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Shiga Toxin (Stx)-producing E. coli (STEC) continue to be a prominent cause of foodborne outbreaks of hemorrhagic colitis worldwide, and can result in life-threatening diseases, including hemolytic uremic syndrome (HUS), in susceptible individuals. Obesity-associated immune dysfunction has been shown to be a risk factor for infectious diseases, although few studies have addressed the role of obesity in foodborne diseases. We hypothesized that obesity may affect the development of HUS through an alteration of immune responses and kidney function. We combined diet-induced obese (DIO) and HUS mouse models to look for differences in disease outcome between DIO and wild-type (WT) male and female C57 B l/6 mice. Following multiple intraperitoneal injections with endotoxin-free saline or sublethal doses of purified Stx2, we examined DIO and WT mice for signs of HUS development. DIO mice receiving Stx2 injections lost more body weight, and had significantly higher (p < 0.001) BUN, serum creatinine, and neutrophil counts compared to WT mice or DIO mice receiving saline injections. Lymphocyte counts were significantly (p < 0.05) lower in Stx2-treated obese mice compared to WT mice or saline-treated DIO mice. In addition to increased Stx2-induced kidney dysfunction, DIO mouse kidneys also had significantly increased expression of IL-1α, IL-1β, IL-6, TNF-α, MCP-1, and KC RNA compared to saline controls (p < 0.05). Serum cytokine levels of IL-6 and KC were also significantly higher in Stx2-treated mice compared to saline controls, but there were no significant differences between the WT and DIO mice. WT and DIO mice treated with Stx2 exhibited significantly higher degrees of kidney tubular dilation and necrosis as well as some signs of tissue repair/regeneration, but did not appear to progress to the full pathology typically associated with human HUS. Although the combined obesity/HUS mouse model did not manifest into HUS symptoms and pathogenesis, these data demonstrate that obesity alters kidney function, inflammatory cells and cytokine production in response to Stx2, and may play a role in HUS severity in a susceptible model of infection., (Published by Elsevier Ltd.)

Evaluating the potential of Salmonella serovars for tissue colonization and egg contamination in laying hens is critical due to widespread consumption of poultry and egg-containing products. The 2009 FDA Egg Rule was implemented to target the eradication of Salmonella enterica Enteritidis (SE) from layers; however, other Salmonella serovars, such as Heidelberg (SH) and Typhimurium (ST), have also been associated with poultry-related outbreaks. We conducted this study to see if serovars other than SE could colonize in laying hens, cause egg contamination, and modulate circulating T-cell populations. Laying hens were orally gavaged with 10 7 colony forming units (CFU) of SE, SH, or ST and assessed for colonization in spleen, ovaries, and oviduct 10 d postchallenge. Splenic colonization was similar for all the serovars; however, colonization of ovaries and oviducts was significantly higher with SH compared to SE and ST. Furthermore, SH challenge resulted in egg contamination, while SE and ST did not result in contaminated eggs. Phenotypic evaluation of peripheral blood lymphocytes showed significant reduction in CD4 cells in SH-challenged birds and lower CD8α and CD8β cells in SE-challenged birds compared to controls. Our data showed that non-SE serovars have equal or higher potential to colonize reproductive tissues of laying hens and may be accompanied by altered lymphocyte populations., (Published by Oxford University Press on behalf of Poultry Science Association 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Internalin A is a surface protein of the facultative intracellular pathogen Listeria monocytogenes that interacts with the human host cell protein E-cadherin to facilitate invasion of epithelial cells. A single amino acid substitution at position 16 in mouse E-cadherin prevents this interaction. Synthetic polypeptides of 30 aa encompassing position 16 of human and mouse E-cadherin were tested for their ability to inhibit in vitro invasion of Caco-2, HepG2 and TIB73 cell lines by L. monocytogenes. Only the human-derived peptide was capable of inhibiting invasion in the human-origin Caco-2 and HepG2 cell lines. These findings demonstrate that small polypeptides can inhibit invasion of biologically relevant cell types by L. monocytogenes in vitro and may be potentially useful as therapeutic agents in vivo.

The objective of the present study was to compare the uptake and killing of Salmonella serovars by murine and avian macrophage cell lines. We used Salmonella enterica serovars Enteritidis (SE338) and Typhimurium (SR11) for this study. Uptake of green fluorescent protein-labeled bacteria was measured using flow cytometry. Cell sorting and plating of viable infected macrophages demonstrated that bacterial clearance was significantly better with J774A.1 compared with HD11 cells. HD11 cells produced significantly higher amounts of nitric oxide (NO) than J774A.1 cells upon infection with SE338 and SR11, whereas J774A.1 cells exhibited greater superoxide production with SR11. Treatment of HD11 cells with recombinant chicken interferon gamma in the absence of bacteria enhanced NO production but did not induce increased levels synergistically with bacteria. Interferon treatment did not influence phagocytosis or increase killing by HD11 cells.

The potential toxicity of dietary soy trypsin inhibitor (TI) was evaluated in neonatal miniature swine. From 1 to 6 weeks of age, two groups of male piglets were artificially reared in an Autosow and automatically fed either TI or control liquid diet. From 6 to 39 weeks of age, these two groups were fed either TI or control chow diet. A third group, sow control (SC), suckled from birth to 6 weeks of age, were also weaned to control chow from 6 to 39 weeks of age. Clinical chemistry and plasma cholecystokinin (CCK) determined at 6, 18, 30 and 39 weeks of age, and serum amylase activity with gross and histopathological analyses of major organs at 6 and 39 weeks of age are reported. TI had no effect on plasma CCK, serum amylase activity, or numerous clinical chemistry values. TI-fed piglets had a larger relative liver weight at 6 weeks of age. Relative pancreas weight decreased with age but was not affected by TI. Gross and histopathological analyses of major organs, except the spleen, were within normal limits. Increased incidence of extramedullary hematopoiesis was noted in the spleen of the TI group at 6 but not at 39 weeks of age. There was no consistent pattern in immunohistochemical foci for secretin, gastrin releasing polypeptide or CCK, and no change in DNA, RNA, mitotic index or nuclear density of pancreatic cells. At 6 weeks of age, TI increased pancreatic protein and amylase activity but not trypsin or chymotrypsin activity. None of the effects suggested that this dose of TI was toxic to either the neonatal or sexually mature miniature male swine.

Mature, female swine were randomly assigned to one of seven dietary groups. Swine in groups 1-3 were fed a cholesterol-rich diet for 55 days while the remaining groups remained on a basal swine diet. At the end of the cholesterol(Chol)-preloading period the swine in groups 1-7 were placed on menhaden oil (MO) and/or corn oil (CO) as follows: groups 1 and 4, 15% CO (control); groups 2 and 5, 0.75% MO+14.25% CO; groups 3 and 7, 15% MO; and group 6, 7.5% MO+7.5% CO. Animals were killed at the end of the approximately 6-month feeding period and portions of liver, pancreas and colon mucosa were analyzed for both ornithine decarboxylase (ODC) and thymidine kinase (TK) activity while polyamine levels were measured in the liver and pancreas. Statistical analyses were carried out by one-way and two-way ANOVA and by trend analysis. In the pancreas, the highest MO group (group 7) had significantly higher ODC levels when compared with the CO control (group 4) and the next to highest MO group (group 6) (one-way ANOVA)-all non-cholesterol preloaded groups. Using a two-way ANOVA (Chol-by-MO), liver ODC was significantly lower in the CO control when compared with the lowest and highest MO groups (groups 5 and 7, respectively), again in the non-cholesterol-preloaded animals. In the colon, the swine in the Chol-low MO group (group 2) had significantly lower TK activity than the Chol/CO control group (group 1) and Chol/Hi MO group (group 3) (one-way ANOVA) and also had significantly lower activity than all groups except the CO control (group 4) (two-way ANOVA). Liver acetylputrescine in the lowest and highest MO groups (groups 5 and 7, respectively) was significantly higher than in the CO group (group 4). Liver spermidine in the Chol-Hi MO group (group 3) was significantly higher than the Chol-Lo MO group (group 2), while the highest MO group (group 7) had a statistically higher level than the other non-cholesterol groups (groups 4-6) (one-way ANOVA). Liver spermine was significantly higher in the Chol-Hi MO group (group 3) when compared to the CO control (group 1) and the Chol-Lo MO group (group 2) (one-way ANOVA). Pancreatic putrescine in the CO control (group 4) was significantly higher than all other groups (two-way ANOVA) while spermine from the 2 Chol-MO groups (groups 2 and 3) was higher than the Chol-CO control (group 1) (one-way ANOVA). Using trend analysis, liver TK, putrescine and spermidine increased in the non-cholesterol preloaded groups with increasing dietary MO, similar to the increase seen in ODC. Thus, of the three organs studied, only liver responded to menhaden oil with changes in both ODC itself or some of its metabolic engendered products and thymidine kinase; at least for one of the parameters, ODC, change associated with dietary MO was dependent on whether the swine were preloaded with cholesterol.

As part of a larger study designed to characterize the early developmental stages of the Hormel-Hanford strain miniature pig, the brain, kidney, liver, pancreas, and spleen from male animals were examined for changes in RNA, DNA, and protein contents from 1 to 196 d after birth. Distinct patterns were found for changes with age in macromolecular levels. Protein levels increased from d 1 to 56 in all organs except spleen, in which little change was noted. Gel electrophoresis showed little qualitative change in the liver protein profile during this period. A fat-free, non-nucleic acid, protein-containing fraction, insoluble in hot alkali, appeared in the brain after approximately 1 wk following birth. DNA concentrations decreased markedly from d 1 to d 196 for brain, kidney, and spleen but decreased more gradually for liver and pancreas. RNA levels declined slightly or remained the same in all organs except pancreas, where a large increase occurred from d 1 to weaning (56 d). Growth proceeded in all organs by increases in cell number (hyperplasia), as evidenced by increases in total (level or concentration x organ weight) DNA, or by hypertrophy, as evidenced by increases in the ratio of protein to DNA or by a combination of both processes. Hypertrophic growth was attained by d 56 and continued to sexual maturity in all organs except spleen. Hyperplastic growth continued to sexual maturity in all organs except brain.

Ornithine decarboxylase (ODC) and fatty acid synthetase (FAS) activities were determined in tissues from male neonate and juvenile miniature swine (Hormel-Hanford strain) at various ages. ODC activity was measured in liver, brain, kidney, pancreas, and spleen at one day and at 1, 4, 8, 12 and between 24 and 32 weeks. Hepatic FAS activity, total lipid, triglyceride, and total cholesterol were measured at 2, 8, 16, and 32 weeks. Generally, tissue ODC activity was highest in the spleen at all ages. Three postnatal patterns of ODC activity were observed for the different organs. The mean values of FAS activity, total lipid, and cholesterol were highest at 8 weeks compared to other sampling periods.

As part of a larger study designed to characterize the early developmental stages of the Hormel-Hanford strain miniature pig, whole body, brain, kidney, liver, pancreas and spleen from male animals were examined for weight increases from one to 196 days, the approximate age of maturity. At 196 days, body weights had increased to 82.5 times the weight at day 1; increases in organ weights were greatest for spleen, less and similar for kidney, liver and pancreas, and the least for brain. Little change in relative organ weights was noted, except for the brain where an almost steady decrease occurred starting from 7 days after birth.

Ornithine decarboxylase (ODC) activity of rat tissues was measured by the standard 14CO2 trapping method after frozen storage (-60 or -70 degrees C) of the tissues or their 105,000g supernatants. True ODC activity was determined by two methods: (a) addition of the inhibitors alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC, or aminooxyacetate (AOA), an inhibitor that blocks the decarboxylation of ornithine by mitochondrial enzymes; and (b) chromatographic analysis of the reaction products. In the frozen supernatants of liver and spleen, ODC activity changed only slightly after 1 day but increased 29 and 14%, respectively, by 30 days; activity in kidney supernatant decreased 17% after 1 day and remained near that level at 30 days. Kidney and spleen ODC activity was inhibited 90-100% by DFMO, but apparent liver ODC activity was inhibited only 60-75%. In the supernatant prepared from tissue stored frozen for 1 day, apparent ODC activity in liver increased 500% over that activity in the freshly prepared supernatant; at 23 days, apparent activity increased 755% for liver and 121% for kidney. After 23 days, DFMO did not inhibit apparent ODC activity in supernatants from frozen liver and inhibited ODC in frozen kidney by only 49%. With AOA, the ODC activities of the fresh and frozen supernatants were similar, indicating that the large increase in apparent ODC activity in frozen tissue was due to artifacts from the metabolism of ornithine via the mitochondrial pathway. HPLC analysis of the reaction products resulting from the incubation of uniformly labeled [14C]ornithine with the fresh and frozen preparations indicated no increase in putrescine with the frozen preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of sodium chloride and 12-O-tetradecanoylphorbol-13-acetate on ornithine decarboxylase (ODC) activity in gastric mucosa of miniature swine was investigated as a model for gastric inflammation. The level of the enzyme was lower in the pylorus than in the fundic or cardiac regions of the stomach in untreated animals. Treatment with sodium chloride at 1 g/kg produced large increases in all three regions, with the greatest relative increase in the pylorus. Treatment with sodium chloride at 0.25 g/kg or the phorbol ester at 2.0 mg/pig produced significant but less dramatic increases. ODC activity in control and treated mucosal extracts was inhibited by the specific ODC inhibitor difluoromethylornithine. Most of the enzyme activity was associated with superficial and exfoliated cells that could be scraped from the mucosal surface. No increase in the inflammatory mediator leukotriene B4 was observed in the mucosal extracts. Ornithine decarboxylase appears to be a useful enzymatic marker for the regenerative events that occur after tissue damage and may correlate with the putative tumor-promoting function of sodium chloride in gastric tissues.

Sprague-Dawley male and female rats were treated with 6-mercaptopurine (6-MP) (2 mg/kg sc) daily from 2 to 22 days of age and killed at 7, 15, 27 and 64 days of age. At 7 and 27 days of age rats were injected with 3H thymidine for measurement of DNA synthesis. Fore- and hindlimb muscles were removed and analyzed for ornithine decarboxylase (ODC) activity (all ages), DNA radioactivity (7 and 27 days), DNA level (27 and 64 days) and RNA level (64 days). As expected, ODC activity and DNA synthesis were higher in muscles of 7-day-old rats than in muscles of the older rats studied. A consistently lower ODC activity was seen in 6-MP-treated vs. control rats for 5-25 days after start of treatment, but the effect was essentially the same for the hindlimb and forelimb muscles. During the 7-27-day time course ODC activity was higher in hindlimb than forelimb muscles. By 27 days of age DNA synthesis was also higher in the hindlimb muscles. DNA synthesis was decreased after 5 days of treatment relative to that of control rats, to an approximately equal extent in forelimb and hindlimb muscles. Five days after the last treatment a trend was seen for slower recovery from inhibition of DNA synthesis in hindlimb muscles, particularly in male rats. DNA levels were reduced in treated rats relative to those in control rats 5 days after the last treatment to approximately the same degree in forelimb and hindlimb muscles. Forty-two days after the last treatment a trend toward increased activity of ODC and increased DNA and RNA levels was seen in muscles of treated rats, probably reflective of recovery processes. These early biochemical effects of 6-MP, which were seen to about the same extent in the forelimb and hindlimb muscles cannot explain by themselves the delayed hindlimb fat atrophy resulting from 6-MP treatment of neonatal rats.

A modified microassay for the determination of metabolically generated 14CO2 is described and is applied to the measurement of ornithine decarboxylase in animal tissue preparations. In this technique, the reaction takes place in a microcentrifuge tube inside a 20-ml scintillation vial that also contains a center well with a CO2-trapping agent. The vial is sealed with a silicone septum-lined plastic screw cap. After the initial incubation period during which the enzymatic reaction occurs, acid is injected through the septum into the reaction tube, and 14CO2 is released during a second incubation period. The reaction vial is then removed, counting solution is added to the scintillation vial, and radioactivity is measured. Linearity is present with respect to both increasing amounts of tissue and incubation time. Recovery of evolved 14CO2 was greater (97.5 vs 91.5%) and variation between replicate samples was less (coefficient of variation 2.7 vs 8.5%) when the modified microassay was compared with an assay system that requires removal of the screw cap from the vial before acid injection. The modification allows greater safety and facilitated assays, which could result in savings of both time and laboratory personnel. Special precautions for the use of NaH14CO3 as the recovery marker are noted.