encoding constitutive resistance to nickel. When 1 M ⁶³Ni²⁺ is added to respiring cells, strain N9A accumulates high, but M243 only negligibly small amounts of nickel. When the cells were preincubated for about 20 h under anoxic conditions and were then exposed to 1 M ⁶³Ni²⁺ anaerobically, both strains accumulated approximately the same amounts of nickel. Aeration of these preloaded cells resulted in rapid efflux by strain M243 but renewed uptake of nickel by N9A. ⁶³Ni²⁺ uptake and efflux are highly sensitive to protonophores such as CCCP, FCCP and TCS but insensitive to DCCD (each at 20 M concentrations). Measurements on the effects of the inhibitors on biosynthetic processes requiring ATP either from substrate phosphorylation or from electron transport phosphorylation made sure that at the concentrations used the inhibitor effects were specific. Thus the results suggest that in the nickelresistant strain M243 under normal aerobic conditions two constitutive energy-dependent nickel transport systems can function concomitantly, a chromosomally-determined specific uptake system and a plasmid-mediated specific nickel efflux system.     

A20/Nrdp1 interaction alters the inflammatory signaling profile by mediating K48- and K63-linked polyubiquitination of effectors MyD88 and TBK1

A20 is a potent anti-inflammatory protein that mediates both inflammation and ubiquitination in mammals, but the related mechanisms are not clear. In this study, we performed mass spectrometry (MS) screening, gene ontology (GO) analysis, and coimmunoprecipitation (co-IP) in a lipopolysaccharide (LPS)-induced inflammatory cell model to identify novel A20-interacting proteins. We confirmed that the E3 ubiquitin ligase Nrdp1, also known as ring finger protein 41 (RNF41), interacted with A20 in LPS-stimulated cells. Further co-IP analysis demonstrated that when A20 was knocked out, degradation-inducing K48-linked ubiquitination of inflammatory effector MyD88 was decreased, but protein interaction-mediating K63-linked ubiquitination of another inflammatory effector TBK1 was increased. Moreover, western blot experiments showed that A20 inhibition induced an increase in levels of MyD88 and phosphorylation of downstream effector proteins as well as of TBK1 and a downstream effector, while Nrdp1 inhibition induced an increase in MyD88 but a decrease in TBK1 levels. When A20 and Nrdp1 were coinhibited, no further change in MyD88 was observed, but TBK1 levels were significantly decreased compared with those upon A20 inhibition alone. Gain- and loss-of-function analyses revealed that the ZnF4 domain of A20 is required for Nrdp1 polyubiquitination. Upon LPS stimulation, the inhibition of Nrdp1 alone increased the secretion of IL-6 and TNF-α but decreased IFN-β secretion, as observed in other studies, suggesting that Nrdp1 preferentially promotes the production of IFN-β. Taken together, these results demonstrated that A20/Nrdp1 interaction is important for A20 anti-inflammation, thus revealing a novel mechanism for the anti-inflammatory effects of A20.     

Rapid clonal propagation of three mulberries,Morus cathayana HemsL,M. lhou Koiz. andM. serrata Roxb., through in vitro culture of apical shoot buds and nodal explants from mature trees

High-frequency bud break and multiple shoots were induced in apical shoot buds and nodal explants ofMorus cathayana, M. lhou andM. serrata on Murashige and Skoog (MS) medium containing 0.5–1.0 mg/l 6-benzylaminopurine (BAP). Addition of gibberellic acid (0.4 mg/l) along with BAP induced faster bud break both in apical shoot buds and nodal explants and also enhanced the frequency of bud break in all three species. Shoot culture initiation was greatly influenced by explant type, explant age and explanting season. The shoots were successfully rooted on half-strength MS medium containing a combination of indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid, each at 1.0 mg/l. The plantlets were successfully acclimated and eventually established in soil.Abbreviations BAP 6-Benzylaminopurine - GA ₃ Gibberellic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - IPA Indole-3-propionic acid - Kn Kinetin - MS Murashige and Skoog (1962) medium - NAA 1-Naphthalene acetic acid     

The glycolysis/HIF-1α axis defines the inflammatory role of IL-4-primed macrophages

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Schistosoma mansoni: Antischistosomal activity of the four optical isomers and the two racemates of mefloquine on schistosomula and adult worms in vitro and in vivo

Recent studies have shown that mefloquine (MQ) reveals interesting antischistosomal properties. We examined the antischistosomal activities of the erythro and threo isomers and racemates of MQ on newly transformed schistosomula (NTS) and adult Schistosoma mansoni in vitro and in mice harbouring adult S. mansoni. The in vitro effects in the presence and absence of haemin were monitored by means of microcalorimetry, scanning electron microscopy and phenotypic evaluation. Incubation of NTS with the erythro derivatives at concentrations of 3 μg/ml and above resulted in convulsions, granularity, decrease in heat flow, and death while NTS incubated with the threo derivatives were only affected at high concentrations (100 μg/ml). Extensive tegumental alterations, decrease in metabolic activity, viability, and death were observed when adult schistosomes had been exposed to 10 μg/ml of the erythro compounds. Moderate tegumental and viability changes but reduced heat production rates were observed with the threo derivatives at 10 μg/ml. In the presence of haemin, all MQ derivatives showed pronounced antischistosomal properties against adult S. mansoni in vitro. In vivo, MQ derivatives achieved statistically significant total and female worm burden reductions ranging between 65.4% and 100%. The highest total worm burden reductions of 93.4% and 90.2% were observed following treatment with the erythro and threo racemates, respectively. In conclusion, the optical isomers and racemates of MQ show only moderate stereoselectivity, in particular in vivo. Our results may enhance our understanding of the mechanism of action and therapeutic profile of MQ derivates on schistosomes.     

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at ≥6 months after birth. We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.