Dennis W. Gaines

BODY AND ORGAN GROWTH OF THE DEVELOPING HORMEL-HANFORD STRAIN OF MALE MINIATURE SWINE

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.

Ecto-5′-Nucleotidase (CD73) Regulates Host Inflammatory Responses and Exacerbates Murine Salmonellosis

Food-borne Salmonella spp., are a major cause of hospitalization and death. Adenosine, an important immune regulator of inflammation, limits tissue damage during infection. CD39 (nucleoside triphosphate dephosphorylase) combined with ecto-5′-nucleotidase (CD73) metabolizes ATP to adenosine. We studied the expressions of CD39 and CD73 in tissues, and T helper cells in mice after Salmonella infection and evaluated the role of CD73 in regulating immune responses and bacterial clearance in wild-type and CD73-deficient (CD73−/−) mice. Both CD39 and CD73 transcript levels declined in the infected wild-type mice. Compared to wild-type mice, tissues from infected CD73−/− mice had significantly higher expression of pro-inflammatory cytokines and reduced anti-inflammatory responses. CD73−/− mice were more resistant to infection and had a greater inflammatory responses and a significantly lower bacterial load in the liver compared to wild-type mice. Thus, CD73 expression attenuates inflammation during murine Salmonellosis and impairs immunity, leading to increased bacterial colonization and prolonged infection.

Survey of undeclared egg allergen levels in the most frequently recalled food types (including products bearing precautionary labelling)

ABSTRACT Since the number of recalls involving undeclared allergens is commonly associated with bakery and snack foods, we aimed to determine the frequency of egg allergens in a large number of these products using two commercial enzyme-linked immunosorbent assay (ELISA) methods. Samples were chosen that either had no egg identified on the product label or which had an egg precautionary statement. Among all samples, egg protein was detected in 5% of products using a Morinaga (MO) kit and 1% of products using a R-Biopharm (RB) kit. For bakery samples, egg protein was detected in 6% of 363 samples with no precautionary labelling (6% by MO and 1% by RB kit) and 12% of 80 samples which had precautionary labelling. For snack samples, egg protein was detected in 2% of 371 samples with no precautionary labelling (2% by MO and < 1% by RB kit) and 5% of 21 samples which had precautionary labelling. The disagreement rates between two methods were 5.2% for bakery products and 2.6% for snack products. The sample repeatability was at an acceptable level for bakery (< 12.5%) and snack foods (< 7.5%) for each method. The relative standard deviation between test kits was high (103.1%) for bakery foods. Four bakery products without precautionary labelling had a higher level of egg protein per serving compared with the eliciting dose (ED10 of 3.7 mg protein) for egg allergic patients. These results highlight the fact that detection methodology plays a vital role for accurate labelling control and mitigation of risk for egg allergic consumers. GRAPHICAL ABSTRACT

Survey of undeclared soy allergen levels in the most frequently recalled food categories with or without precautionary labelling

ABSTRACT A comprehensive study was designed to determine the frequency and levels of soy allergen in packaged bakery and snack food products. A representative sample of products with no soy allergen disclosed on the label was analysed using two widely used enzyme-linked immunosorbent assay (ELISA) methods. Samples were chosen that either had no soy identified on the product label or which had a soy precautionary statement. Among 558 bakery and snack products, soy protein was detected in 17% of the products using the Neogen (NE) kit and 11% of the products using the Elisa Systems (ES) kit. The disagreement rates between kits were 8.8% for bakery products and 3.3% for snack products. Overall soy protein was detected at higher frequency in bakery products than in snack foods. Among 284 bakery samples, soy protein was detected in 25% of the samples with no precautionary statement and 19% of the samples which had a precautionary statement. Among 274 snack samples, soy protein was detected in 11% of the samples with no precautionary statement and 9% of the samples which had a precautionary statement. The sample repeatability was at an acceptable level (< 9%) for each method and food commodity. The reproducibility between kits was 23% for bakery foods and 36% for snack foods. None of the bakery (21) and snack (6) products without precautionary labelling (measured level > 5 ppm) had a higher level of soy protein per serving compared with the eliciting dose10 (ED10) of 10.6 mg for soy allergic patients. But the level of soy protein per serving may be clinically relevant to a subpopulation of soy allergic patients if a more stringent eliciting dose is applied. These findings emphasise that suitable detection methodologies and references doses are crucial for labelling accuracy and the safety of soy allergic consumers. GRAPHICAL ABSTRACT

INTERACTION OF AFLATOXINS AS MEASURED BY THEIR BIOCHEMICAL ACTION ON RAT LIVER SLICES AND HEPATOCYTES

The possible interaction between pairs of aflatoxins com monly occurring together in foods was evaluated by determining their biochem ical actions on the liver and hepatocytes of young mature rats. RNA synthesis, a sensitive and 14 early target of the effects of aflatoxin B1, was m easured with [ C]orotic acid or 3 [ H]uridine used as substrates. In the first study (comprised of four replicate experiments), liver slices treated with aflatoxin B1 at 0, 120, 240, and 480 ng/ ml were incubated in the presence and absence of aflatoxin B2 at 120 ng/ ml. In the second study, liver slices were treated with the sam e concentrations of B1 incubated in the presence and absence of aflatoxin G1 at 120 ng/ m l (comprised of four replicate experim ents) or at 240 ng/ ml (comprised of two replicate experim ents). In the third study (single experim ent), isolated hepatocytes were treated with aflatoxin B1 at 0, 60, 120, 240, and 480 ng/ ml in the presence and absence of aflatoxin G1 at 120 ng/ m l. Leakage of lactic acid...

Diet-induced obesity precipitates kidney dysfunction and alters inflammatory mediators in mice treated with Shiga Toxin 2

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.

Ornithine decarboxylase, fatty acid synthetase, and lipid levels in selected organs of the postnatal developing male miniature pig

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.

Induction of ornithine decarboxylase in the stomach mucosa of swine with NaCl or 12-O-tetradecanoylphorbol-13-acetate

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.

Macromolecular Levels, Dna Synthesis and Ornithine Decarboxylase Activity in Leg Muscles From 6-Mercaptopurine-Treated Rats

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.