Viscera Of Mice Research Articles

Gigantol restores the sensitivity of mcr carrying multidrug-resistant bacteria to colistin

BackgroundThe emergence and wide spread of plasmid-mediated colistin resistance gene (mcr-1) and its mutants have immensely limited the efficacy of colistin in treating multidrug-resistant (MDR) Gram-negative bacterial infections. The development of synergistic combinations of antibiotics with a natural product that coped with the resistance of MDR bacteria was an economic strategy to restore antibiotics activity. Herein, we investigated gigantol, a bibenzyl phytocompound, for restoring in vitro and in vivo, the sensitivity of mcr-positive bacteria to colistin. MethodsThe synergistic activity of gigantol and colistin against multidrug-resistant Enterobacterales was studied via checkerboard assay and time-killing curve. Subsequently, the transcription and protein expression levels of mcr-1 gene were determined by RT-PCR and Western blots. The interaction of gigantol and MCR-1 was simulated via molecular docking and verified via site-directed mutagenesis of MCR-1. Hemolytic activity and cytotoxicity assay were used to evaluate the safety of gigantol. Finally, the in vivo synergistic effect was evaluated via two animal infection models. ResultsGigantol restored the activity of colistin against mcr-positive bacteria E.coli B2 (MIC from 4 μg/ml to 0.25 μg/ml), Salmonella 15E343 (MIC from 8 μg/ml to 1 μg/ml), K. pneumoniae 19-2-1 (MIC from 32 μg/ml to 2 μg/ml) carrying mcr-1, mcr-3, mcr-8, respectively. Mechanistic studies revealed that gigantol down-regulated the expression of genes involved in LPS-modification, reduced the MCR-1 products and inhibited the activity of MCR-1 by binding to amino acid residues Tyr287 and Pro481 in its D-glucose-binding pocket. Safety evaluation showed that the addition of gigantol relieves the hemolysis caused by colistin. Compared with monotherapy, the combination of gigantol and colistin significantly improved the survival rate of Gallgallella mellonella larvae and mice infected by E.coli B2. Moreover, there was a considerable decrease in the bacterial load present in the viscera of mice. ConclusionOur results confirmed that gigantol was a potential colistin adjuvant, and could be used to tackle multi-drug resistant Gram-negative pathogen infections combined with colistin.

Leptospiral lipopolysaccharide-induced expression of inflammatory cytokines and mononuclear-macrophage infiltration in viscera of mice with Leptospira interrogans infection

Objective To understand the role of leptospiral lipopolysaccharide (L-LPS) in inducing human monocytes or mouse mononuclear-macrophages to produce inflammatory cytokines and to analyze the infiltration of peripheral blood-derived mononuclear-macrophages in mice with Leptospira interrogans (L.interrogans) infection. Methods Phenol-water method was used to extract L-LPS from L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai. The endotoxic activity of L-LPS was determined by using limulus test. Cytokine antibody array was used to detect the cytokines produced by human THP-1 monocytes or mouse J774A.1 mononuclear-macrophages after treatment with L-LPS or infection with L.interrogans strain Lai. The infiltration of peripheral blood-derived mononuclear-macrophages in lung, liver and kidney tissues, and the cytokines in serum samples collected from L.interrogans strain Lai-infected C3H/HeJ mice were detected by immunohistochemical examination using HRP-labeled peripheral blood-derived mononuclear-macrophage-specific antibody against CD68 and cytokine antibody array, respectively. Results The activity of L-LPS was about 1/5 of that of Escherichia coli LPS (E-LPS) with respect to solidifying limulus amebocyte lysate. Treating THP-1 and J774A.1 cells with L-LPS significantly increased the production of 29 and 9 cytokines (P<0.05 or P<0.01), respectively, including the major proinflammatory cytokines IL-6 and TNF-α and multiple chemokines of mononuclear-macrophages. The results of cytokine detection in the supernatants of L.interrogans-infected cells and the serum samples collected from L.interrogans-infected mice were similar to those in L-LPS-treated cells. A great number of peripheral blood-derived mononuclear-macrophages were presented in the lung, liver and kidney tissues of the infected mice after infection with the L.interrogans strain Lai. Conclusion L-LPS played a critical role in inducing the expression of multiple inflammatory cytokines by mononuclear-macrophages and in promoting the expression of mononuclear-macrophage chemokines. Mononuclear-macrophages were the major inflammatory cells infiltrating the viscera of L.interrogans-infected mice. Key words: Leptospira interrogans; Lipopolysaccharide; Inflammatory cytokine; Mononuclear-macrophage; Infiltration

Cross-species genetic exchange between visceral and cutaneous strains of Leishmania in the sand fly vector

Genetic exchange between Leishmania major strains during their development in the sand fly vector has been experimentally shown. To investigate the possibility of genetic exchange between different Leishmania species, a cutaneous strain of L. major and a visceral strain of Leishmania infantum, each bearing a different drug-resistant marker, were used to coinfect Lutzomyia longipalpis sand flies. Eleven double-drug-resistant progeny clones, each the product of an independent mating event, were generated and submitted to genotype and phenotype analyses. The analysis of multiple allelic markers across the genome suggested that each progeny clone inherited at least one full set of chromosomes from each parent, with loss of heterozygosity at some loci, and uniparental retention of maxicircle kinetoplast DNA. Hybrids with DNA contents of approximately 2n, 3n, and 4n were observed. In vivo studies revealed clear differences in the ability of the hybrids to produce pathology in the skin or to disseminate to and grow in the viscera, suggesting polymorphisms and differential inheritance of the gene(s) controlling these traits. The studies, to our knowledge, represent the first experimental confirmation of cross-species mating in Leishmania, opening the way toward genetic linkage analysis of important traits and providing strong evidence that genetic exchange is responsible for the generation of the mixed-species genotypes observed in natural populations.

Isolation and characterization of a toxic phospholipase A2 from the venom of the Taiwan habu (Trimeresurus mucrosquamatus).

A toxic phospholipase A2 (PLA2) was isolated from Taiwan-habu-snake (Trimeresurus mucrosquamatus) venom by sequential chromatographies on CM-52, Sephadex G-75 and S-Sepharose columns. This basic PLA2 has a single polypeptide with an estimated M(r) of 15,000. The PLA2 activity was Ca(2+)-dependent and inactivated by p-bromophenacyl bromide and its specific antibody. The toxic PLA2-induced myotoxic and direct haemolytic effects as well as respiratory distress in mice with an intraperitoneal LD50 of 1.17 micrograms/g body weight. The histological examination showed it caused haemorrhage and congestion in the viscera of mice. It was also cytotoxic to myeloma cells (NS-1), baby-hamster-kidney (BHK) cells and human umbilical endothelial cells. By neutralization experiments with a specific antibody against toxic PLA2, it was found that the enzymic activity of toxic PLA2 is essential for its myotoxicity, but it is not the only factor responsible for the lethal toxicity.

Asymmetric distribution of dense bands in muscle cells of mammalian arterioles

Arterioles from viscera of mice, rats, guinea pigs, rabbits, sheep, and pigs were examined by electron microscopy. In all the organs and species investigated the muscle cells of the tunica media are arranged in tight helices and make several full turns. The myofilaments also are arranged approximately circumferentially, although there is often a little divergence between bundles of myofilaments in different areas of the same cell. The dense bands are conspicuous and anchor the myofilaments to the cell membrane; they are markedly larger and more numerous on the regions of the cell membrane facing the adventitia than on those facing the intima. This asymmetrical distribution is interpreted as essential to an efficient mechanism for constriction of the lumen.

The nature and physical characteristics of a transmissible agent from human sarcoid tissue.

In controlled experiments normal and immunologically deficient CBA mice were inoculated in footpads, intraperitoneally, or intravenously with whole fresh, fresh supernatant, and fresh filtered supernatant (0.2 mu) or with each of these homogenates after autoclaving or their further exposure to 60Co irradiation (2.5 MR). As controls, mice were inoculated with identically prepared fresh nonsarcoid homogenates by these same routes. Epithelioid- and giant-cell granulomas were present in viscera of mice given each fresh sarcoid homogenate by any of these routes 15 months after inoculation, but were not present in mice given nonsarcoid autoclaved sarcoid, or 60Co-irradiated sarcoid homogenate. Successful passages were achieved following the inoculation of whole or filtered supernatant (0.2 mu) sarcoid mouse tissue homogenate into footpads, intraperitoneally, or intravenously. The epithelioid- and giant-cell granulomas evolved slowly over a period of many months following the inoculation of sarcoid tissue or passage homogenates and persisted thereafter in association with Kveim reactivity. The transmissible agent is inactivated when homogenate prepared from human sarcoid or mouse sarcoid tissue is autoclaved or 60Co-irradiated, can be repeatedly passaged, and has been shown to pass an 0.2-mu membrane filter. It is therefore presumably viable and must approximate to the size of a virus or be capable of being deformed so as to pass a filter of such a pore size (0.2mu), L forms.

Effects of 8-Azaguanine on the Pathogenesis of Experimental Chagas' Disease

8-Azaguanine does not inhibit the growth of the Tulahuen strain of Trypanosoma cruzi in vivo, or produce any curative effects. Instead it produces an intensified infection in C3H mice when the dosage is 75 mg of 8-azaguanine/kg/day, which may be due to antibody suppression in the host by the drug. The parasitemias of the experimental animals were significantly greater than the controls, and eight tissues were significantly more parasitized. These tissues were: adrenal cortex, bone marrow, brown fat, colon, diaphragm, duodenum, pancreas, and spleen. It is interesting that the most heavily parasitized tissue, brown fat, and the adrenal cortex also contain the most steroids. Nakamura and James (1952) experimented with 10 purine derivatives, not including 8azaguanine, against Trypanosoma cruzi in vitro. They found that oxygen consumption and growth were inhibited by all of the derivatives studied. They indicated that further study of the effects of purine and pyrimidine derivatives on T. cruzi seemed desirable. Jaffe (1963), in experiments with Trypanosoma equiperdum, found that infected mice showed an 8% increase in survival time when treated with 20 mg 8-azaguanine/kg/day. From early experiments with 8-azaguanine on Tetrahymena geleii it was postulated that the protozoan substituted 8-azaguanine for the normal guanine and incorporated it into nucleic acids, with a resulting metabolic disturbance and growth failure (Kidder and Dewey, 1949). Since then the analog has been isolated from the RNA of 8azaguanine-inhibited cultures of Tetrahymena (Heinrich et al., 1952), and from the nucleic acids of tumors and viscera of mice treated with 8-azaguanine (Mitchell et al., 1950). 8-Azaguanine has been shown to be substituted for guanine in bacterial, viral, and mammalian RNA replacing up to 5% of the guanine residues (Lasnitzki, 1954). Smith and Matthews (1957) found that 40% of total RNA guanine in Bacillus cereus can be replaced, and Levin (1963) demonstrated that most of this 8azaguanine was incorporated into sRNA. Parks (1962) demonstrated that at daily dosages of 75 mg/kg the average survival time of mice with mammary adenocarcinoma was almost twice that of untreated control animals. In Received for publication 13 August 1968. general, investigations in many laboratories all point to inhibition of protein synthesis as an important or major effect of 8-azaguanine on tissues (Parks, loc. cit.). The 8-azaguanine inhibition of antibody synthesis was demonstrated by Dutton et al. (1958) in spleen cells of rabbits, and by Malmgren et al. (1952) who found reduced hemolytic and precipitin antibody titers in several strains of inbred mice. The present report deals with investigations designed to demonstrate effects of 8-azaguanine on the pathogenesis of acute experimental Chagas' disease. MATERIALS AND METHODS The Tulahuen strain of T. cruzi was the infective agent. The strain was virulent when sent to us by Dr. Frans C. Goble, Ciba Pharmaceutical Company, Summit, New Jersey, and remained virulent throughout the experiments. The trypanosomes were maintained in HM medium (Warren, 1960). Adult C.H mice (mean weight 23 g) were used and were maintained on a diet of Purina Laboratory Chow. The experiments (1 to 4) were run separately in the sequence in which they are presented. A total of four different tubes of medium (each containing 10 ml of fluid) were used, one tube for each experiment. Care was taken to insure equal distribution of the cycle stages to experimental and control mice in each experiment by using inocula from the same tube of medium. At the time of the inoculations the trypanosomes had been in culture 14 days. Special care was taken to prevent concomitant bacterial infections by examining the medium before use in each experiment and following aseptic techniques throughout all of the inoculations. Each animal was inoculated subcutaneously with 0.1 ml of medium containing a total of ca. 500,000 trypanosomes as counted with a hemocytometer counting chamber. Fourteen to 18% of the total trypanosomes counted were infective metacyclic stages making a total of 70,000 to 90,000