Reduced Smad3 protein expression and altered transforming growth factor-beta1-mediated signaling in cystic fibrosis epithelial cells.

Cystic fibrosis (CF) is a disease characterized by an aggressive inflammatory response in the airways. Given the antiinflammatory properties of transforming growth factor (TGF)-beta1, it was our goal to examine components of TGF-beta1-mediated signaling in both a cultured cell model and a mouse model of CF. A CF-related reduction of protein levels of the TGF-beta1 signaling molecule Smad3 was found in both of these model systems, whereas Smad4 levels were unchanged. Functional effects of reduced Smad3 expression are manifest in our cultured cell model, as reduced basal and TGF-beta1-stimulated levels of luciferase expression using the TGF-beta1-responsive reporter construct 3TP-Lux in the CF-phenotype cells compared with control cells. However, TGF-beta1-stimulated responses using the A3-Luc reporter construct were normal in both cell lines. These results suggest that select TGF-beta1-mediated signaling pathways are impaired in CF epithelial cells. This selective loss of Smad3 protein expression in CF epithelium may also influence inflammatory responses. Our data demonstrate that both CF-phenotype cells lacking Smad3 expression, and A549 cells expressing a dominant-negative Smad3, are unable to support TGF-beta1-mediated inhibition of either the interleukin (IL)-8 or the NOS2 promoter. We conclude that a CF-related reduction in Smad3 protein expression selectively alters TGF- beta1-mediated signaling in CF epithelium, potentially contributing to aggressive inflammatory responses.

In vivo alterations of IFN regulatory factor-1 and PIAS1 protein levels in cystic fibrosis epithelium.

Inducible nitric oxide synthase-2 (NOS2) expression has been shown to be reduced in cystic fibrosis (CF) epithelial cells. Reduced NOS2 expression is unexpected, given the inflammatory nature of CF airway disease, and is an indication that cell-signaling mechanisms necessary for proper NOS2 regulation are probably altered in CF epithelium. Therefore, we examined the expression levels of regulatory factors necessary for NOS2 expression in CF epithelium and showed that IFN regulatory factor-1 (IRF-1) is necessary for full NOS2 expression. Mice lacking IRF-1 expression have diminished epithelial NOS2 expression, as well as reduced NO-dependent chloride transport across the nasal epithelia. Furthermore, IRF-1 protein expression is reduced in nasal and intestinal epithelial cells from CF mice, suggesting a possible mechanism for the CF-related reduction of epithelial NOS2 expression. Active signal transducer and activator of transcription-1 (Stat1) is necessary for both NOS2 and IRF-1 expression. We found that protein levels of Stat1 were increased in CF cells, but that the active phosphorylated form of Stat1 was bound to the protein inhibitor of activated Stat1 (PIAS1). We propose that increased levels of PIAS1 diminish certain cell-signaling pathways, resulting in reduced IRF-1 and NOS2 expression in CF epithelial cells.

Cystic fibrosis transmembrane conductance regulator-dependent regulation of epithelial inducible nitric oxide synthase expression.

Recent evidence has shown that the inducible form of nitric oxide (NO) synthase (NOS2) has reduced expression in airway epithelia of patients with cystic fibrosis (CF) despite the presence of chronic inflammation. The goal of this paper is to determine whether NOS2 expression is regulated by the presence of functional CF transmembrane conductance regulator (CFTR). Using a human trachea epithelial cell line in which CFTR activity is blocked by the overexpression of the CFTR regulatory domain, we found that loss of CFTR activity reduces NOS2 messenger RNA expression as determined by reverse transcriptase/polymerase chain reaction and reduces overall NO production compared with mock-transfected controls. An in vivo model using mice lacking CFTR expression (cftr -/-), wild-type mice (cftr +/+), and cftr -/- mice that have had human CFTR introduced to the intestinal epithelium using the fatty acid binding protein (FABP) promoter (FABP-hcftr) was also examined. Electrical characterization confirmed that FABP-hcftr mice had corrected electrophysiologic properties compared with cftr -/- mice in the ileum, but FABP-hcftr nasal transepithelial potential difference measurements were identical to cftr -/- values showing specific intestinal correction. NOS2-specific immunostaining revealed that NOS2 expression is evident in sections of ileum and nasal epithelium of cftr +/+ mice but is absent in both tissues in cftr -/- mice. FABP-hcftr mice, however, show strong NOS2 staining in epithelial cells of the ileum but reduced staining in the nasal epithelium, suggesting a CFTR-related influence in the regulation of NOS2 expression in epithelial cells.

Nitric oxide-mediated regulation of transepithelial sodium and chloride transport in murine nasal epithelium.

Transepithelial ion transport is regulated by a variety of cellular factors. In light of recent evidence that nitric oxide (NO) production is decreased in cystic fibrosis airways, we examined the role of NO in regulating sodium and chloride transport in murine nasal epithelium. Acute intervention with the inducible NO synthase (iNOS)-selective inhibitor S-methylisothiourea resulted in an increase of amiloride-sensitive sodium absorption observed as a hyperpolarization of nasal transepithelial potential difference. Inhibition of iNOS expression with dexamethasone also hyperpolarized transepithelial potential difference, but only a portion of this increase proved to be amiloride sensitive. Chloride secretion was significantly inhibited in C57BL/6J mice by the addition of both S-methylisothiourea and dexamethasone. Mice lacking iNOS expression [NOS2(-/-)] also had a decreased chloride-secretory response compared with control mice. These data suggest that constitutive NO production likely plays some role in the downregulation of sodium absorption and leads to an increase in transepithelial chloride secretion.

Lactic Acid Bacteria in Cheddar Cheese Made with Sodium Chloride, Potassium Chloride or Mixtures of the Two Salts.

Three different split lots of Cheddar cheese curd were prepared with added sodium chloride (NaCl) potassium chloride (KCl) or mixtures of NaCl/KCl (2:1 1:1 1:2 and 3:4 all on wt/wt basis) to achieve a final salt concentration of 1.5 or 1.75%. At intervals during ripening at 3±1°C samples were plated with All-Purpose Tween (APT) and Lactobacillus Selection (LBS) agar. Isolates were obtained of bacteria that predominated on the agar media. In the first trial ( Lactococcus lactis subsp. lactis plus L. lactis subsp. cremoris served as starter cultures) L. lactis subsp. lactis Lactobacillus casei and other lactobacilli were the predominant bacteria regardless of the salting treatment Received by the cheese. In the second trial ( L. lactis subsp. lactis served as the starter culture) unclassified lactococci L. lactis subsp. lactis unclassified lactobacilli and L. casei predominated regardless of the salting treatment given the cheese. In the third trial ( L. lactis subsp. cremoris served as the starter culture) unclassified lactococci unclassified lactobacilli L. casei and Pediococcus cerevisiae predominated regardless of the salting treatment applied to the cheese Thus use of KCl to replace some of the NaCl for salting cheese had no detectable effect on the kinds of lactic acid bacteria that developed in ripening Cheddar cheese.

Microflora of Cheddar Cheese Made with Sodium Chloride, Potassium Chloride, or Mixtures of Sodium and Potassium Chloride.

Cheddar cheese samples from three different split lots of cheese curd were prepared with added NaCl, KCl, or mixtures of NaCl/KCl (2:1, 1:1, 1:2 and 3:4, all on wt/wt basis) to achieve a final salt concentration of 1.5 or 1.75%. Cheeses were stored at 3 ± 1°C and their microbiological characteristics were evaluated over a 36-week ripening period. Populations of aerobic microorganisms, lactic acid bacteria, nonstarter lactic acid bacteria, aerobic spores, coliforms, and yeasts and molds in cheeses made with KCl or NaCl/KCl mixtures were not significantly (P>0.05) different from those of control cheeses made with NaCl. Staphylococcus aureus and Escherichia coli were not detected in any of the test or control cheeses.

Thermal Destruction of Listeria monocytogenes in Ground Pork Prepared with and without Soy Hulls.

D-values and z-values were determined for Listeria monocytogenes Scott A cells heated in raw ground pork prepared with and without soy hulls and in a soy hull/water mixture. Products inoculated with ca. 107 colony-forming units (CFU) per g were sealed in glass vials, immersed in a water bath, and held at 50, 55, 60, or 62°C for predetermined times. Survival was determined by testing heated samples with McBride listeria agar. The D-values for L. monocytogenes cells at 50, 55, and 60°C were 108.81, 9.80, and 1.14 min, respectively, when heating was in ground pork and 113.64, 10.19, and 1.70 min, respectively, when heating in ground pork with added soy hulls. At 62°C L. monocytogenes cells were inactivated too rapidly to permit determination of the D-value. The D-values for L. monocytogenes in the soy hull/water mixture at 50 and 55°C were 19.84 and 3.94 min, respectively. L. monocytogenes cells were inactivated too quickly to determine the D-value at 60°C. The z-values for L. monocytogenes in ground pork prepared with and without soy hulls were 5.45 and 5.05°C, respectively. If ground pork naturally contains 102 L. monocytogenes cells per g and if we want to assure safety with a 4-D Listeria cook (reducing the L. monocytogenes population by four orders of magniatude), then according to results of this study, ground pork must be heated to an internal temperature of 60°C for at least 4.6 min and ground pork with added soy hulls for at least 6.8 min.

Sensitivity of Listeria monocytogenes to Selected Spices.

Ten spices were added separately to tryptose broth which was then inoculated to contain, per ml, 105 or 107 Listeria monocytogenes strain Scott A or V7, respectively, and held at 4°C for 7 d. Strain Scott A appeared to be more sensitive to effects of spices than was strain V7. The population of strain Scott A was decreased to <10/ml in 1 d by 1% sage, in 4 d by 1% allspice, and in 7 d by 1% cumin, garlic powder, paprika, and red pepper. Black pepper and mace at 1% reduced but did not completely inactivate the population of strain Scott A, whereas 1% white pepper enhanced its growth. At 1%, only sage reduced the population of strain V7 to <10, but 7 d rather than 1 d of storage were required. At 3 and 5%, some activity against strain V7 was exhibited by allspice, mace, and nutmeg. Generally, the antilisterial effect of spices increased as the concentration in the medium increased from 1 to 3 to 5% .

Fate of Listeria monocytogenes During the Manufacture of Mozzarella Cheese.

Mozzarella cheese was made from a mixture of pasteurized whole and skim milk which was inoculated to contain 104-105 CFU Listeria monocytogenes (strain Ohio, California, or V7) per ml. Temperature of milk was maintained at 40°C (104°F) for 30 min when curd became resilient and the pH reached 5.90-5.93. Populations of L. monocytogenes changed at different rates during the various phases of making Mozzarella cheese. During the early stages of curd formation, numbers of L. monocytogenes were ca. 4-fold greater in curd than in whey. Numbers of L. monocytogenes in freshly cut curd were 25 to 38% greater than in inoculated milk. Cooking curd at 40°C for ca. 30 min caused a decrease of ca. 38% as compared to numbers of the pathogen in curd after cutting. During cheddaring of curd, numbers of L. monocytogenes increased by ca. 25%, over numbers at the end of cooking. Placing of curd in hot water [77°C (170°F)] and stretching for 3-4 min caused complete demise of the pathogen, as determined by our methods. The curd temperature during stretching was 58 to 65°C (136 to 149°F). Results of cold enrichments were all negative for stretched and brined curd. L. monocytogenes failed to survive during the making of Mozzarella cheese as done in this study.

Behavior of Listeria monocytogenes in the Presence of Sodium Propionate Together with Food Acids.

Tryptose broth (TB) containing 0.00, 0.05, 0.15, or 0.3% sodium propionate was adjusted to pH 5.0 or 5.6 with acetic, tartaric, lactic, or citric acid, inoculated to contain ca. 103 CFU of Listeria monocytogenes /ml and incubated at 13 or 35°C. The bacterium grew in all controls (free of propionate) under all conditions; however, only slight growth occurred at 13°C when the pH was adjusted to 5.0 with acetic or tartaric acid. Growth also occurred at 13 and 35°C when TB adjusted to pH 5.6 with acetic or tartaric acid contained 0.05 or 0.15% propionate, but 0.3% inhibited growth of the pathogen. When the pH of TB was adjusted to 5.0 with the same acids, L. monocytogenes was inhibited or inactivated by 0.15 or 0.3% propionate. The pathogen grew at 13 or 35°C in TB that contained 0.05 or 0.15% propionate and was adjusted to pH 5.6 with lactic or citric acid although the lag phase was prolonged as the concentration of propionate was increased. Under these conditions, propionate at 0.3% occasionally inhibited growth of L. monocytogenes . Growth was reduced and sometimes inhibited completely by 0.15 or 0.3% propionate when the pH of TB was adjusted to 5.0 with the same acids.

Freezing of Listeria monocytogenes and Other Microorganisms: A Review.

When the temperature of microbes is lowered rapidly, some are injured through thermal shock. Frozen cells can be injured mechanically by intra- and extracellular ice crystals. During freezing, as water is removed, there is a concentration of cell solutes which can lead to dissociation of cellular lipoprotein. Warming of frozen cells can be accompanied by growth of ice crystals which then can physically affect cells. Freeze-thaw injury of microbes is manifested by an increase in fastidiousness and by changes in cellular morphology, release of materials from the micro- and macrostructure of cells, and denaturation of macromolecules. Given the proper environmental conditions, cells can repair such injury. Cryoprotectants minimize damage to cells during freezing and frozen storage. Death and injury of Listeria monocytogenes were greater when cells were frozen and stored at -18°C rather than -198°C. Tryptose broth was more protective of cells than a phosphate buffer solution when freezing and storage were at -18°C; the reverse was true at -198°C. Repeated freezing (-18°C) and thawing (35°C) were more detrimental to cells of L. monocytogenes than were repeated freezing at -198°C and thawing at 35°C. Freezing cells at -198°C and storing them at -18°C caused more injury and death than did freezing and storage at -198°C. Glycerol was an effective cryoprotectant for L. monocytogenes . Less effective were milk fat, lactose, and casein. The extent of injury and death varied among strains of L. monocytogenes given the same treatment. Freezing and thawing increased susceptibility of L. monocytogenes to effects of lipase and lysozyme.

Thermal Inactivation of Listeria monocytogenes in Chicken Gravy.

Heat resistance of Listeria monocytogenes strains V7 and Scott A in chicken gravy and changes in heat resistance during refrigerated storage were studied. After chicken gravy was made, it was cooled to 40°C, inoculated with 105 CFU L. monocytogenes per ml of gravy, and then stored at 7°C for 10 d. Gravy was heated at 50, 55, 60, and 65°C immediately after inoculation and after 1, 3, 5, and 10 d of refrigerated storage. The D values for strains Scott A and V7 in gravy heated at 50°C at day 0 were 119 and 195 min and at day 10 they were 115 and 119 min, respectively, whereas at 65°C comparable values at day 0 were 0.48 and 0.19 min and at day 10 they were 0.014 and 0.007 min. Heat resistance (expressed as D values) was greater at day 0 than at the end of refrigerated storage. The z values ranged from 3.41 to 6.10°C and were highest at the early stages of chill storage and then decreased at the later stages. Strain V7 was more heat resistant than Scott A at 50°C. Strain Scott A always had a higher z value than did strain V7 at the same storage interval. A heat treatment greater than the 4-D process recommended by the U.S. Department of Agriculture was required to inactivate the large numbers of L. monocytogenes that developed in chicken gravy during refrigerated storage.

Transmission Electron Microscopy of Unfrozen and Frozen/Thawed Cells of Listeria monocytogenes Treated With Lipase and Lysozyme.

Unfrozen cells of Listeria monocytogenes typically contained no preplasma space exterior to the plasma membrane (PM) when viewed by transmission electron microscopy. Cells of L. monocytogenes strains Scott A, V7, and California (CA), after freezing and frozen storage, exhibited one or more of the following when viewed with transmission electron microscopy: (a) retraction of cytoplasm and infolding of the PM to form mesosomes, (b) extra-and intracellular rupture of the cell wall (CW), (c) formation of intracellular "bubbles," and (d) damage to the CW and PM that could have resulted from autolysin activity. Type and degree of effect depended on frozen storage time and strain of L. monocytogenes . Lysozyme treatment of unfrozen or frozen/stored (19 d)/thawed cells of strains Scott A, V7, and CA resulted in protoplast formation and damage to the CW. Three stages of protoplast formation were observed when cells of strain CA were frozen, stored 2 weeks, thawed, and treated with lysozyme. More damage to the CW and PM occurred when frozen storage time was extended for up to 6 weeks before treatment with lysozyme. Lipase and lysozyme treatment of unfrozen or frozen/stored (19 d)/thawed cells of strain Scott A resulted in protoplast formation with some damage to the PM and irregularity in shape of cells. Damage to the PM increased with increasing frozen storage time for up to 6 weeks. Some cells of strain CA resisted freezing, frozen storage for 6 weeks, thawing, and treatment with lipase and lysozyme.

Lysozyme and Lipase Alter Unfrozen and Frozen/Thawed Cells of Listeria monocytogenes.

Unfrozen and frozen/thawed cells of Listeria monocytogenes strains Scott A, V7, and California were treated with lipase and/or lysozyme. Cells of strain Scott A were more susceptible to the lytic action of lysozyme than were cells of strains V7 and California. Treatment of unfrozen cells with lipase before exposure to lysozyme enhanced cellular lysis. This also was true for cells held frozen for up to 6 weeks before they were thawed and treated with enzymes. Some variation existed among strains of L. monocytogenes in their susceptibility to effects of lysozyme. Frozen storage of cells of all three strains increased their susceptibility to lysis by lipase, and this was related inversely with the percentage of cells that survived freezing and frozen storage. Transmission electron microscopy showed some enzyme-treated cells formed protoplasts.

Ultrafiltration and Reverse Osmosis in Dairy Technology: A Review.

Ultrafiltration and reverse osmosis processes can be useful in the dairy foods industry. When milk is processed, milk fat and casein are rejected fully (e.g., are in retentate) and thus are concentrated by ultrafiltration and reverse osmosis membranes. Lactic cultures are slow to reduce the pH of retentate because of its increased buffering capacity since concentrated calcium phosphate and proteins are present. Conditions for growth of pathogenic microorganisms and inhibition of such bacteria in ultrafiltered milk differ from those of unfiltered milk. The principal advantage of using ultrafiltered milk for conversion into such cheeses as Cheddar, cottage, Havarti, Feta, brick, Colby, and Domiati is an increase in yield of product. Additional benefits claimed for use of ultrafiltered milk in cheese making include reduction in costs of energy, equipment, and labor; improved consistency of cheese flavor; and possible production of new byproducts.

Survival of Borrelia burgdorferi in Whole Milk, Low Fat Milk, and Skim Milk at 34°C and in Skim Milk at 5°C.

Autoclaved whole milk, low-fat milk, protein-fortified skim milk and regular skim milk were inoculated to contain ca. 105 to 106 Borrelia burgdorferi strains 35210, 35211, or EBNI/ml and stored at 34°C for 16 d. Similarly inoculated skim milk also was held at 5°C for 46 d. Numbers of survivors were estimated by the Most Probable Number (MPN) technique. In all instances, numbers of B. burgdorferi decreased over the storage period. At 34°C, no strain of B. burgdorferi was detected after day 12. The mean D-values, at 34°C, for strains 35210, 35211, and EBNI were 2.2, 2.4, and 2.2 d, respectively. The mean D-values, at 34°C, for all strains in whole milk, low-fat milk, protein-fortified skim milk, and regular skim milk were 2.4, 2.3, 1.9, and 2.4 d, respectively. At 5°C, spirochete numbers in regular skim milk decreased, but all three strains remained at a detectable level for 46 d. The mean D-values, at 5°C, for strains 35210, 35211, and EBNI were 12, 15, and 12 d, respectively.

Growth of Listeria monocytogenes at 4, 32, and 40°C in Skim Milk and in Retentate and Permeate from Ultrafiltered Skim Milk.

Pasteurized skim milk and retentate (concentrated fivefold or twofold by volume) and permeate from ultrafiltered skim milk were inoculated with Listeria monocytogenes strains California or V7 and incubated at 4, 32, or 40°C. Changes in populations of the pathogen were determined, growth curves were derived, and generation times and maximum populations calculated for each combination of strain, product, and temperature. Both strains grew faster and achieved higher (ca. 1 to 2 orders of magnitude) populations at 4°C in retentate of either concentration than in skim milk. The pathogen grew in permeate at 4°C and attained maximum populations of ca. 106 to 107/ml. Tyndallized samples of skim milk and retentate and permeate from ultrafiltered skim milk were inoculated with the same strains of L. monocytogenes and incubated at 32 or 40°C. Populations achieved by the pathogen at these temperatures, ca. 107 to 108/ml, were similar in skim milk, retentate, and permeate.

Behavior of Listeria monocytogenes in the Presence of Flavobacteria in Skim Milk at 7 or 13°C.

Sterile samples of skim milk were inoculated with Listeria monocytogenes (strain Scott A, California, or V7), Flavobacterium lutescens or Flavobacterium species, or a combination of L. monocytogenes plus flavobacteria and incubated at 7 or 13°C for 8 weeks. McBride Listeria agar was used to determine populations of L. monocytogenes at 0, 7, 14, 28, 42, or 56 d, and plate count agar was used to enumerate flavobacteria at the same time intervals. Growth of L. monocytogenes was significantly (P<0.05) enhanced during incubation at 7 or 13°C in mixed cultures with F. lutescens . When tested with Flavobacterium sp. ATCC 21429, numbers of L. monocytogenes decreased insignificantly (P<0.05) in mixed cultures compared to pure cultures. L. monocytogenes did not affect growth of flavobacteria; also, no marked changes in pH of the milk were caused either by L. monocytogenes alone or by mixed cultures.