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PubMed1973

Differences in the response of rabbit small intestine to heat-labile and heat-stable enterotoxins of Escherichia coli.

The response of adult rabbit small intestine to the heat-stable (ST) and heat-labile (LT) enterotoxins of Escherichia coli has been investigated by employing the ligated loop technique. Fluid accumulation was determined in relation to enterotoxin dose and duration of gut exposure. The individual responses to ST and LT differed in a characteristic manner. Onset of net fluid accumulation in response to ST appeared to be immediate even at the lowest dose tested. Onset of net fluid accumulation in response to LT was rapid at high doses but delayed at low doses. Maximum volume per length ratios elicited by ST occurred between 4 and 6 h after injection of loops over the entire range of doses tested. However, maximum ratios elicited by LT occurred no less than 10 h after injection even at low doses. Fluid accumulation elicited by LT increased in duration with increasing dose; high doses of LT producing a response which was sustained for at least 18 h. The net effect of these differences in reaction characteristics is a sharp increase in the proportion of the cumulative net secretory response attributable to LT with time. Therefore, a 6-h assay time is appropriate for the titration of ST, whereas an 18-h assay is not. The 18-h assay was found more appropriate for toxin-antitoxin neutralization studies since only LT was neutralized by anti-enterotoxin serum. LT of E. coli (swine) strain P-263 and (human) strain 334 was neutralized by antibody stimulated by enterotoxin from E. coli (human) strain H-10407. LT was labile to mild acid conditions, whereas ST was not.

Animals↗
PubMed1980

Cloning of deoxyribonucleic acid regions encoding a heat-labile and heat-stable enterotoxin originating from an enterotoxigenic Escherichia coli strain of human origin.

A heat-labile and heat-stable enterotoxin (LT+ ST+) plasmic (62.7 kilobases in size) was isolated from an enterotoxigenic Escherichia coli human strain, H10407, and used for analysis of the LT+ and ST+ deoxyribonucleic acid (DNA) regions. A DNA segment containing the LT+ and ST+ DNA regions, which consisted of two restriction endonuclease EcoRI fragments (E1 and E2), was inserted into the cloning vehicle ColE1::Tn5 by EcoRI digestion and subsequent ligation. Further cloning experiments localized the LT+ DNA region on a 5.1-kilobase restriction endonuclease PstI fragment present over the junction between the E1 and E2 fragments, as seen in the original LT+ ST+ plasmid, and the ST+ DNA region on a 1.5-kilobase PstI fragment present in either the E1 or E2 fragment. A change in the relative orientation of the E1 and E2 fragments resulted in altered levels of LT production. The relative orientation of the ColE1::Tn5 fragment to the E1 and E2 fragments also markedly influenced both LT and ST production levels. The LT+ ST+ E1-E2 region contained two unique DNA sequences consisting of a DNA segment flanked by inverted repeats which were readily distinguished from each other by size. The cloned ST+ PstI fragment was structurally very similar to one of these unique DNA sequences present in the LT+ ST+ E1-E2 region.

Bacterial Toxins↗
PubMed1984

Rapid differentiation of enterotoxigenic Escherichia coli that produce heat-stable and heat-labile toxins by frequency-pulsed electron capture gas-liquid chromatography analysis of diarrheal stool specimens.

Thirty-three stool specimens from infants in the village of Tamooh near Cairo, Egypt, were studied by frequency-pulsed electron capture gas-liquid chromatography (FPEC-GLC). In 13 of the diarrheal cases, the suspected causative agent isolated was Escherichia coli which produced heat-stable toxin (ST), and in 10 other cases E. coli that produced heat-labile toxin (LT) were isolated. Ten control stool samples, collected from infants from whom no pathogenic organisms were isolated, were analyzed at the same time. Comparisons also were made against healthy control stools from individuals in the United States who had been previously analyzed by FPEC-GLC (Brooks et al., J. Clin. Microbiol. 20:549-560, 1984). The stools were suspended in water and centrifuged, and the supernatant was extracted with organic solvents and derivatized to form electron-capturing derivatives of carboxylic acids, hydroxy acids, alcohols, and amines. Results from the study showed distinct differences among the FPEC-GLC profiles of E. coli ST-positive stools, of E. coli LT-positive stools, and of the control stool samples. An unidentified compound appearing in the ether-soluble hydroxy acid fraction from E. coli ST-positive stools was tentatively identified by mass spectrometry as 6-methoxy-2-hydroxyhexanoic acid. 6-Methoxy-2-hydroxyhexanoic acid was found in all stools that contained E. coli ST but was not present either in stools from which E. coli LT was isolated or in control samples. 6-Methoxy-2-hydroxyhexanoic acid may prove to be an important marker for use in the identification of E. coli ST. In addition to 6-methoxy-2-hydroxyhexanoic acid, the carboxylic acid, alcohol, and amine FPEC-GLC profiles obtained from stools were very different between these two organisms. The data indicate that FPEC-GLC analysis of diarrheal stool specimens might be a rapid way to distinguish diarrhea caused by E. coli ST, E. coli LT, Clostridium difficile, and rotavirus.

Bacterial Toxins↗
PubMed1990

Trivalent heat-labile- and heat-stable-enterotoxin probe conjugated with horseradish peroxidase for detection of enterotoxigenic Escherichia coli by hybridization.

A 1,268-bp polynucleotide probe for heat-labile and heat-stable enterotoxins (LTh, STIa, STIb) was conjugated with horseradish peroxidase (HRP). The HRP-conjugated trivalent probe was applied to the detection of enterotoxigenic Escherichia coli (ETEC) by colony and stool hybridizations. The binding of the probe to its targets was assayed by the addition of HRP substrates hydrogen peroxide and luminol in the presence of an enhancer, and the chemiluminescence was recorded by exposure to X-ray film. Slot blot hybridization demonstrated that the HRP-conjugated trivalent probe specifically hybridized with the DNA isolated from ETEC strains. The trivalent probe also specifically identified bacterial colonies of ETEC that produced LTh, STIa, STIb, LTh-STIa, or LTh-STIb. Treatment of targets with sodium dodecyl sulfate and proteinase K remarkably reduced nonspecific hybridization to DNAs of non-ETEC strains. Furthermore, this probe was able to detect stool specimens seeded with 10(2) original ETEC cells per 5 mg of feces. These results suggest that the HRP-conjugated trivalent probe is a candidate for use in the clinical laboratory to detect ETEC.

Bacterial Toxins↗
PubMed1998

Associations between heat-stable (O) and heat-labile (HL) serogroup antigens of Campylobacter jejuni: evidence for interstrain relationships within three O/HL serovars.

A comparative examination of the heat-stable (O) and heat-labile (HL) serogrouping results for 9,024 sporadic human isolates of Campylobacter jejuni revealed conserved associations between specific O and HL antigens (O/HL serovars). Forty-nine percent of the isolates which grouped for both O and HL antigens belonged to one of three serovars: O 4 complex/HL 1 (17.9%), O 1/HL 2 (16.8%), or O 50/HL 7 (14.5%). Other common serovars were O 2/HL 4 (8.3%), O 6/HL 6 (8.1%), O 53/HL 11 (4.5%), O 19/HL 17 (3.3%), O 5/HL 9 (3.3%), O 9/HL 9 (3.2%), and O 23/HL 5 (3.1%). These 10 serovars accounted for 83.1% of the serogroupable isolates. A large number of strains (41.3%) could be typed by only one of the two methods or could not be serogrouped (11%). Strains belonging to three serovars, O 2/HL 4, O 50/HL 7, and O 23/HL 5, were further characterized by combining data from expressed features (O/HL serogroups, phage groups, and biotypes) with restriction fragment length polymorphism genotypes. These polyphasic data demonstrated that within each serovar, individual isolates showed substantial conservation of both genomic and phenotypic characteristics. The essentially clonal nature of the three serovars confirmed the potential of combined O and HL serogrouping as a practical and phylogenetically valid method for investigating the epidemiology of sporadic C. jejuni infection.

Animals↗
PubMed2004

Real-time fluorescence PCR assays for detection and characterization of heat-labile I and heat-stable I enterotoxin genes from enterotoxigenic Escherichia coli.

To facilitate the diagnosis of enterotoxigenic Escherichia coli (ETEC) infections in humans, we developed and evaluated real-time fluorescence PCR assays for the Roche LightCycler (LC) against the enterotoxin genes commonly present in strains associated with human illness. Separate LC-PCR assays with identical cycling conditions were designed for the type I heat-labile enterotoxin (LT I) and the type I heat-stable enterotoxin (ST I) genes, using the LC hybridization probe format. A duplex assay for ST I with two sets of amplification primers and three hybridization probes was required to detect the major nucleotide sequence variants of ST I, ST Ia and ST Ib. LC-PCR findings from the testing of 161 E. coli isolates of human origin (138 ETEC and 23 non-ETEC) were compared with those obtained by block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the LT I and ST I genes. The LC-PCR and block cycler PCR assays were also compared for their abilities to detect LT I and ST I genes in spiked stool specimens with different methods of sample preparation. Findings from these experiments revealed that the limits of detection for the LC-PCR assays were the same or substantially lower than those observed for the block cycler PCR assay. Melting curve analysis of the amplified LT I and ST I genes revealed sequence variation within each gene, which for the ST I genes correlated with the presence of ST Ia and ST Ib. The rapidity, sensitivity, and specificity of the LC-PCR assays make them attractive alternatives to block cycler PCR assays for the detection and characterization of ETEC.

Bacterial Toxins↗
PubMed1980

Intracerebroventricular injection of sympathomimetic drugs inhibits both heat production and heat loss mechanisms in the rat.

The effects of intracerebroventricular (i.c.v.) injections of sympathomimetic drugs on thermoregulatory functions in conscious rats maintained at low (8 decrees C), moderate (22 degrees C), and high (30 degrees C) ambient temperatures were assessed. Norepinephrine, tyramine, and ephedrine each produced hypothermia at ambient temperature (Ta) 8 degrees C and hyperthermia at Ta 22 and 30 degrees C. At Ta 8 degrees, the hypothermia in response to norepinephrine, tyramine, and ephedrine was due to decreased metabolic rate (M) whereas at Ta 22 degrees C the hyperthermia was due to cutaneous vasoconstriction. AT Ta 22 degrees C, the hyperthermia in response to norepinephrine and tyramine was due to cutaneous vasoconstriction whereas the hyperthermia in response to ephedrine was brought about by increased M (due to behavioral excitation). Intracerebroventricular injection of epinephrine produced hypothermia followed by hyperthermia at Ta 8 and 22 degrees C. The hypothermia was due to decreased M whereas the hyperthermia was due to cutaneous vasoconstriction and increased M. AT Ta 30 degrees C, epinephrine led to a reduction in cutaneous temperature and hyperthermia. Furthermore, i.c.v. administration of phenylephrine produced a decreased M and hypothermia Ta 8 degrees C and an increased M (due to behavioral excitation) and hyperthermia at Ta 30 degrees C. At Ta 22 degrees C, phenylephrine produced hyperthermia (due to cutaneous vasoconstriction and increased M) preceded by hypothermia (due to decreased M). Moreover, the temperature effects induced by norepinephrine were antagonized by pretreatment with the adrenoceptor antagonist phentolamine. In general, the data indicate that activation of central adrenoceptors with sympathomimetic drugs inhibits both heat production and heat loss mechanisms in the rat.

Animals↗
PubMed1999

Light masking of circadian rhythms of heat production, heat loss, and body temperature in squirrel monkeys.

Whole body heat production (HP) and heat loss (HL) were examined to determine their relative contributions to light masking of the circadian rhythm in body temperature (Tb). Squirrel monkey metabolism (n = 6) was monitored by both indirect and direct calorimetry, with telemetered measurement of body temperature and activity. Feeding was also measured. Responses to an entraining light-dark (LD) cycle (LD 12:12) and a masking LD cycle (LD 2:2) were compared. HP and HL contributed to both the daily rhythm and the masking changes in Tb. All variables showed phase-dependent masking responses. Masking transients at L or D transitions were generally greater during subjective day; however, L masking resulted in sustained elevation of Tb, HP, and HL during subjective night. Parallel, apparently compensatory, changes of HL and HP suggest action by both the circadian timing system and light masking on Tb set point. Furthermore, transient HL increases during subjective night suggest that gain change may supplement set point regulation of Tb.

Animals↗
PubMed2003

Matching the heart to heat-induced circulatory load: heat-acclimatory responses.

Heat acclimation enhances cardiac efficiency by increasing stroke volume and decreasing heart rate. These adaptations involve biochemical changes in the contractile apparatus, switched on by altered expression of genes coding contractile and calcium-regulatory proteins and partially mediated by persistent low thyroxine. Heat acclimation also produces cross-tolerance to oxygen deprivation, thus reinforcing cardiac adaptation to oxygen demand/supply mismatching via energy-sparing pathways.

Acclimatization↗
PubMed2001

Small heat shock protein of a hyperthermophilic archaeum, Thermococcus sp. strain KS-1, exists as a spherical 24 mer and its expression is highly induced under heat-stress conditions.

Small heat shock proteins (sHsps) are the most ubiquitous molecular chaperones. Several sHsps have been shown to exhibit chaperone activity and protect proteins from thermal and chemical aggregation. We have characterized a small heat shock protein from a hyperthermophilic archaeum, Thermococcus sp. strain KS-1. Electron microscopy revealed that the protein exists as a spherical oligomer with a diameter of 14+/-1 nm. The molecular weight of the oligomer was determined to be 478.6 kDa by size exclusion chromatography-multiangle laser light scattering. Thus, the Thermococcus sHsp is likely to exist as a spherical 24meric oligomer with almost the same structure as the Methanococcus jannaschii sHsp. The Thermococcus sHsp homo-oligomer protected porcine heart citrate synthase from thermal aggregation. It also slightly enhanced the refolding of acid-denatured green fluorescent protein. While the Thermococcus sHsp could not be detected in cells grown at the optimal growth temperature or lower, the expression of the protein was highly induced when the cells were grown at temperatures higher than the optimal growth temperature. Since only group II chaperonins and sHsps exist in hyperthermophilic archaea as molecular chaperones, sHsps should have an important role in protecting cells from lesions caused by aggregates of thermally denatured cellular proteins.

Journal Article↗
PubMed1996

Comparison of the effects of heat and moisture exchangers and heated humidifiers on ventilation and gas exchange during weaning trials from mechanical ventilation.

Heat and moisture exchangers (HME) are increasingly used to warm and humidify inspired gases in intubated ventilated patients. But these devices add dead space that may alter the alveolar ventilation. This could impair the efficiency of spontaneous ventilation (SV) during weaning trials from mechanical ventilation. Fifteen patients were tested with an HME (Hygrobac-DAR) and a heated humidifier (HH) (Fischer-Paykel MR 450) in a random order during weaning trials in SV with inspiratory pressure support. Minute ventilation VE, tidal volume), and respiratory rate were recorded and arterial blood was sampled for blood gas analysis with each device. The HME gave a significantly greater VE than the HH (9.3 +/- 0.8 L/min vs 8.1 +/- 0.8 L/min; p < 0.005), because of increased respiratory rate (21 +/- 2/min vs 19 +/- 2/min; p < 0.05). Tidal volume was unchanged for HME and HH (470 +/- 32 mL vs 458 +/- 39 mL). The higher PaCO2 with HME than with HH (44 +/- 2 mm Hg vs 42 +/- 2 mm Hg; p < 0.005) revealed an insufficient alveolar ventilation response to the increase in dead space. Arterial Po2 rose with the HME, but not significantly above the HH values (103 +/- 6 mm Hg vs 97 +/- 6 mm Hg; p = 0.055), possibly because of a positive end-expiratory pressure effect of the HME. The need to increase VE in SV when an HME is used should be taken into account during difficult weaning from mechanical ventilation.

Adult↗
PubMed1997

A prospective, randomized comparison of an in-line heat moisture exchange filter and heated wire humidifiers: rates of ventilator-associated early-onset (community-acquired) or late-onset (hospital-acquired) pneumonia and incidence of endotracheal tube occlusion.

PURPOSE: To compare the performance of an in-line heat moisture exchanging filter (HMEF) (Pall BB-100; Pall Corporation; East Hills, NY) to a conventional heated wire humidifier (H-wH) (Marquest Medical Products Inc., Englewood, Colo) in the mechanical ventilator circuit on the incidence of ventilator-associated pneumonia (VAP) and the rate of endotracheal tube occlusion. METHODS: This report describes a prospective, randomized trial of 280 consecutive trauma patients in a 20-bed trauma ICU (TICU). All intubated patients not ventilated elsewhere in the medical center prior to their TICU admission were randomized to either an in-line HMEF or a H-wH in the breathing circuit. Ventilator circuits were changed routinely every 7 days, and closed system suction catheters were changed every 3 days. HMEFs were changed every 24 h, or more frequently if necessary. A specific endotracheal tube suction and lavage protocol was not employed. Patients were dropped from the HMEF group if the filter was changed more than three times a day or the patient was placed on a regimen of ultra high-frequency ventilation. The Centers for Disease Control and Prevention (CDC) criteria for diagnosis of pneumonia were used; early-onset, community-acquired pneumonia was defined if CDC criteria were met in < or =3 days, and late-onset, hospital-acquired pneumonia was defined if criteria were met in >3 days. Laboratory and chest radiograph interpretation were blinded. RESULTS: The patient ages ranged from 15 to 95 years in the HMEF group and 16 to 87 years in the H-wH group (p=not significant), with a mean age of 46 years and 48 years, respectively. The male to female ratio ranged between 78 to 82%/22 to 18%, respectively, and 55% of all admissions were related to blunt trauma, 40% secondary to penetrating trauma, and 5% to major burns. There was no difference in Injury Severity Score (ISS) between the two groups. Moreover, there was no significant difference in mean ISS among those who did not develop pneumonia and those patients who developed either early-onset, community-acquired or late-onset, hospital-acquired pneumonia. The HMEF nosocomial VAP rate was 6% compared to 16% for the H-wH group (p<0.05), and total ventilator circuit costs (per group) were reduced. There were no differences in duration of ventilation (mean+/-SD) if the patient did not develop pneumonia or if the patient developed an early-onset, community-acquired or a late-onset, hospital-acquired pneumonia. Moreover, total TICU days were reduced in the HMEF group. In addition, the incidence of partial endotracheal tube occlusion was not significantly different between the H-wH and the HMEF groups. CONCLUSIONS: The HMEF used in this study reduced the incidence of late-onset, hospital-acquired VAP, but not early-onset, community-acquired VAP, compared to the conventional H-wH circuit. This was associated with a significant reduction in total ICU stay. Disposable ventilator circuit costs in the HMEF group were reduced compared to the H-wH group in whom circuit changes occurred at 7-day intervals. CLINICAL IMPLICATIONS: The use of the HMEF is a cost-effective clinical practice associated with fewer late-onset, hospital-acquired VAPs, and should result in improved resource allocation and utilization.

Adolescent↗
PubMed1976

Microcalorimetric measurements of heat production in human erythrocytes. Heat effect during methylene blue stimulation.

The metabolic activity in human erythrocytes during stimulation with 10(-4) mol/l methylene blue has been studied by a microcalorimetric method. Simultaneous measurements were performed on cells from the same preparation suspended in different media. Mean values for the ratios between heat effect values were 7.1 +/- 1.0, 7.4 +/- 0.8, and 10.2 +/- 1.7 (+/- S.D.) for cells suspended in plasma, serum, and glucose phosphate buffer, respectively. All heat effect values were corrected to pH 7.40 using the correction factor found in the present work (0.4 % per 0.01 pH unit). The present calorimetric results are in qualitative agreement with previous reports of other investigators concerning the stimulating effect of methylene blue and the influence of pH on the pentose phosphate pathway.

Blood↗
PubMedDate not supplied

Hypothermia following whole-body heating of mice: effect of heating time and temperature.

We have observed an acute and prolonged lowering of body temperature (hypothermia) following whole body heating (WBH) of mice. This phenomenon of heat-induced hypothermia and the subsequent recovery of normal temperatures have been systematically investigated. The hypothermic period can be characterized by two parameters: Tnadir and a recovery time constant (tau). For treatment temperatures below 41 degrees C and treatment durations of 1 h or less, a mild hypothermia (Tnadir greater than 33 degrees C) and fast recovery (tau less than 1 h) occur. Tnadir and tau vary slightly with treatment temperature and are almost independent of treatment length. At treatment temperatures 41 degrees C and above for up to 1 h, we observed acute hypothermia (Tnadir as low as 28 degrees C) and slow recovery (tau = several hours). This region of prolonged hypothermia is characterized by a rapid change of Tnadir and tau with temperature, and a much less rapid change with treatment duration. The WBH temperature-time range causing prolonged hypothermia is very narrow, and if exceeded results in lethality. Critical lethal temperatures have been estimated for several treatment durations from the time constant data. Post-WBH hypothermia can be minimized by keeping the animals in a 37 degrees C environment. However, we find that neither survivability nor intestinal cell repopulation is enhanced by this procedure.

Anesthesia↗
PubMed1998

Use of a multiplex PCR system for the simultaneous detection of heat labile toxin I and heat stable toxin II genes of enterotoxigenic Escherichia coil in skim milk and porcine stool.

Enterotoxigenic Escherichia coli (ETEC) strains which produce heat labile and/or heat stable toxins (LT and ST) may cause diarrhea in humans and farm animals. Using PCR primers specific for the LT I and ST II genes, a multiplex PCR system which allows detection of LT 1-and ST 11-producing ETEC strains was developed. When skim milk was used for a PCR assay, it was found that if target cells in the sample were precultured in MacConkey broth for 8 h prior to PCR as few as 10(0) cells per ml of the sample could be detected. Without the preculture step, 10(4) CFU of target cells per 0.2 g of porcine stool specimen were required to generate visible PCR products. The multiplex PCR system can be used for rapid testing of fecal specimens, food and possibly environmental samples for the presence of ETEC strains.

Animals↗
PubMed1991

Heat-stable and heat-labile components of nonspecific acid phosphatase detected in Pseudomonas pseudomallei.

In a whole cell assay system with p-nitrophenyl phosphate as substrate, strains of Pseudomonas pseudomallei showed a two-peak pattern in pH activity curve of acid phosphatase, suggesting the presence of two enzyme components different in pH optimum (4.2 and 5.2). The component of 5.2 pH optimum was detected in the outer membrane fraction and the activity was resistant to heating at 70 C for 30 min. The other component of 4.2 pH optimum was heat-labile. No substantial difference was observed in the enzymatic activity between R and S type colonies.

Acid Phosphatase↗
PubMedDate not supplied

An investigation of heat transfer to the implant-bone interface related to exothermic heat generation during setting of autopolymerizing acrylic resins applied directly to an implant abutment.

Excessive heat generation at the implant-bone interface may cause bone damage and compromise osseointegration. Autopolymerizing acrylic resins are commonly used intraorally to join impression copings and suprastructure components for soldering. The effect of heat generation at the implant surface related to the exothermic setting reaction of autopolymerizing acrylic resins applied to an attached abutment was examined in vitro. Two brands of autopolymerizing acrylic resin, Duralay and GC Pattern Resin, were compared. Acrylic resin was applied to a titanium alloy abutment connected to a titanium alloy cylindric implant in varying controlled volumes, with both bulk application and brush paint-on techniques. The implant was embedded in an acrylic resin mandible in a 37 degrees C water bath. Temperature changes were recorded via embedded thermocouples at the cervical and apical of the implant surface. Analysis of variance for repeated measures was used to compare treatment groups. A mean maximum increase in temperature of 4 to 5 degrees C was seen at the implant cervical for both materials, with a maximum temperature increase of 6 degrees C. No difference between Duralay and GC Pattern Resin was seen, except for bulk application to medium-sized copper bands at the implant cervical (P < .05). No difference between the bulk and brush techniques was seen for all options, except for GC, where bulk application to medium-sized copper bands produced higher temperatures than the brush technique (P < .05). Spray coolant reduced temperatures for bulk application of both Duralay and GC (P < .05).

Acrylic Resins↗
PubMedDate not supplied

Effects of heat--stress in rats: Critical period and reduced survival of post-implantation heat-stress inducted in early gestation.

High ambient temperature (34.4 C degrees and 60-65% relative humidity (RH) did not affect foetal survival when bred female rats were exposed between day-8 and -18 (Day-0=day of breeding). Heat-stress at this intensity given on day-0 was critical for embryonic and/or foetal survival rate and more so, if the ambient temperature was increased to 36.6 C degrees and 70-75% RH than on day-1. Postimplantation foetal survival was reduced due to heat-stress given only during early gestation period.

Animals↗