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Biomedical subjects

G Ramsay

Publications and source records attributed to G Ramsay.

At least 73 records · Page 4Linked to original sources
PubMed1993

A broadly cross-protective monoclonal antibody binding to Escherichia coli and Salmonella lipopolysaccharides.

During the last decade, episodes of sepsis have increased and Escherichia coli has remained the most frequent clinical isolate. Lipopolysaccharides (LPS; endotoxin) are the major toxic and antigenic components of gram-negative bacteria and qualify as targets for therapeutic interventions. Molecules that neutralize the toxic effects of LPS are actively investigated. In this paper, we describe a murine monoclonal antibody (MAb; WN1 222-5), broadly cross-reactive and cross-protective for smooth (S)-form and rough (R)-form LPS. As shown in enzyme-linked immunosorbent assay and the passive hemolysis assay, WN1 222-5 binds to the five known E. coli core chemotypes, to Salmonella core, and to S-form LPS having these core structures. In immunoblots, it is shown to react with both the nonsubstituted core LPS and with LPS carrying O-side chains, indicating the exposure of the epitope in both S-form and R-form LPS. This MAb of the immunoglobulin G2a class is not lipid A reactive but binds to E. coli J5, an RcP+ mutant which carries an inner core structure common to many members of the family Enterobacteriaceae. Phosphate groups present in the inner core contribute to the epitope but are not essential for the binding of WN1 222-5 to complete core LPS. Cross-reactivity for clinical bacterial isolates is broad. WN1 222-5 binds to all E. coli clinical isolates tested so far (79 blood isolates, 80 urinary isolates, and 21 fecal isolates) and to some Citrobacter, Enterobacter, and Klebsiella isolates. This pattern of reactivity indicates that its binding epitope is widespread among members of the Enterobacteriaceae. WN1 222-5 exhibits biologically relevant activities. In vitro, it inhibits the Limulus amoebocyte lysate assay activity of S-form and R-form LPS in a dose-dependent manner and it neutralizes the LPS-induced release of clinically relevant monokines (interleukin 6 and tumor necrosis factor). In vivo, WN1 222-5 blocks endotoxin-induced pyrogenicity in rabbits and lethality in galactosamine-sensitized mice. The discovery of WN1 222-5 settles the long-lasting controversy over the existence of anti-core LPS MAbs with both cross-reactive and cross-protective activity, opening new possibilities for the immunotherapy of sepsis caused by gram-negative bacteria.

Animals↗
PubMed1993

Comparison of HA-1A and E5 monoclonal antibodies to endotoxin in rats with endotoxaemia.

OBJECTIVE: To investigate the ability of two anti-endotoxin IgM antibodies (10 mg/kg) to prevent the hyperdynamic response induced by infusion of Escherichia coli rough mutant endotoxin core types R1 and R2 in rats. DESIGN: Randomised blind laboratory study. SETTING: University department of surgery. MATERIAL: 8 groups of 5 male Sprague-Dawley rats and 10 control animals. MAIN OUTCOME MEASURE: Percentage change in cardiac output during the period of observation. RESULTS: E5 provided significant protection against R1 (p = 0.04) but not against R2. HA-1A gave significant protection against R2 (p < 0.04) but not against R1. In clinical isolates of E. coli the R1 type predominates (about 70%). CONCLUSION: These results may reflect true differences in specificity between the antibodies, but there remains the possibility that these are non-specific effects. Further controls with irrelevant murine and humanized IgM monoclonal antibodies are in progress.

Animals↗
PubMed1992

Retroperitoneal necrotizing fasciitis.

Necrotizing fasciitis is a mixed infection of the skin and subcutaneous tissues with a characteristic clinical and pathological appearance. Early radical surgical excision of all affected tissue is the treatment of choice. In a series of 19 patients with necrotizing fasciitis, bacteriological assessment in 15 confirmed the mixed nature of the infection, with Bacteroides sp. isolated from ten patients. All 12 patients who underwent radical surgical excision survived. A subgroup of patients was identified in whom the appearance of necrotizing fasciitis in the abdomen or perineum was indicative of more extensive disease in the retroperitoneal tissues. Surgical resection of all affected tissue was not feasible in these cases and the outcome was uniformly fatal, giving an overall mortality rate for the series of 37 per cent.

Aged↗
PubMed1992

Prevention of cardiovascular effects of endotoxaemia by monoclonal antibodies specific for core endotoxin.

Passive immunization with antibody to the core region of endotoxin (core lipopolysaccharide (LPS)) has been reported to reduce mortality in severe sepsis. A rat model of endotoxaemia that reproduces the hyperdynamic cardiovascular state seen in early sepsis was developed to test monoclonal antibodies specific for core LPS. A thermodilution technique of measuring cardiac output was adapted for use in rats. Twenty-five animals were anaesthetized and mechanically ventilated with monitoring of central venous pressure and mean arterial pressure. Fluid replacement was adjusted to maintain the central venous pressure. Controls (n = 10) and antibody-treated animals (n = 5) showed no significant change in cardiac output. Animals given 0.1 mg kg-1 R2 endotoxin over 1 h (n = 5) showed a significant rise in cardiac output of 65 per cent (P < 0.01). This was abolished in rats given both antibody and endotoxin (n = 5). This study provides evidence that a monoclonal antibody against core LPS abolishes the hyperdynamic state induced by endotoxin infusion.

Animals↗
PubMed1992

Selective decontamination of the digestive tract in intensive care.

Nosocomial infection in intensive care unit (ICU) practice is a common problem and is associated with abnormal carriage of Gram-negative aerobic bacilli in the gastrointestinal tract, resulting in endogenous infections. Selective decontamination of the digestive tract (SDD) is a regimen aimed at preventing or eradicating this abnormal carriage. A large number of trials examining SDD in ICU practice have been published, the vast majority showing a significant reduction in the incidence of nosocomial, Gram-negative infection. However, the impact on morbidity and mortality is much less certain. A recent meta-analysis has suggested a 10-20% reduction in mortality (3-6% absolute difference) with SDD. A discussion of these results is presented together with potential criticisms of SDD.

Anti-Bacterial Agents↗
PubMed1990

Linked thermal and solute perturbation analysis of cooperative domain interactions in proteins. Structural stability of diphtheria toxin.

The temperature and guanidine hydrochloride (GuHCl) dependence of the structural stability of diphtheria toxin has been investigated by high-sensitivity differential scanning calorimetry. In 50 mM phosphate buffer at pH 8.0 and in the absence of GuHCl, the thermal unfolding of diphtheria toxin is characterized by a transition temperature (Tm) of 54.9 degrees C, a calorimetric enthalpy change (delta H) of 295 kcal/mol, and a van't Hoff to calorimetric enthalpy ratio of 0.57. Increasing the GuHCl concentration lowers the transition temperature and the calorimetric enthalpy change. At the same time, the van't Hoff to calorimetric enthalpy ratio increases until it reaches a value of 1 at 0.3 M GuHCl and remains constant thereafter. At low GuHCl concentrations (0-0.3 M), the thermal unfolding of diphtheria toxin is characterized by the presence of two transitions corresponding to the A and B domains of the protein. At higher GuHCl concentrations (0.3-1 M), the A domain is unfolded at all temperatures, and only one transition corresponding to the B domain is observed. Under these conditions, the most stable protein conformation at low temperatures is a partially folded state in which the A domain is unfolded and the B domain folded. A general model that explicitly considers the energetics of domain interactions has been developed in order to account for the stability and cooperative behavior of diphtheria toxin. It is shown that this cooperative domain interaction model correctly accounts for the temperature location as well as the shape and area of the calorimetric curves. Under physiological conditions, domain-domain interactions account for most of the structural stability of the A domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗
PubMed1990

Selective decontamination in intensive care practice: a review of clinical experience.

Acquired infection is a common problem in intensive care and in a general ICU the infection rate can exceed 80% in patients ventilated beyond 5 days. SDD, adapted from regimes used in neutropenic patients, was first introduced to the ICU situation in Groningen. This article reviews 10 published trials of SDD in ICU. The trial designs vary but all show a significant reduction in both colonisation rates and acquired infection rates. Infection rates were reduced from 10%-78% to 3%-10% in the SDD treated groups. Of the 10 trials 2 showed an overall reduction in mortality 2 showed a reduction in infection-related mortality and 1 showed a reduction in mortality amongst trauma patients. Although further evaluation of trials is required SDD now appears to be of proven efficacy in certain groups of high risk patients within ICU.

Anti-Bacterial Agents↗
PubMed1989

Energetics of diphtheria toxin membrane insertion and translocation: calorimetric characterization of the acid pH induced transition.

The pH and temperature stabilities of diphtheria toxin and its fragments have been studied by high-sensitivity differential scanning calorimetry. These studies demonstrate that the pH-induced conformational transition associated with the mechanism of membrane insertion and translocation of the toxin involves a massive unfolding of the toxin molecule. At physiological temperatures (37 degrees C), this process is centered at pH 4.7 at low ionic strength and at pH 5.4 in the presence of 0.2 M NaCl. At pH 8, the thermal unfolding of the nucleotide-bound toxin is centered at 58.2 degrees C whereas that of the nucleotide-free toxin is centered at 51.8 degrees C, indicating that nucleotide binding (ApUp) stabilizes the native conformation of the toxin. The unfolding profile of the toxin is consistent with two transitions most likely corresponding to the A fragment (Tm = 54.5 degrees C) and the B fragment (Tm = 58.4 degrees C), as inferred from experiments using the isolated A fragment. These two transitions are not independent, judging from the fact that the isolated A fragment unfolds at much lower temperatures (Tm = 44.2 degrees C) and that the B fragment is insoluble in aqueous solutions when separated from the A fragment. Interfragment association contributes an extra -2.6 kcal/mol to the free energy of stabilization of the A fragment. Whereas the unfolding of the entire toxin is irreversible, the unfolding of the A fragment is a reversible process. These findings provide a thermodynamic basis for the refolding of the A fragment after reexposure to neutral pH immediately following translocation across the lysosomal membrane.

Calorimetry, Differential Scanning↗
PubMed1988

Triple regimen of selective decontamination of the digestive tract, systemic cefotaxime, and microbiological surveillance for prevention of acquired infection in intensive care.

All 324 patients admitted over sixteen months to a general intensive therapy unit (ITU) were prospectively studied to assess the effect of a novel prophylactic antibiotic regimen on the incidence of acquired infection. Consecutive control (161 patients) and test (163 patients) groups were analyzed. In the control group, antibiotic administration was determined by clinical and microbiological evidence of infection. In the test group, treatment consisted of a triple regimen of selective decontamination of the digestive tract (polymyxin E, tobramycin, and amphotericin B) administered throughout the ITU stay, systemic cefotaxime administered for the initial four days, and regular microbiological screening of multiple sites. The test group showed a striking and consistent reduction in colonisation of the digestive tract with aerobic gram-negative bacilli, and there was a substantial reduction in the incidence of acquired infection (24% to 10%). Mortality in certain categories of patients was also reduced. There is now a considerable body of evidence to justify the more widespread use of this selective parenteral and enteral anti-sepsis regimen (SPEAR) in general intensive care practice.

Adult↗
PubMed1988

Endotoxaemia in multiple organ failure due to sepsis.

Patients with multiple organ failure secondary to intraabdominal sepsis are often blood culture negative despite exhibiting the features of septic shock. This study examined the possible central role of endotoxin in such patients. In 15 consecutive intensive care patients with the above clinical picture endotoxin was measured by a chromogenic limulus (LAL) assay; on admission and thereafter 4 hourly. Regular blood cultures and cultures of any primary septic focus were also performed and liver function was assessed by measurement of indocyanine-green clearance from plasma (ICGC). All 15 patients had significant endotoxaemia at least intermittently. No significant difference was observed between survivors (n = 5) and non-survivors (n = 10) in either initial or peak endotoxin levels, although the pattern of endotoxaemia differed with non-survivors exhibiting consistently high or steadily increasing levels. Of 5 patients with an intra-abdominal (I/A) septic focus only one had a positive blood culture while 5 of 10 patients with extra-abdominal (E/A) infection had positive cultures. Despite this the I/A group had higher initial and peak endotoxin levels. 3 patients with Gram-positive septicaemia had significant endotoxaemia in the absence of any gram-negative infection. Changes in ICGC appeared to be of useful prognostic significance. ICGC was significantly lower in the I/A group and in both groups there was a significant negative correlation between ICGC and the level of endotoxaemia. These results suggest that endotoxin may play a central role in the syndrome of multiple organ failure and further suggest that the endotoxin is endogenous (gut-derived) secondary to failure of hepatic filtration.

Adult↗
PubMed1987

Definition of regions in human c-myc that are involved in transformation and nuclear localization.

To study the relationship between the primary structure of the c-myc protein and some of its functional properties, we made in-frame insertion and deletion mutants of the normal human c-myc coding domain that was expressed from a retroviral promoter-enhancer. We assessed the effects of these mutations on the ability of c-myc protein to cotransform normal rat embryo cells with a mutant ras gene, induce foci in a Rat-1-derived cell line (Rat-1a), and localize in nuclei. Using the cotransformation assay, we found two regions of the protein (amino acids 105 to 143 and 321 to 439) where integrity was critical: one region (amino acids 1 to 104) that tolerated insertion and small deletion mutations, but not large deletions, and another region (amino acids 144) to 320) that was largely dispensable. Comparison with regions that were important for transformation of Rat-1a cells revealed that some are essential for both activities, but others are important for only one or the other, suggesting that the two assays require different properties of the c-myc protein. Deletion of each of three regions of the c-myc protein (amino acids 106 to 143, 320 to 368, and 370 to 412) resulted in partial cytoplasmic localization, as determined by immunofluorescence or immunoprecipitation following subcellular fractionation. Some abnormally located proteins retained transforming activity; most proteins lacking transforming activity appeared to be normally located.

Animals↗