Modulation of Cellular Immune Response by Orbifloxacin in Noninfected and E. coli-Infected Mice

The studies were conducted on noninfected and Escherichia (E) coli-infected mice treated with orbifloxacin administered orally 10 times at 24-hr intervals at a dose of 2.5 mg/kg. Orbifloxacin did not change the activity of peritoneal macrophages in noninfected mice. Administration of orbifloxacin in E. coli-infected mice modulated the effects of infection on the percentage of phagocyting macrophages, the percentage of NBT-positive cells, and nitric oxide production. Orbifloxacin did not affect the synthesis and release of interleukin-1 by macrophages. Orbifloxacin exerted a modulating effect on the subsets of lymphocytes in thymus, spleen, and mesenteric lymph node cells in noninfected and E. coli-infected mice.     

Profile of Chicken Macrophage Functions After Exposure to Catecholamines In Vitro

The effects of catecholamines (CA) on various chicken macrophage functions were examined. Macrophage monolayers were exposed to .01, .1, .25, 1, 2, and 5 (μg/mL of dopamine (DA), norepinephrine (NE) and epinephrine (E) for 1 hr. All CA were toxic for macrophages at 1 -5 μg dose range resulting in 25-50% cell death. All CA at the .1 and .25 μg/mL level increased E. coli and sheep red blood cells (SRBC) phagocytosis by macrophages. the percentage of Fc-receptor positive macrophages increased after CA exposure. Prolonged exposure of macrophages (3 hr) reduced SRBC phagocytosis by DA-treated but not in NE-and E-treated macrophages. However, after 1 hr exposure and 3 hr recovery period, CA-induced changes were reversed in all but DA-treated cultures. Apomorphine and metoclopromide blocked DA whereas propranolol blocked NE and E effects suggesting specificity of the observed effects via catecholaminergic receptors on chicken macrophages. Dopamine and NE (.25 μg/mL) did not affect but E exposure enhanced LPS-induced tumoricidal factor production. These findings suggest that CA modulate chicken macrophage effector functions.     

A new, simple and rapid method for enumerating human T lymphocytes in full blood: the E. coli (ATCC 11303) rosette test

A new bacterial rosette technique for enumerating T lymphocytes is described. E. coli (strain B; ATCC 11303), fixed in formaldehyde after overnight growth in thioglycolate medium, are mixed with washed whole blood cells (100 μl) and after incubation at 4°C, slides are made, stained and counted. The nature of the lymphocytes forming E. coli rosettes was demonstrated by comparing their cytochemical staining characteristics with those of E rosetted lymphocytes, and by mixed E. coli and E, mouse E rosette and Fc receptor tests, and by mixed E. coli rosette tests and anti-Ig staining. E. coli and E rosette tests in controls and pediatric patients were also compared. The results show that T_μ and T_γ cells rosette with E. coli.     

Identification and characterization of "pathoadaptive mutations" of the cadBA operon in several intestinal Escherichia coli

The dysenteric Shigella spp. and enteroinvasive Escherichia coli (EIEC) have evolved from commensal E. coli by the acquisition of a virulence plasmid and inactivation of genes of the cad locus encoding lysine decarboxylase (LDC) by so-called pathoadaptive mutation. As horizontal gene transfer and recombination occurs frequently in E. coli we were interested to see if similar pathoadaptive mutations are commonly present in other intestinal pathotypes. Therefore, we examined 140 intestinal E. coli strains of various pathotypes and the ECOR collection for their ability to decarboxylate lysine, and identified 25 strains that were unable to do so. Complementation of a Shiga toxin-producing E. coli and two enteropathogenic E. coli strains, both LDC-negative, with the intact cad locus restored LDC activity and resulted in a reduction in adherence to tissue culture cells. We investigated the cad locus for possible alterations by using hybridization and PCR techniques and compared the results with the alterations reported for Shigella spp. and EIEC strains. Interestingly, the alterations of the cad genes were similar to those previously reported, pointing towards a parallel evolution of LDC silencing in different intestinal E. coli pathotypes.     

Immunoreactivity of Recombinant Carcinoembryonic Antigen Proteins Expressed in Escherichia Coli

Immunoreactivities of recombinant carcinoembryonic antigen (CEA) proteins expressed in Escherichia coli (E. coli) were analyzed in relation to the CEA domain structure [domains N, I (A1—B1), II (A2—B2), III (A3—B3) and M). We reconstructed in a prokaryotic expression vector, pUCPL-cI, the cDNAs for CEA-N, CEA-I. CEA-II, and CEA-III-M. The latter three were expressed as fusion products with bacterial β-galactosidase. The recombinant proteins were solubilized by sonication in 1% sodium dodecyl sulfate (SDS) and purified by preparative SDS-polyacrylamide gel electrophoresis followed by electroelution. Their molecular weights Judged from Western blotting coincided with those calculated from their cDNA sequences, respectively. By solid-phase enzyme immunoassays, the immunoreactivities of the purified recombinant proteins were tested with 21 distinct anti-CEA monoclonal antibodies (MAbs) which had been found to recognize the peptide epitopes of the CEA molecule and to be reactive with the recombinant CEA proteins expressed in Chinese hamster ovary (CHO) cells. Fourteen of the 21 MAbs reacted with the recombinant CEA proteins expressed in E. coli and confirmed the localization of the epitopes identified by using the recombinant CEA proteins expressed in CHO cells. The reactivities of 5 MAbs with the recombinant proteins expressed in E. coli were remarkably low when compared with those of the proteins expressed in CHO cells but also confirmed the localization of the epitopes identified with the recombinant CEA proteins expressed in CHO cells. The remaining 2 MAbs did not react with any recombinant protein expressed in E. coli. These results indicate that the fusion CEA-proteins expressed in E. coli are useful in the localization of the epitopes on the polypeptide chains when they reacted with the MAbs tested. However, one third of the epitopes of CEA peptides may be profoundly affected by the presence of disulfide bonds and/or sugar chains which do not seem to be formed well in E. coli.     

Effect of Freezing Colostrum on Resistance of Neonatal Lambs to Experimental Infection with Escherichia coli

Effects of freezing and thawing colostrum on resistance of neonatal lambs to experimental infection with Escherichia coli were evaluated using 16 newborn lambs. Eight sets of twins were fed colostrum from the ewe at 3.3 and 15.4 h of age. Colostrum was obtained from the ewe and divided into two equal portions. One portion was frozen in liquid nitrogen and then thawed in a water bath prior to feeding. The second portion was held at approximately 39°C in a water bath. Four sets of twins were orally inoculated with 3 ×10⁸ to 10¹¹ cfu of enterotoxigenic E. coli at 24 h of age. Blood was sampled at 0 and 24 h for IgG and differential leukocyte counts. Freezing and thawing reduced cell viability in colostrum from 43.1 to 10.1%. Neither freezing and thawing colostrum nor E. coli inoculation affected plasma IgG or total or differential leukocyte counts, fecal scores, respiration rates, rectal temperatures, fecal coliform excretion or intake. Shedding of K99+ E. coli was increased and body weight gain from 7 to 14 d was decreased when lambs were inoculated with E. coli. Results of this study suggest that freezing and thawing colostrum does not destroy components that provide resistance to E. coli challenge in newborn lambs.     

An ultrastructural study of enteropathogenic Escherichia coli infection in human infants

Over the past 2 years, we have studied and treated 18 infants with protracted diarrhea due to an enteropathogenic Escherichia coli serogroup 0119. All patients had persistent stool escretion and jejunal overgrowth with this pathogenic E. coli. Jejunal biopsy revealed atrophy of villi with a chronic inflammatory cell infiltrate in the lamina propria. E. coli 0119 adhered to the luminal surface of enterocytes. Electron microscopy showed disappearance of glycocalyx and microvilli at the areas of bacterial adherence. Intracellular damage was indicated by dilatation of rough endoplasmic reticulum, mitochondrial changes, and cytoplasmic pallor. Similar changes in histology and ultrastructure occurred in ileal epithelial cells. Glandular crypt epithelium showed prominent subnuclear vacuolation and separation of lateral intercellular junctions throughout the small intestine. Rectal mucosal biopsy showed mucus depletion and irregular atrophy of the epithelium, with E. coli 0119 adherent to the luminal surface. Ultrasuctural damage paralleled that in the small intestine. E. coli 0119 causes damage to epithelial cells throughout the infant intestinal tract. This damage leads to atrophy of villi and a marked reduction in absorptive surface area, resulting in protracted diarrhea.     

基于XGBoost方法的大肠杆菌-NC膜复合电极阻抗模型研究

将机器学习方法用于分析大批量大肠杆菌菌液浓度以及临界低阈值抗生素抑菌效果评价.采用XGBoost机器学习算法,构建金电极上大肠杆菌-NC膜贴附模型,用于检测不同浓度大肠杆菌的电化学阻抗谱.在此基础上,分析不同浓度抗生素硫酸阿米卡星作用于标准浓度大肠杆菌所对应的阻抗谱变化.根据Randles等效电路,使用ZView软件拟...     

Environmental factors influence the production of enterobactin, salmochelin, aerobactin, and yersiniabactin in Escherichia coli strain Nissle 1917

The probiotic Escherichia coli strain Nissle 1917 produces four siderophores: the catecholates enterobactin and salmochelin, the hydroxamate aerobactin, and the mixed-type siderophore yersiniabactin. We studied the influence of pH, temperature, and carbon source on the production of these four siderophores. Yersiniabactin and salmochelin were maximally produced under neutral to alkaline conditions (pH 7.0 and 7.6, respectively), whereas aerobactin was maximally produced at a more acidic pH (pH 5.6), which agrees with the slightly higher complex stability of hydroxamates at acidic pH values compared to the catecholates. Under nearly all conditions studied, catecholate siderophore production was higher with glycerol than with glucose as the carbon source. Yersiniabactin production was also higher with glycerol as the carbon source at pH 7.0. At 42 °C, strain Nissle 1917 grew poorly or not at all because of the iron-limiting conditions. In a competition experiment between wild-type strain Nissle 1917 and a mutant of this strain with a deletion in the yersiniabactin operon, the wild-type overgrew the mutant at pH 7.0 and 7.6 and not at pH 5.6. These results agree with yersiniabactin production being of greater advantage at neutral and slightly alkaline pH values. The production of four siderophores may help the probiotic E. coli Nissle 1917 to compete with other E. coli strains in the colon. The probiotic strain Nissle 1917 used in our experiments has many characteristics in common with uropathogenic E. coli and other pathogenic strains which also secrete these siderophores. Uropathogenic E. coli strains may need the multitude of siderophores to adapt to the pH of urine, which varies between pH 4.6 and 8.0.     

Effect of Saccharomyces boulardii and Bacillus cereus var. toyoi on the humoral and cellular response of mice to vaccines

The effect of Saccharomyces boulardii and Bacillus cereus var. toyoi on the humoral and cellular response of mice to the simultaneous inoculation of a tetravalent E. coli bacterin and a viable vaccine against Canine Parvovirus is reported. Thirty-six isogenic BALB-c female mice, seven weeks old, were vaccinated with 1/20th of the dose used for pigs and dogs, respectively, and randomly divided into three groups. Group 1 received non-supplemented feed, group 2 was supplemented with S. boulardii, and group 3 with Bacillus cereus var. toyoi. The humoral response was quantified through ELISAs using specific antigens, and the cellular response quantifying IL-4 and IFNg through ELISA. Means of seroconversions to the bacterin were always higher in supplemented mice than in controls, varying from 1.6-1.8 for group 2 and from 1.2-1.4 for group 3. Seroconversions against Parvovirus for group 2 were 5.2-12 times higher, and those of group 3 were 6.8-9.1 times higher than those of controls. IL-4 was produced by spleen cells of S. boulardii supplemented mice stimulated with E. coli fimbriae.     

Specific regions of genome plasticity and genetic diversity of the commensal Escherichia coli A0 34/86

Escherichia coli A0 34/86 (O83:K24:H31) is a commensal strain that has been used for prophylactic and therapeutic colonization of the intestine of newborn infants. To identify traits specific for E. coli A0 34/86, we used a minimal tiling set of 148 BAC clones of A0 34/86 genomic DNA, to construct restriction-digested BAC arrays. Hybridization with genomic DNA from four E. coli strains (CFT073; O157:H7; K12 and Nissle 1917) allowed selection of two BAC clones that were sequenced to identify A0 34/86-specific regions. Genes for the yersiniabactin siderophore system, several proteins homologous to Salmonella enterica serovar Typhimurium vitamin B₁₂ synthesis proteins, as well as genes necessary for the degradation of propanediol, the pix fimbriae determinant and genes coding for a putative phosphoglycerate transport system present also on pathogenicity island V of E. coli strain 536 were all identified in E. coli A0 34/86. This comparative analysis underlines the important genome heterogeneity between E. coli strains.     

Bone-marrow derived macrophages as targets for the replication of mouse hepatitis virus type 3

Bone-marrow (BM) derived macrophages are sensitive target cells for replication of mouse hepatitis virus type 3 (MHV3). These cells can be grown in large numbers and the percentage of defined macrophages increased until day 10 when 100% of the cells represented macrophages. MHV3 replicated within these cells to high titers and caused the formation of multi-nucleated giant cells. This effect was seen with very low virus inocula in BM macrophages of C57BL/6 mice that are highly susceptible to in vivo infection with MHV3 whereas macrophages from resistant A/J mice did not show a cytopathic effect at these virus doses. 1000-fold higher virus doses, however, caused the cytopathic effect in macrophages of both C57BL/6 and A/J mice.     

Aspects of genome plasticity in pathogenic Escherichia coli

The species Escherichia coli comprises not only non-pathogenic or commensal variants that belong to the normal intestinal flora of most mammals, but also various pathogenic strains causing diverse intestinal and extraintestinal infections in man and animals. Virulence factors and mechanisms involved in pathogenesis have been successfully analyzed for many years resulting in a wealth of knowledge about many E. coli pathotypes. However, our knowledge on the genome content, diversity and variability between pathogenic and also non-pathogenic subtypes is only slowly accumulating. Pathotypes have been largely defined by the presence or absence of particular DNA segments that in most cases appear to have been acquired via horizontal gene transfer events. As these regions are frequently subjected to excisions, rearrangements, and transfers they contribute to the previously unexpected and underestimated rapid evolution of E. coli variants resulting in the development of novel strains and even pathotypes. In these studies various novel aspects of genome diversity and plasticity in extraintestinal and intestinal pathogenic E. coli pathotypes have been addressed and the results have been directly applied for the improvement of diagnostic methods.     

Massive destruction of malaria-parasitized red blood cells despite spleen closure

It is currently accepted that malaria-parasitized red blood cells (pRBC) are eliminated, like senescent erythrocytes, phagocytically by macrophages in the red pulp of the spleen. Here, however, we show that self-healing Plasmodium chabaudi malaria activates spleen closure in C57BL/6 mice. Confocal laser scanning microscopy revealed that spleen closing was manifested by elimination of entry into the red pulp of 3-microm polystyrol particles, pRBC, and nonparasitized red blood cells but not of bovine serum albumin. This spleen closure did not reflect a reduction in the number of phagocytic cells, as shown by flow cytometry, whereas marginal zone macrophages (MZM) were lost and red pulp macrophages entered the white pulp. Splenic trapping of pBRC was strongly reduced in the absence of MZM and marginal metallophilic macrophages (MMM), as it is in noninfected mice with a disrupted lymphotoxin beta receptor (LTbetaR(-/-)), and it was still significantly reduced when the number of MZM and MMM was diminished, as in tumor necrosis factor alpha-deficient (TNF-alpha(-/-)) mice. Moreover, mice deficient in TNF-alpha, tumor necrosis factor receptor I (TNFRI(-/-)), and LTbetaR exhibited progressive impairment in malaria-induced spleen closing. Treatment of C57BL/6 mice with TNF-alpha induced loss of MZM and spleen closing by about 20%. Our data indicate that TNF/TNFRI signaling is involved in regulating malaria-induced spleen closure, which is maximal during crisis, when parasitemia declines more than 100-fold. Consequently, the vast majority of pRBC cannot be destroyed by the spleen during crisis, suggesting that the known sophisticated sequestration system of Plasmodium parasites did not evolve to avoid splenic clearance.     

The deposition of C5b-9 complexes and its precursors on E. coli J5 during complement activation enhances uptake and toxicities of gentamicin

The complement system provides the host with protection against pathogenic agents and in some cases can result in damage to host tissue. However, the exact mechanism of how complement kills Gram-negative bacteria in lysozyme-neutralized and or lysozyme-depleted serum is still under active investigation. In previous studies, it has been demonstrated that inner membrane damage by the membrane attack complex contributes to depolarization and the subsequent collapse of the membrane potential. In these studies we have shown that the membrane attack complex and its precursors provide additional protective effect by the enhanced uptake of antibiotics in the death of E. coli J5. Specifically, the deposition of C5b fragments from C6 neutralized Pooled Normal Human Serum (PNHS) and C5b6 complexes from C7 neutralized PNHS on E. coli J5 contribute to antibiotic uptake and killing. Since C5b and C5b6 do not form pores, we suggest that disturbances and or cracks in the outer membrane by the deposited complexes accelerates uptake of the antibiotics and enhanced killing of E. coli J5 employed in these studies.     

Programmed aging or error catastrophe? An exmination by two-dimensional polyacrylamide gel electrophoresis

We have examined newly synthesized proteins in the young adult and in older populations of the nematode Caenorhabditis elegans using two-dimensional polyacrylamide gel electrophoresis (2D PAGE). A temperature-sensitive mutant strain, DH26, with a mean life span of about 15 days, under our conditions, was used to block progeny development. Nematodes of several different ages were pulse-labeled for 5 h, in vivo, with ³⁵S-labeled E. coli, A subsequent 30-min chase with unlabeled E. coli served to rid the worms of endogenous labeled E. coli proteins. We resolve 700 or more proteins by 2D PAGE polyacrylamide gel electrophoresis of extracts of young nematodes. The patterns of these proteins are highly reproducible in comparisons of independent repeats of identical experiments. No new major proteins are synthesized at any time during the adult phase (4–22 days) nor are any of the most abundant proteins not made during this period. At our level of detectability (estimated as a satellite spot containing 4% of the amount of label in a major spot) we see no misincorporation of radioactive amino acids into newly synthesized proteins. These data are inconsistent with predictions by any one of several, so called, “error catastrophe” models of senescence and also show that modulation of the highest abundancy classes of proteins are also not involved in senescence.     

Spirulina Platensis Exposure Enhances Macrophage Phagocytic Function in Cats

Bronchoalveolar lavage macrophages isolated from cats were cultured on glass coverslips. Macrophages were exposed to a water-soluble extract of Spirulina platensis in concentration range of 0 to 60 ug per mL for two hours. Spirulina-extract exposure did not cause significant macrophage cytotoxicity over untreated Control cultures. Macrophage monolayers from treated and control cultures were incubated with sheep red blood cells (SRBC) as well as viable Escherichia coli. The percentages of phagocytic macrophages for both of these particulate antigens were higher (a two-fold increase in SRBC phagocytosis and over 10% increase in Escherichia coli uptake) in cultures treated with various concentrations of Spirulina-extract. However the numbers of either types of particles internalized by phagocytic macrophage were not different between the control and treated cultures. These data which showed that Spirulina platensis extract enhances macrophage phagocytic function imply that dietary Spirulina supplementation may improve the disease resistance potential in cats.     

Cytotoxicity of mouse serum and peritoneal fluid for Ascaris suum infective larvae

Serum from BALB/c mice infected with Ascaris suum resulted in 51Cr release from A. suum infective larvae during in vitro incubation. Peritoneal fluid from infected or noninfected BALB/c mice also produced in vitro release of 51Cr from A. suum larvae. 51Cr release did not occur when anti-A. suum serum or peritoneal fluid from infected or noninfected mice were heat-inactivated. Serum and peritoneal fluid from infected or noninfected AKR-C5 deficient mice did not generate 51Cr release from A. suum larvae.     

Modulation of Leukocyte Phagocytic and Oxidative Burst Responses by Human Seminal Plasma

The typical absence of immune responses to spermatozoa in the female reproductive tract at the time of insemination, despite the presence of a marked leukocytic infiltrate into the cervical mucus is intriguing. It may be that localised immunoregulatory mechanisms exist and this study used whole blood flow cytometry to determine the effects of human seminal plasma on neutrophil and monocyte function. Seminal plasma inhibited the proportion of neutrophils and monocytes phagocytosing E.coli, and the intensity of neutrophil phagocytosis, but enhanced the magnitude of the phagocytic response of those monocytes that escaped inhibition relative to PBS treated controls. Oxidative burst responses to E.coli were also inhibited and this effect was mediated by low molecular weight species, as dialysis totally abrogated the inhibitory activity. Seminal plasma had no effect on the neutrophil burst response to fMLP when compared to the controls, however there was a significant difference between the responses of undialysed and dialysed seminal plasma treated samples. Undialysed seminal plasma significantly inhibited the proportion of monocytes undergoing the burst response to fMLP and there were significant differences between the proportion of cells responding and their intensity in undialysed and dialysed seminal plasma treated samples. In summary, this study reports differential modification of neutrophil and monocyte function by human seminal plasma. The residual capacity of these cells to undergo phagocytosis and generate oxidative burst responses suggests that localised innate immune function remains intact and is possibly enhanced in the female reproductive tract at the time of insemination. Other mechanisms must protect inseminated sperm at this time.     

Immune Response of E. cuniculi Infected Mice to Aflatoxin B1

The immunomodulatory effects of aflatoxin B₁ (AFB₁) on mice experimentally infected with Encephalitozoon cuniculi (E. cuniculi) were studied. Mice inoculated intraperitoneally with spores of E. cuniculi drank a daily solution of AFB₁ (0.2 mg kg^-1 of body weight) for 27 days. Application of AFB₁ to mice demonstrated a decrease of immunocompetent cells. On the other hand, the mice infected with E. cuniculi and given AFB₁ showed significant increased number of both monocytes and CD8+ T cells, and tendency to a decrease in CD4+ T cells. Aflatoxin B₁ revealed to merely modulate systemic immune response of E. cuniculi infected mice.