Unusually high illness severity and short incubation periods in two foodborne outbreaks of Salmonella Heidelberg infections with potential coincident Staphylococcus aureus intoxication.

We describe the investigation of two temporally coincident illness clusters involving salmonella and Staphylococcus aureus in two states. Cases were defined as gastrointestinal illness following two meal events. Investigators interviewed ill persons. Stool, food and environmental samples underwent pathogen testing. Alabama: Eighty cases were identified. Median time from meal to illness was 5·8 h. Salmonella Heidelberg was identified from 27 of 28 stool specimens tested, and coagulase-positive S. aureus was isolated from three of 16 ill persons. Environmental investigation indicated that food handling deficiencies occurred. Colorado: Seven cases were identified. Median time from meal to illness was 4·5 h. Five persons were hospitalised, four of whom were admitted to the intensive care unit. Salmonella Heidelberg was identified in six of seven stool specimens and coagulase-positive S. aureus in three of six tested. No single food item was implicated in either outbreak. These two outbreaks were linked to infection with Salmonella Heidelberg, but additional factors, such as dual aetiology that included S. aureus or the dose of salmonella ingested may have contributed to the short incubation periods and high illness severity. The outbreaks underscore the importance of measures to prevent foodborne illness through appropriate washing, handling, preparation and storage of food.

'One Health' investigation: outbreak of human Salmonella Braenderup infections traced to a mail-order hatchery - United States, 2012-2013.

Human salmonellosis linked to contact with live poultry is an increasing public health concern. In 2012, eight unrelated outbreaks of human salmonellosis linked to live poultry contact resulted in 517 illnesses. In July 2012, PulseNet, a national molecular surveillance network, reported a multistate cluster of a rare strain of Salmonella Braenderup infections which we investigated. We defined a case as infection with the outbreak strain, determined by pulsed-field gel electrophoresis, with illness onset from 25 July 2012-27 February 2013. Ill persons and mail-order hatchery (MOH) owners were interviewed using standardized questionnaires. Traceback and environmental investigations were conducted. We identified 48 cases in 24 states. Twenty-six (81%) of 32 ill persons reported live poultry contact in the week before illness; case-patients named 12 different MOHs from eight states. The investigation identified hatchery D as the ultimate poultry source. Sampling at hatchery D yielded the outbreak strain. Hatchery D improved sanitation procedures and pest control; subsequent sampling failed to yield Salmonella. This outbreak highlights the interconnectedness of humans, animals, and the environment and the importance of industry knowledge and involvement in solving complex outbreaks. Preventing these infections requires a 'One Health' approach that leverages expertise in human, animal, and environmental health.

Cytotoxicity of root canal irrigating solutions and photodynamic therapy using curcumin photosensitizer.

BACKGROUND: Photodynamic therapy (PDT) has shown satisfactory antibacterial effects. However, little information regarding the cytotoxicity potential of PDT using curcumin as a photosensitizer (PS) on fibroblasts are found. The aim of this in vitro study was to evaluate the cytotoxicity of root canal irrigating solutions and photodynamic therapy with curcumin PS on the L-929 cell line. METHODS: Healthy mouse skin fibroblast cells were distributed into the following 7 experimental groups: G1 - culture medium DMEM (control group); G2 - 0.9% sodium chloride; G3 - 2.5% sodium hypochlorite (NaOCl); G4 - 5% NaOCl; G5 - PDT with curcumin PS at 500 mg/L + blue LED; G6 - PDT with curcumin PS at 750 mg/L + blue LED; and G7 - PDT with curcumin PS at 1000 mg/L + blue LED. All experimental groups which underwent PDT action were submitted to blue LED for 4 min, with a wavelength of 480 nm and energy fluency of 75 J/cm². The cultures were maintained under standard cell culture conditions (37°C, 100% humidity, 5% CO2). Cell viability analysis was performed using the colorimetric method to evaluate the periods of 6, 24, and 48 h. Data were subjected to the Kruskal-Wallis test, followed by the Dunn test to compare groups and Friedman test to compare periods (α = 0.05). RESULTS: When comparing the periods, no significant differences were observed for any of the experimental groups analyzed (p > 0.05), except for the NaOCl2.5 group that exhibited higher cell viability at 6 h compared to the period of 48 h (p = 0.0489). In the comparisons of the experimental groups, there were no statistically significant differences between the control group compared to all disinfection protocols, regardless of the period evaluated (p > 0.05), except for the PDT + C1000 group that showed lower cell viability (P < 0.05). CONCLUSIONS: PDT with curcumin at 1000 mg/L was cytotoxic on L-929 fibroblast cell culture. However, laser-activated curcumin at a concentration of 500 mg/L presented no influence on L-929 fibroblast cell viability in in vitro conditions.

Glial cell degeneration and hypomyelination caused by overexpression of myelin proteolipid protein gene.

Myelin proteolipid protein (PLP), the major myelin protein in the CNS, has been thought to function in myelin assembly. Thus, mutations within the gene coding for PLP (Plp) cause hypomyelination, such as the jimpy phenotype in mice and Pelizaeus-Merzbacher disease in humans. However, these mutants often exhibit premature death of oligodendrocytes, which form CNS myelin. To elucidate the functional roles of Plp gene products in the maturation and/or survival of oligodendrocytes, we produced transgenic mice overexpressing the Plp gene by introducing extra wild-type mouse Plp genes. Surprisingly, transgenic mice bearing 4 more Plp genes exhibited dysmyelination in the CNS, whereas those with 2 more Plp genes showed normal myelination at an early age (3 weeks after birth), but later developed demyelination. Overexpression of the Plp gene resulted in arrested maturation of oligodendrocytes, and the severity of arrest was dependent on the extent of overexpression. Overexpression also led to oligodendrocyte cell death, apparently caused by abnormal swelling of the Golgi apparatus. Thus, tight regulation of Plp gene expression is necessary for normal oligodendrocyte differentiation and survival, and its overexpression can be the cause of both dys- and demyelination.

Saccharomyces cerevisiae PHO5 promoter region: location and function of the upstream activation site.

Saccharomyces cerevisiae repressible acid phosphatase (PHO5) is induced when inorganic phosphate in the culture medium is depleted. To study the mechanism of this regulation, we constructed various deletions in the 5'-flanking region of the PHO5 gene. Two elements were revealed by this analysis: an upstream activation site (UAS) and a downstream element, both playing parts in the expression of this gene. The UAS is located between -384 and -292 upstream of the initiation codon and activates expression of the gene when inorganic phosphate is depleted. It consists of two homologous regions (UAS I and UAS II) that contain CTGCACAAATG and an adenine-plus-thymine-rich sequence, either one of which suffices for the effect. The downstream element includes a putative TATA box at -100 from the ATG codon and is necessary for efficient transcription and expression of the normal-sized PHO5 transcript. The distance between the UAS and the downstream element can be altered without causing loss of expression efficiency, and the action of the UAS is not affected by its orientation. These results are consistent with a model wherein UAS acts as a site of activation for transcription by interaction with a protein factor(s) that becomes active when inorganic phosphate is depleted from the culture medium.

Stimulation of prostaglandin cyclooxygenase and prostacyclin synthetase activities by estradiol in rat aortic smooth muscle cells.

The effects of estradiol on the arachidonic acid pool and prostacyclin biosynthetic activity in rat aortic smooth muscle cells were studied. Estradiol has no significant effect on the distribution of [14C]arachidonic acid in cells with respect to prostacyclin production assay, the endogenous fatty acid (specifically, arachidonic acid) composition of cellular phospholipid fractions and cellular phospholipase (or/and lipase) activities. However, estradiol significantly stimulates both prostaglandin cyclooxygenase and prostacyclin synthetase activities of cells, and induction of new protein biosynthesis is involved in the effect of estradiol on the stimulation of prostacyclin biosynthetic activity.

Stimulation of prostacyclin biosynthetic activity by estradiol in rat aortic smooth muscle cells in culture.

Effects of estradio on cell proliferation and prostacyclin biosynthetic activity in cultured rat aortic smooth muscle cells are investigated. Prostacyclin is the major product formed from arachidonic acid by the cells, both in a cell-free homogenate system and in intact cells. When cells are treated with estradiol, no effect on cell proliferation is observed. However, a significantly stimulatory effect on prostacyclin biosynthetic activity in cells is evident. The maximal effect of estradiol on the stimulation of prostacyclin biosynthetic activity is observed after 5-day treatment of the cells with estradiol at its physiological concentration (10(-9) M). Our results support the idea that estradiol would be a protective hormone in atherosclerotic heart disease.

Age-related decrease in prostacyclin biosynthetic activity in rat aortic smooth muscle cells.

Prostaglandin biosynthetic activity in cultured rat aortic smooth muscle cells was investigated as a function of age by both intact cell and cell-free homogenate assays. The total cyclooxygenase activity for prostaglandin biosynthesis is the same in cells from young and old rats. However, cells from young rats proceduce more prostacyclin than prostaglandin E2, and cells from old rats produce more prostaglandin E2 than prostacyclin. Age-related decrease in prostacyclin biosynthesis is also found when protaglandin H2 is used as substrate. Lower prostacyclin and higher prostaglandin E2 biosynthetic activity in aortic smooth muscle cells from aged rats may contribute to the direct explanation of pathogenesis of spontaneous atherosclerosis and artial thrombosis in aged humans and other mammals.

Effects of estradiol on the metabolism of arachidonic acid by aortas and platelets in rats.

We have previously reported that estradiol treatment stimulates prostacyclin production by cultured rat aortic smooth muscle cells, through the stimulation of fatty acid cyclooxygenase and prostacyclin synthetase activities. In order to see whether estradiol stimulates the fatty acid cyclooxygenase activity in platelets, intact rats were treated with estradiol, and thromboxane biosynthesis in platelets and prostacyclin production by aortas were investigated. Estradiol significantly stimulates prostacyclin production by aortas. However, no significant effect on thromboxane biosynthesis in platelets is observed. Our present results support the idea that estradiol would be a protective hormone in atherosclerotic heart disease.

Stimulatory effect of insulin on aortic smooth muscle cell migration induced by 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid and its modulation by elevated extracellular glucose levels.

In investigations on the role of insulin on migration of rat aortic smooth muscle cells, migration of the cells was measured by a modified Boyden chamber technique with 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) as a chemoattractant. Insulin itself was not a chemoattractant for these cells, and insulin added just before the migration assay did not affect cell migration in the presence or absence of 12-HETE. Cells pretreated with insulin in culture dishes for a long period, however, showed a significant increase in migration induced by 12-HET, and the increase depended on the insulin concentration: concentrations of insulin of greater than 50 microIU/ml caused about twofold increase in cell migration. On the other hand, long-term incubation with various concentrations of insulin (0.15-1000 microIU/ml) did not affect nonspecific cell migration in the absence of 12-HETE. The stimulatory effect of insulin on cell migration gradually increased with the duration of insulin treatment, reaching a plateau after 4 days. Thus, insulin stimulated 12-HETE-induced smooth muscle cell migration in a time- and dose-dependent manner. When the extracellular D-glucose concentration in the Boyden chamber was increased from 100 to 300 mg/dl, the stimulatory effect of insulin on 12-HETE-induced cell migration was augmented. This modulation by D-glucose was not due to an increase in the osmotic pressure of the medium, since addition of mannitol to increase the osmotic pressure did not enhance the effect of insulin on cell migration.(ABSTRACT TRUNCATED AT 250 WORDS)

Possible role of stem cell factor as a serum factor: monoclonal anti-c-kit antibody abrogates interleukin-6-dependent colony growth in serum-containing culture.

The monoclonal rat anti-c-kit antibody (ACK2), which abrogates colony growth supported by stem cell factor (SCF), significantly inhibited the interleukin-6 (IL-6)-dependent growth of hematopoietic progenitors derived from spleen cells of normal and 5-fluorouracil (5-FU)-treated mice and from bone marrow cells of normal mice in serum-containing culture. The numbers and types of colonies supported by IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF), however, were not influenced by the addition of ACK2 to the cultures of the bone marrow cells from normal mice. In replating experiments with pooled blast cells, ACK2 caused a partial, but significant, inhibition of GM colony growth supported by a combination of IL-6 and fetal bovine serum (FBS), which suggests that FBS is one source of the SCF activity. Conversely, the addition of SCF or FBS with IL-6 to a serum-free culture had significant synergistic effects on the total number of colonies derived from post-5-FU spleen cells and from pooled blast cells. The dose response study showed that the ability of 30% FBS to interact with IL-6 on the colony growth by post-5-FU spleen cells was equivalent to that of approximately 5 ng/mL SCF. These findings suggest that c-kit plays an important role in the growth of hematopoietic progenitors responding to IL-6, and that SCF in the serum affects the development of hematopoietic progenitors in serum-containing cultures.

The conformation of mature human alpha-amylase conditions its secretion from yeast.

The yeast Saccharomyces cerevisiae expresses the cloned cDNA (Amy) encoding human salivary alpha-amylase (Amy) under control of the yeast PHO5 promoter, and secretes the active enzyme into the culture medium. Two approaches were utilized to define the moiety of Amy, which is required for proper secretion and glycosylation. In one approach, chimeras were constructed with a variety of secretion signal sequences (yeast mating factor precursor sequence, yeast acid phosphatase signal sequence and human gastrin signal sequence) fused to the secretion signal-deleted Amy cDNA. The other approach involved analysis of a set of deletion series and a set of point mutations in the Amy-encoding region. The results showed that heterologous signal sequences were sufficient for proper secretion in yeast, irrespective of the insertion of some extra amino acids. In most cases, enzymes with deletions and Cys-465 substitution were not secreted, even though they had complete secretion signal sequences. Instead, they accumulated in the cell in a glycosylated form. Thus, proper secretion seems to require an appropriate conformation in the polypeptide moiety to be secreted.

Age-related increase in the migration of aortic smooth muscle cells induced by 12-L-hydroxy-5,8,10,14-eicosatetranoic acid.

Studies were made on how age influenced the migration of aortic smooth muscle cells. For this, aortic cells from rats of various ages were established in culture in vitro and migration of the cells was measured by the filter membrane technique in modified Boyden chambers. 12-L-Hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), which is mainly produced by platelets and leukocytes and is a strong chemoattractant for rat aortic smooth muscle cells, was used at a concentration of 6 X 10(-15) g/ml in this work. Proliferation of smooth muscle cells decreased significantly with age, but the plating efficiency and cell size did not. In contrast, cell migration induced by 12-HETE showed an age-related increase: The 12-HETE-induced cell migration activities of cells from 6- and 25-month-old rates were 226.7 +/- 35.4 and 223.2 +/- 37.9 cells/10 high-power fields (HPF), respectively, which were significantly higher than that of 121.4 +/- 20.1 cells/10 HPF for cells from 2-month-old rats (P less than 0.05). There was no age-related change in the dose-response curve to 12-HETE for migration of smooth muscle cells. The observed changes are closely associated with the processes of development and maturation of rat aorta.

Low serum estradiol levels in subjects with arterial calcification.

The relationship between arterial calcification and serum estradiol levels was studied in 72 postmenopausal female and 59 male subjects. In both sexes, subjects with iliac artery calcification had rather lower serum estradiol levels (8.4 +/- 1.4 pg/ml in the females, and 19.2 +/- 2.5 pg/ml in the males) than controls (16.1 +/- 1.6 pg/ml in the females, and 29.7 +/- 2.4 pg/ml in the males). The bone mineral content of females with iliac artery calcification (0.44 +/- 0.02 g/cm2) was lower than controls (0.52 +/- 0.01 g/cm2); a positive correlation between serum estradiol levels and bone mineral content was found in the females. However, bone mineral content did not significantly differ between males with and without arterial calcification (0.67 +/- 0.03 g/cm2 in the former, and 0.65 +/- 0.02 g/cm2 in the latter). These results indicate that arterial calcification and increased bone resorption are both individual results of estrogen deficiency.

Elastinolytic activity in the rat aortic smooth muscle cells in culture.

Elastinolytic activity was examined in cultured rat aortic smooth muscle cells, using Congo Red elastin as a substrate. Elastinolytic activity was demonstrated in the soluble fraction of sonicated smooth muscle cells, with an optimal pH around 10.0. The soluble fraction also showed elastase-like esterolytic activity against the synthetic substrate, N-succinyl-trialanyl-paranitroanilide.

Platelets stimulate aortic smooth muscle cell migration in vitro. Involvement of 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid.

The migration of rat aortic smooth muscle cells was measured in modified Boyden chambers. Smooth muscle cells were motile in vitro and their migration was stimulated (time- and dose-dependently) by a platelet-derived factor. Treatment of platelets with indomethacin resulted in a significant increase in smooth muscle cell migration, whereas treatment with 5,8,10,14-eicosatetraenoic acid inhibited it. Purified 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid at a very low concentration (6 x 10(-15)-6 x 10(-13) g/ml) significantly stimulated smooth muscle cell migration. The locomotion induced by 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid was chemokinetic. These findings point to the physiological importance of a platelet 12-lipoxygenase product of arachidonic acid in the early phase of atherosclerosis.

Testosterone inhibits prostacyclin production by rat aortic smooth muscle cells in culture.

The effects of testosterone on cell proliferation and prostacyclin production were investigated using rat aortic smooth muscle cells in culture. Testosterone at 10(-10)-10(-6) M did not have any significant effect on cell proliferation, but it significantly inhibited prostacyclin production by the cells. Maximal inhibition of prostacyclin production (70%) was observed when cells were treated with a physiological concentration of 19(-8) M testosterone for 5 consecutive days. These results suggest that testosterone may stimulate thrombus formation and accelerate atherosclerosis by suppressing prostacyclin production in arterial smooth muscle cells.

Triiodothyronine stimulates prostacyclin production by rat aortic smooth muscle cells in culture.

The effect of triiodothyronine (T3) on prostacyclin production in rat aortic smooth muscle cells was investigated by using an intact cell assay system. T3 at its physiological concentrations has no significant effect on smooth muscle cell proliferation, but it does significantly stimulate prostacyclin production by the cells. Maximum stimulation of prostacyclin production is obtained when cells are treated with T3 for 4 consecutive days. The dose--response curve shows a linear relationship between the stimulation of prostacyclin production and T3 concentrations in the range 0.007--10 microgram/dl. The maximal prostacyclin production by T3-treated cells (at a concentration of 10 microgram/dl), is 270% compared with control cells. T3 treatment shows no significant effect on phospholipase (and/or lipase) activities. Our results suggest that thyroid hormone might play an important physiological role in the protection of arteries from atherosclerotic changes by stimulating prostacyclin production in arterial smooth muscle cells.

Calcium dependency of aortic smooth muscle cell migration induced by 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid. Effects of A23187, nicardipine and trifluoperazine.

We have previously reported that 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), a 12-lipoxygenase product of arachidonic acid in platelets, is a potent chemoattractant for rat aortic smooth muscle cells. In the present study, the mechanism involved in 12-HETE-associated smooth muscle cell migration was investigated in relation to calcium mobilization in the cells. Migration of smooth muscle cells was measured by a filter membrane technique in modified Boyden chambers. Smooth muscle cell migration induced by 12-HETE increased with the increase of extracellular Ca2+ concentration and became maximal at the physiological Ca2+ concentration of 1.25 mM. The calcium ionophore A23187, at concentrations of 0.2 and 2.0 microM, significantly stimulated cell migration. Nicardipine, a potent calcium-entry blocker, significantly inhibited 12-HETE-associated smooth muscle cell migration at concentrations from 10(-9) to 10(-5) M. Concentrations of trifluoperazine from 10(-9) to 10(-5) M and W-7 at 10(-5) M, which are specific inhibitors of calmodulin, also significantly inhibited cell migration induced by 12-HETE. Cytochalasin B at 1.0 and 10 microM, and colchicine at 0.1 and 1.0 microM concentrations drastically inhibited cell migration, indicating that actin-containing microfilaments and microtubules are involved in smooth muscle cell migration. These findings indicated that the stimulation of smooth muscle cell migration by 12-HETE is a highly calcium-dependent process and suggest that 12-HETE might act at the initial stage of smooth muscle cell migration through enhancing calcium influx through plasma membrane and thus stimulating cell migration.

Comparative effect of lipoxygenase products of arachidonic acid on rat aortic smooth muscle cell migration.

We investigated the effects of mono-hydroxyeicosatetraenoic acids (HETEs) and N-formyl-methionyl-leucyl-phenylalanine (F-Met-Leu-Phe) on rat aortic smooth muscle cell migration in modified Boyden chambers. 12-HETE showed the most potent stimulatory effect on smooth muscle cell migration among the mono-HETEs tested. The optimal concentrations for cell migration were 3 X 10(-15) and 3 X 10(-13) g/ml for 12-HETE and 10(-8) g/ml for 15-HETE, 5-HETE and F-Met-Leu-Phe were inactive with these cells. As 12-HETE is biosynthesized from arachidonic acid by the 12-lipoxygenase pathway in platelets and macrophages, and 15-HETE by the 15-lipoxygenase pathway in granulocytes, the present results indicate an important role for such cells in the early phase of atherosclerosis.