Survival of chlorine-injured enterotoxigenic Escherichia coli in an in vitro water system

Survival of chlorine-injured and noninjured subpopulations of enterotoxigenic Escherichia coli was compared in KH2PO4-buffered water and chlorine-neutralized tap water. Injured cells were no less persistent than noninjured cells and did not exhibit limited survival as a consequence of chlorine injury. At high inoculum densities, some injured cells were able to repair, apparently owing to the accumulation of materials arising from the chlorination procedure.     

Model studies on a membrane filtration method for the enumeration of coagulase-positive staphylococci in swimming-pool water using rabbit plasma-bovine fibrinogen agar

The recovery of Staphylococcus aureus from swimming-pool water by membrane filtration was studied in model experiments. On the nonselective medium tryptone soya agar (TSA) there was no difference in counts of noninjured S. aureus with all membrane filters tested and with pour plates. Chlorine-injured S. aureus was enumerated most efficiently on TSA by Gelman Tuffryn HT-450 and Sartorius SM 13806 filters. Tuffryn filters were also most productive when used in combination with the selective medium rabbit plasma - bovine fibrinogen agar (RPFA). Other filters, particularly Gelman GN-6 and Millipore HAWP, when used on RPFA were shown to have a synergistic inhibitory effect on both noninjured and chlorine-injured S. aureus. This effect was not found on Baird-Parker agar. Using Tuffryn filters, counts on RPFA were equal to those on TSA for noninjured S. aureus and 0.1-2.0 log units less for chlorine-injured S. aureus. Despite this, the possibility for reading the in situ coagulase reaction for individual colonies on RPFA is considered such an advantage of this medium that its general use for enumeration of S. aureus in swimming pools is recommended. Further studies should be carried out to allow better resuscitation.     

Chlorine-induced damage to surface adhesions during sublethal injury of enterotoxigenic Escherichia coli.

A comparison of the adhesive ability of noninjured and chlorine-injured enterotoxigenic Escherichia coli was made by in vitro attachment to human peripheral leukocytes. Chlorination selected for noninjured cells with greater capabilities for colonizing the small intestine. Injured populations exhibited reduced association with leukocytes. Maximum reduction was seen in populations with greater than 80% injury. These cells demonstrated less adhesive ability than nonpiliated populations. Electron micrographs suggested that reduced adhesive ability was due to the loss of surface structures as a consequence of sublethal chlorination. The data imply a reduced ability among chlorine-injured pathogens to colonize the small intestine and initiate disease.     

Effect of chlorine injury on heat-labile enterotoxin production in enterotoxigenic Escherichia coli

Heat-labile enterotoxin (LT) production was examined in chlorine-injured and noninjured populations of enterotoxigenic Escherichia coli (ETEC) by passive immune hemolysis and Y-1 mouse adrenal tumor cell assays. Sublethally injured populations showed reduced LT production after 1, 2.5, and 4 h incubation in trypticase soy broth plus 0.25% glucose, pH 8.0. Reduction was observed during injury, resuscitation, and for at least 1.5 h following repair. LT levels comparable with that present in noninjured cells were found after 24 h incubation in the same medium, indicating delayed toxigenesis rather than permanent damage. Chlorinated populations failed to incorporate [14C]glucose until repair was completed suggesting a possible explanation for delayed toxin production. The results indicate a temporary loss of virulence among sublethally injured ETEC in chlorinated waters.     

Transplantation of embryonic neuroectodermal progenitor cells into the site of a photochemical lesion: immunohistochemical and electrophysiological analysis

GFP labeled/NE-4C neural progenitor cells cloned from primary neuroectodermal cultures of p53- mouse embryos give rise to neurons when exposed to retinoic acid in vitro. To study their survival and differentiation in vivo, cells were transplanted into the cortex of 6-week-old rats, 1 week after the induction of a photochemical lesion or into noninjured cortex. The electrophysiological properties of GFP/NE-4C cells were studied in vitro (8-10 days after differentiation induction) and 4 weeks after transplantation using the whole-cell patch-clamp technique, and immunohistochemical analyses were carried out. After transplantation into a photochemical lesion, a large number of cells survived, some of which expressed the astrocytic marker GFAP. GFP/GFAP-positive cells, with an average resting membrane potential (Vrest) of -71.9 mV, displayed passive time- and voltage-independent K+ currents and, additionally, voltage-dependent A-type K+ currents (KA) and/or delayed outwardly rectifying K+ currents (KDR). Numerous GFP-positive cells expressed NeuN, betaIII-tubulin, or 68 kD neurofilaments. GFP/betaIII-tubulin-positive cells, with an average Vrest of -61.6 mV, were characterized by the expression of KA and KDR currents and tetrodotoxin-sensitive Na+ currents. GFP/NE-4C cells also gave rise to oligodendrocytes, based on the detection of oligodendrocyte-specific markers. Our results indicate that GFP/NE-4C neural progenitors transplanted into the site of a photochemical lesion give rise to neurons and astrocytes with membrane properties comparable to those transplanted into noninjured cortex. Therefore, GFP/NE-4C cells provide a suitable model for studying neuro- and gliogenesis in vivo. Further, our results suggest that embryonic neuroectodermal progenitor cells may hold considerable promise for the repair of ischemic brain lesions.     

Relationship of life stress to injury in intercollegiate volleyball

The present study examined whether male and female National Collegiate Athletic Association (NCAA) Division I intercollegiate volleyball players with high life stress would be at greater risk for the occurrence of injury. Life stress was measured with the Social and Athletic Readjustment Rating Scale (SARRS) and the Athletic Life Experiences Survey (ALES). Regardless of how the data were analyzed (injured v noninjured, high stress v low stress, severity of injury), no relationship was found between life stress and injury, indicating that previous findings for football players were not duplicated for intercollegiate volleyball players. Also, different levels of coping resources among volleyball players did not mediate the life stress to injury rate but did differentiate injured from noninjured players.     

An autoradiographic evaluation of the regeneration of corneal epithelium in Bufo marinus.

Epithelial regeneration following scraping of a central portion of corneal epithelium of the toad, Bufo marinus, was studied. Wound closure occurred by migration of a single layer of epithelial cells from adjacent noninjured areas. Autoradiography indicated that following wound closure at two days after scraping, DNA synthesis began in these epithelial cells covering the former scraped area, and was even more pronounced three days after scraping. At four days the former denuded area was covered by an epithelium two to four cells thick and by the fifth day regeneration was complete. These studies also indicated that mitotic time in this organism is rapid.     

A novel strictly anaerobic recovery and enrichment system incorporating lithium for detection of heat-injured Listeria monocytogenes in pasteurized milk containing background microflora.

Heat-injured cells of Listeria monocytogenes were recovered from heated raw milk containing noninjured Enterococcus faecium by combining a simple method for obtaining strict anaerobiosis with a novel enrichment broth, Penn State University broth (PSU broth). Strictly anaerobic conditions were rapidly achieved by adding 0.5 g of filter-sterilized cysteine per liter to PSU broth and then purging the preparation with N2 gas. Little resuscitation or growth occurred in strictly anaerobic PSU broth without lithium chloride because of overgrowth by E. faecium. The growth of E. faecium decreased dramatically with increasing LiCl concentration; LiCl concentrations of 8 and 10 g/liter were completely bacteriostatic. The mechanism of inhibition by LiCl appeared to involve competition with the divalent cations Ca2+ and Mg2+. Heat-injured L. monocytogenes consistently recovered and grew rapidly in strictly anaerobic PSU broth containing 4, 6, or 7 g of LiCl per liter. The use of strictly anaerobic PSU broth containing 7 g of LiCl per liter permitted detection of severely heat-injured L. monocytogenes in one simple recovery-enrichment step by eliminating oxygen toxicity and inhibiting the growth of background microflora, without preventing the resuscitation and subsequent growth of heat-injured L. monocytogenes. L. monocytogenes heated in raw milk at 62.8 degrees C for 10, 15, and 20 min could be consistently recovered from strictly anaerobic PSU broth enrichment cultures at 30 degrees C after 48, 96, and 144 h, respectively, and hence, use of PSU broth may result in better recovery of both injured and noninjured cells from foods than currently used U.S. Department of Agriculture and Food and Drug Administration preenrichment procedures.     

Mechanism for the suppression of gamma-interferon responsiveness in mice after thermal injury

As reported previously, gamma-interferon production was decreased after the administration of inducers to thermally injured mice as compared with noninjured controls. Similarly, spleen cells from injured mice had decreased ability to produce interferon in vitro after stimulation with inducers. The present study demonstrated that the decrease in interferon production was associated with the presence of suppressor cells in the spleen of burned mice that were capable of inhibiting interferon production by normal splenic lymphocytes in vitro. Passive transfer of spleen cells containing suppressor cell activity derived from injured mice induced suppression in normal mice, and the time of the appearance of suppressor cell activity in injured mouse spleens closely approximated the time of the appearance of the suppression of interferon production observed in mice after thermal injury. The suppressor cells were characterized as a population of macrophages by the following: they adhered to plastic surface and could be removed from spleen cells by carbonyl-iron treatment; treatment of plastic-adherent cells with anti-Thy-1.2 and anti-mouse immunoglobulin antisera followed by complement failed to abrogate the suppression produced by these cells.     

Comparative study of membrane filtration and enrichment media for the isolation and enumeration of Pseudomonas aeruginosa from sewage, surface water, and swimming pools

The recovery of Pseudomonas aeruginosa on several selective culture media was tested using raw sewage and secondary sewage effluent samples as well as spiked chlorinated imitation swimming water and samples from whirlpools. mPA-medium B gave good recovery of both vital and chlorine-injured P. aeruginosa and selectivity was greater than 90% when analysing whirlpool samples. It is therefore the medium recommended for examination of chlorinated swimming pools. When analysing sewage polluted water with the mPA-B medium, reduced selectivity was noted from low verification rates and from overgrowth by competitive flora. A modified medium (mPA-D; addition of cetrimide, omission of sulphapyridine and actidione) was more selective and sufficiently recovered noninjured cells. Chlorine-injured cells were completely inhibited, however. C-390 (9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan) was confirmed to be highly selective for P. aeruginosa when used in spread plates at a concentration of 30 micrograms/mL; P. aeruginosa was slightly inhibited. However, the medium could not be used with conventional membrane filtration techniques, because cellulose ester filters interfered with the selective action of C-390. Selectivity could be improved by using Gelman Tuffryn (polysulphone) filters and increasing the C-390 concentration to 120 micrograms/mL. At this concentration, however, the medium was strongly inhibitory to P. aeruginosa; resuscitation only partially improved recovery. Two other membrane filtration media were tested. Both cetrimide - nalidixic acid agar and Drake's medium No. 19 were inhibitory to chlorine-injured cells. Several types of membrane filters were tested and there was little difference between them.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tracheal venous blood and lymph collection: a model to study airway injury in sheep

Airway injury is a frequent result of the inhalation or aspiration of toxic material. Although upper airway damage can be identified endoscopically, pathophysiological changes are difficult to evaluate. This paper describes an animal model in which changes in tracheal blood and lymph flow rates, wet-to-dry weight ratios, and lymph-to-plasma protein ratios can be evaluated after injury. In this model, 12 cm of the cervical trachea were isolated using a double-cuffed endotracheal tube and injured with cotton smoke at near room temperature. Injury to the trachea was evaluated in twenty-five anesthetized sheep 4 (n = 3), 8 (n = 3), 24 (n = 3), 48 (n = 3), 96 (n = 3), and 192 (n = 2) h after smoke exposure and compared with sham control animals (n = 8). A significant increase in tracheal venous blood flow from 1.3 +/- 0.4 (SD) ml.min-1.cm-1 for the noninjured trachea to 2.8 +/- 1.2 was noted 24 h after injury (P less than 0.01). Lymph flow significantly increased from 1.3 +/- 0.4 microliters.min-1.cm-1 for the noninjured trachea to 9.8 +/- 3.3 24 h after injury while wet-to-dry weight ratios were elevated from 3.0 +/- 0.2 for noninjured trachea to 4.6 +/- 0.9 from 4 to 24 h after injury (P less than 0.01) and decreased to 3.7 +/- 0.5 by 96 h. Cast material consisting of airway exudate, cellular debris, and intact ciliated epithelial cells was both expectorated and found in the trachea when the animals were killed.(ABSTRACT TRUNCATED AT 250 WORDS)     

c-fos Expression in Gracilothalamic Tract Neurons after Electrical Stimulation of the Injured Sciatic Nerve in the Adult Rat

The number of c-fos protein-like immunoreactive (Fos-LI) cells in the gracile nucleus was determined after electrical stimulation at Aα/Aβ-fiber strength of the normal and of the previously injured sciatic nerve in adult rats. No Fos-LI cells were seen after electrical stimulation of the noninjured sciatic nerve, or after sciatic nerve injury without electrical stimulation. However, stimulation 21 days after sciatic nerve transection resulted in numerous Fos-LI cells in the ipsilateral gracile nucleus. Combined Fos immunocytochemistry and retrograde labeling from the thalamus showed that the majority (76%; range = 70–80%) of the cells in the gracile nucleus that expressed Fos-LI after nerve injury projected to the thalamus. The results indicate that morphological, biochemical, and physiological alterations in primary sensory central endings and second-order neurons, which have earlier been demonstrated in the dorsal column nuclei after peripheral nerve injury, are accompanied by changes in the c-fos gene activation pattern after stimulation of the injured sciatic nerve. A substantial number of the c-fos-expressing neurons project to the thalamus.     

Studies on corneal endothelial growth and repair. IV. Changes in the surface during cell division as revealed by scanning electron microscopy

Changes in the surface morphology of regenerating rabbit, rat and frog corneal endothelial cells in vivo have been investigated by scanning electron microscopy. In adult tissue these cells do not normally divide unless given a stimulus, such as injury. Surfaces of quiescent rabbit and rat cells are devoid of microvilli but display globular projections and surface pits up to 300 nm in diameter. However, regenerating endothelia are characterized by the appearance of microvilli which attain their greatest length when the cells are rounded. At this stage, cells also possess filopodia and broad processes. In cytokinesis, the microvilli have shortened and blebs and ruffles appear for the first time. In contrast to rabbits and rats, frog endothelial cells of noninjured tissue are covered by microvilli and smaller surface pits of 60-70 nm diameters. During regeneration, these cells have reduced numbers of microvilli and extensive foldings of the membrane. Neither blebs nor filopodia occur during the mitotic cycle and ruffles are not detected until cytokinesis.     

Ultrastructural immunocytochemical localization of fibronectin deposition during corneal endothelial wound repair. Evidence for cytoskeletal involvement

The distribution of the extracellular matrix (ECM) protein, fibronectin (FN), has been examined ultrastructurally in noninjured and injured rat corneal endothelium in vivo and in vitro by immunoperoxidase cytochemistry. In noninjured endothelia, FN was observed within the rough endoplasmic reticulum (RER) cisternae but not along the cell-Descemet's membrane (DM) interface. Twenty-four and 48 h after a circular freeze injury, immunoperoxidase reaction product was detected at the cell-DM interface as well as within cytoplasmic vesicles and intercellular spaces. By 1 and 2 wk post-injury, a line of reaction product could still be demonstrated at the cell-DM interface and evidence for newly deposited basement membrane material was observed in this region. In order to understand whether fibronectin deposition during wound repair was dependent on cytoskeletal influences, organ culture experiments were performed in which the media was supplemented with either 10(-8) M colchicine or 2.5 X 10(-3) M cytochalasin B. Without inhibitors, injured corneas cultured for 24 h had FN deposition at the cell-DM interface similar to the in vivo results. Corneas cultured in the presence of cytochalasin B also showed FN deposition at the cell-DM interface. However, when injured endothelia were cultured in the presence of colchicine, no reaction product was observed at the cell-DM interface, although it could be detected intracellularly within RER. Incubating the tissues in the presence of puromycin abolished all extracellular and intracellular staining. These results indicate that during wound repair, corneal endothelial cells produce fibronectin and deposit it upon Descemet's membrane by a mechanism that may be mediated by microtubules.     

Psychosocial factors in athletic injuries: development and application of the social and athletic readjustment rating scale (SARRS).

The possible role of psychosocial factors in athletics, namely football injuries, is examined. Initially Holmes and Rahe's Social Readjustment Rating Scale (SRRS) was modified to the Social and Athletic Readjustment Scale (SARRS). Additions to the scale and differences in football players from the general population are discussed. Life change scores over one- and two-year intervals were obtained for college varsity football players. Players suffering major time loss injuries had significantly higher predictive scores than noninjured players.     

Trans-spinal direct current enhances corticospinal output and stimulation-evoked release of glutamate analog, D-2,3-³H-aspartic acid

Trans-spinal direct current (tsDC) stimulation is a modulator of spinal excitability and can influence cortically elicited muscle contraction in a polarity-dependent fashion. When combined with low-frequency repetitive cortical stimulation, cathodal tsDC [tsDC(-)] produces a long-term facilitation of cortically elicited muscle actions. We investigated the ability of this combined stimulation paradigm to facilitate cortically elicited muscle actions in spinal cord-injured and noninjured animals. The effect of tsDC-applied alone or in combination with repetitive spinal stimulation (rSS) on the release of the glutamate analog, D-2,3-(3)H-aspartate (D-Asp), from spinal cord preparations in vitro-was also tested. In noninjured animals, tsDC (-2 mA) reproducibly potentiated cortically elicited contractions of contralateral and ipsilateral muscles tested at various levels of baseline muscle contraction forces. Cortically elicited muscle responses in animals with contusive and hemisectioned spinal cord injuries (SCIs) were similarly potentiated. The combined paradigm of stimulation caused long-lasting potentiation of cortically elicited bilateral muscle contraction in injured and noninjured animals. Additional analysis suggests that at higher baseline forces, tsDC(-) application does not increase the rising slope of the muscle contraction but causes repeated firing of the same motor units. Both cathodal and anodal stimulations induced a significant increase of D-Asp release in vitro. The effect of the combined paradigm of stimulation (tsDC and rSS) on the concentration of extracellular D-Asp was polarity dependent. These results indicate that tsDC can powerfully modulate the responsiveness of spinal cord neurons. The results obtained from the in vitro preparation suggest that the changes in neuronal excitability were correlated with an increased concentration of extracellular glutamate. The combined paradigm of stimulation, used in our experiments, could be noninvasively applied to restore motor control in humans with SCI.     

Host range susceptibility of Enterococcus sp. strains isolated from diseased turbot: possible routes of infection.

Experiments were conducted to assess the pathogenicity of Enterococcus sp. strains isolated from diseased turbot for several fish species (turbot, salmon, trout, and seabream), as well as for mice. The intraperitoneal injection assays indicated that the tested strains showed host specificity for turbot, with a high degree of virulence (50% lethal dose of 10(4) cells per g of fish). The Spanish Enterococcus sp. isolates were nonpathogenic for the other fish species studied and for mice. The possible routes of infection were determined by bath exposure (with and without prior abrasion of the skin) and by intragastric inoculations with food and feces contaminated with the pathogen. The bath challenges indicated that the Enterococcus isolates were able to overcome the defense mechanisms present on the surface of the turbot only if the skin was abraded prior to the exposure. The antibacterial activities of components of a glycoprotein nature present in the turbot skin mucus are probably responsible in part for the resistance in noninjured fish to infection. On the other hand, we demonstrated the capacity of this pathogen to overcome adverse conditions in the stomachs of fish when associated with food or fecal material, since it is able to establish an infective state and to produce mortalities after 16 to 20 days postingestion. From all of these findings, we can conclude that horizontal transmissions through water and the fecal-oral route are the main avenues of infection of turbot streptococcosis.     

Site of reovirus replication in liver is determined by the type of hepatocellular insult.

Reovirus type 1 strain Lang is restricted from replicating in adult murine livers. In noninjured livers, approximately 1% of hepatocytes express reovirus antigen during infection. However, hepatocytes can be induced to express reovirus antigen if challenged with either toxins or trauma. We used selective hepatotoxins or surgical trauma to demonstrate that reovirus antigen localization in liver is determined by the site of hepatocellular insult and the timing of the virus inoculum.