Search PubMed⌕ Search

Biomedical subjects

D L Stevens

Publications and source records attributed to D L Stevens.

At least 91 records · Page 5Linked to original sources
PubMed1994

Clinical courses of seven survivors of Clostridium septicum infection and their immunologic responses to alpha toxin.

Clostridium septicum bacteremia typically portends a fulminant disease associated with high mortality. We describe the clinical courses of seven survivors of C. septicum infection and their antibody responses to the alpha toxin produced by C. septicum. Three patients had clinical syndromes ranging from uncomplicated bacteremia to early typhlitis, and three patients had syndromes ranging from abscess to myonecrosis and septic shock. In addition, an AIDS patient who developed septic shock and who had extensive gas in the retroperitoneal musculature did not undergo surgery but survived after receiving antimicrobial therapy and intensive supportive care. Both immunocompetent patients with myonecrosis had detectable IgG to alpha toxin by immunoblot analysis. IgG to alpha toxin was not detected in the four immunocompetent patients who had C. septicum bacteremia but who did not have myonecrosis or in the AIDS patient with myonecrosis. Therefore, humoral responses to alpha toxin during C. septicum infection may be related to the host's clinical syndrome and immune status.

Adult↗
PubMed1994

Persistent acylation of high-molecular-weight penicillin-binding proteins by penicillin induces the postantibiotic effect in Streptococcus pyogenes.

Penicillin at 10X MIC induced a postantibiotic effect (PAE) of 2.1 h in Streptococcus pyogenes. Progressive increases in the densities of penicillin-binding proteins (PBPs) 1-3 of the bacterium were detected at 30, 60, and 90 min during the postantibiotic phase. The increase in colony-forming units during this phase paralleled the kinetics of incorporation of lysine into proteins, suggesting that growth was triggered by de novo synthesis of PBPs. The question was raised as to whether the progressive increases in densities of PBPs were due to the restoration of preexisting PBPs or to synthesis of new PBPs. With 10X MIC of clindamycin to inhibit PBP synthesis during the postantibiotic phase, the temporal increase in densities of PBPs 1-3 were totally inhibited. These results suggest that the PAE of penicillin in S. pyogenes is caused by irreversible binding of penicillin to PBPs 1-3 and represents the time necessary for synthesis of new PBPs required for normal growth.

Acylation↗
PubMed1994

Analysis of circulating phagocyte activity measured by whole blood luminescence: correlations with clinical status.

The present study was undertaken to determine if measurement and analysis of phagocyte function are useful for diagnosis and staging of infection. Circulating phagocyte activity was measured in healthy volunteers and sequentially in patients with acute infections of different types and severity, including those with diabetes mellitus or human immunodeficiency virus (HIV) infection. Using an automated luminescence system, these phagocyte functions were measured in whole blood: basal and phorbol 12-myristate 13-acetate (PMA)-stimulated oxidase activity, basal and PMA-stimulated simple dioxygenation (e.g., oxidase-driven haloperoxidase activity), and circulating and primed opsonin receptor-dependent dioxygenation. Multiple discriminant analysis of these data showed significant differences between healthy controls, diabetic patients, HIV-positive subjects, and patients with pneumonia or sepsis syndromes. Longitudinally, circulating phagocyte function correlated with clinical condition, severity of infection, and outcome. This methodology provides rapid, objective, and sensitive diagnostic and monitoring information for patients with infections.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Investigation of the role of the disulphide bond in the activity and structure of staphylococcal enterotoxin C1.

The goal of this study was to investigate the role of the disulphide bond of staphylococcal enterotoxin C1 (SEC1) in the structure and activity of the toxin. Mutants unable to form a disulphide bond were generated by substituting alanine or serine for cysteine at positions 93 and/or 110. Although we did not directly investigate the residues between the disulphide linkage, tryptic lability showed that significant native structure in the cystine loop is preserved in the absence of covalent bonding between residues 93 and 110. Since no correlation was observed between the behaviour of these mutants with regard to toxin stability, emesis and T cell proliferation we conclude that SEC1-induced emesis and T cell proliferation are dependent on separate regions of the molecule. The disulphide bond itself is not an absolute requirement for either activity. However, conformation within or adjacent to the loop is important for emesis. Although mutants with alanine substitutions were not emetic, those with serine substitutions retained this activity, suggesting that the disulphide linkage stabilizes a crucial conformation but can be replaced by residues which hydrogen bond.

Amino Acid Sequence↗
PubMed1994

Identification and molecular analysis of a locus that regulates extracellular toxin production in Clostridium perfringens.

The anaerobic bacterium Clostridium perfringens mediates clostridial myonecrosis, or gas gangrene, by producing a number of extracellular toxins and enzymes. Transposon mutagenesis with Tn916 was used to isolate a pleiotropic mutant of C. perfringens that produced reduced levels of phospholipase C, protease and sialidase, and did not produce any detectable perfringolysin O activity. Southern hybridization revealed that a single copy of Tn916 had inserted into a 2.7 kb HindIII fragment in the C. perfringens chromosome. A 4.3kb PstI fragment, which spanned the Tn916 insertion site, was cloned from the wild-type strain. When subcloned into a shuttle vector and introduced into C. perfringens this fragment was able to complement the Tn916-derived mutation. Transformation of the mutant with plasmids containing the 2.7 kb HindIII fragment, or the 4.3 kb PstI fragment, resulted in toxin and enzyme levels greater than or equal to those of the wild-type strain. The PstI fragment was sequenced and found to potentially encode seven open reading frames, two of which appeared to be arranged in an operon and shared sequence similarity with members of two-component signal transduction systems. The putative virR gene encoded a protein with a deduced molecular weight of 30,140, and with sequence similarity to activators in the response regulator family of proteins. The next gene, virS, into which Tn916 had inserted, was predicted to encode a membrane-spanning protein with a deduced molecular weight of 51,274. The putative VirS protein had sequence similarity to sensor proteins and also contained a histidine residue highly conserved in the histidine protein kinase family of sensor proteins. Virulence studies carried out using a mouse model implicated the virS gene in the pathogenesis of histotoxic C. perfringens infections. It was concluded that a two-component sensor regulator system that activated the expression of a number of extracellular toxins and enzymes involved in virulence had been cloned and sequenced. A model that described the regulation of extracellular toxin production in C. perfringens was constructed.

Amino Acid Sequence↗
PubMed1994

Predictions of T-cell receptor- and major histocompatibility complex-binding sites on staphylococcal enterotoxin C1.

We have focused on regions of staphylococcal enterotoxin C1 (SEC1) causing immunomodulation. N-terminal deletion mutants lacking residues 6 through 13 induced T-cell proliferation similar to that induced by native toxin. However, mutants with residues deleted between positions 19 and 33, although nonmitogenic themselves, were able to inhibit both SEC1-induced T-cell proliferation and binding of the native toxin to major histocompatibility complex (MHC) class II. Presumably, these deletions define a part of SEC1 that interacts with the T-cell receptor. Three synthetic peptides containing residues located in a region analogous to the alpha 5 groove of SEC3 had residual mitogenic activity or blocked T-cell proliferation induced by SEC1 and appear to recognize the same site as SEC1 on a receptor for the toxin, presumably MHC class II. We conclude that isolated portions of the SEC1 molecule can retain residual mitogenic activity but that the entire protein is needed to achieve maximal superantigenic stimulation. Our results, together with the results of other investigators, support a model in which SEC1 binds to an alpha helix of MHC class II through a central groove in the toxin and thereby promotes or stabilizes the interaction between antigen-presenting cells and T cells.

Amino Acid Sequence↗
PubMed1994

[Streptococcal myositis in children: four case histories].

We report about four children, who suffered from myositis caused by beta-hemolytic group A streptococci (GAS). The cases were observed during the last 12 months, and differed much in severity. Soft tissue infections caused by GAS are reported with increasing frequency from the USA, Australia and Europe. They occur in hitherto healthy children and young adults, mostly without a predisposing trauma. In children, a preceding varicella infection is often found. Some patients develop a streptococcal toxic shock syndrome with a letality of 20-50%. The bacteria, which can be isolated from normally sterile body sites, are morphologically inconspicuous, and are mostly of the serological type M1 or M3.

Child↗
PubMed1993

The in vitro antibacterial activity of ceftriaxone against Streptococcus pyogenes is unrelated to penicillin-binding protein 4.

The in vitro activities of penicillin and ceftriaxone were compared against 29 strains of Streptococcus pyogenes with the result that ceftriaxone showed greater activity than penicillin. The morphological changes induced by 1/2 and 1x MIC concentrations of penicillin and ceftriaxone, respectively, were very similar using scanning electron microscopy. Competitive binding studies using 'cold' penicillin or ceftriaxone as inhibitors of radiolabeled penicillin binding demonstrated that ceftriaxone had a very low affinity for penicillin binding protein (PBP) 4 compared to that of penicillin. Since ceftriaxone had greater antibacterial activity, this suggests that PBP 4 may not be important to the in vitro activity of ceftriaxone. In contrast, the IC50 for ceftriaxone was much lower (> 200 fold) for PBPs 2 and 3 compared to PBP 4, suggesting greater avidity of these high molecular mass PBPs for ceftriaxone. These data may at least in part explain the superior in vitro activity of ceftriaxone compared to penicillin against S. pyogenes. These data, together with the observation that PBP 1 was saturated at a lower concentration of penicillin than any of the other PBPs, suggest that the inhibition of PBPs 1, 2, and 3 mediates the bactericidal activity of beta-lactam antibiotics against group A streptococci.

Bacterial Proteins↗
PubMed1993

Comparison of oral cefpodoxime proxetil and cefaclor in the treatment of skin and soft tissue infections.

This multicenter, randomized, double-blind study was designed to compare the safety and efficacy of cefpodoxime proxetil and cefaclor in the treatment of skin and soft tissue infections. Patients were aged > or = 12 years with acute (< or = 7 days duration), single-site skin or skin-structure infections. The 7- to 10-day treatment regimens were cefpodoxime proxetil (400 mg cefpodoxime) orally with food twice a day with cefaclor-matched placebo (orally, fasting, three times a day); or cefaclor (Ceclor; 500 mg anhydrous equivalent) orally, fasting, three times a day, with cefpodoxime-matched placebo (orally with food twice a day). Clinical progress and cultures were evaluated upon admission to the study; on study days 7-10 and 15-18; and 2-3 weeks after treatment. Cefpodoxime had lower minimum inhibitory concentrations against the majority of Staphylococcus species than did cefaclor. Both treatments were highly effective (99% pathogen eradication and 86% cure rate). These high eradication rates were not unexpected in this study of minor infections in which patients with resistant pathogens were excluded. Cefaclor had a higher failure rate [2 (4%) of 57], than did cefpodoxime [2 (1%) of 139; p not significant]. Most patients in both groups completed treatment as planned: 185 (74%) of 249 cefpodoxime-treated patients and 91 (75%) of 122 cefaclor-treated patients. Both treatments were well tolerated and considered safe and effective in the treatment of skin and skin structure infections. However, the twice-a-day dosing regimen for cefpodoxime proxetil compared with the three-times-a-day regimen for cefaclor may result in better patient compliance.

Administration, Oral↗
PubMed1993

Induction and rejoining of DNA double-strand breaks in V79-4 mammalian cells following gamma- and alpha-irradiation.

The induction and rejoining of DNA double-strand breaks (dsbs) in V79-4 mammalian cells following irradiation by 60Co gamma-rays and 238Pu alpha-particles (average LET 120 keV microns-1) under aerobic conditions have been determined using both the sucrose sedimentation and filter elution techniques under non-denaturing conditions. Cellular inactivation was also determined. The dependence of the initial yield of dsbs at 277 K on dose under aerobic conditions is linear with a relative biological effectiveness (RBE) for alpha-particles of 0.85 +/- 0.14 (sedimentation) and 0.68 +/- 0.12 (elution) compared with 60Co gamma-rays. The ability of the cells to rejoin dsbs at 310K is significantly reduced for alpha-irradiations with only 30-50% rejoined for a 3-h incubation period. With low LET radiation, > 90% of the dsbs are rejoined within 3 h at a dose of 20 Gy. The RBE for cellular inactivation was determined to be 4.0 at the 1% survival level. From the cellular dimensions and the D0-value for cellular inactivation by alpha-particles, it is determined that, on average, 4.7 tracks traverse the cell nucleus per lethal lesion. Under hypoxic conditions, the RBE values for induction of dsbs and cellular inactivation (10% level) by alpha-particles are approximately 3.0 and approximately 11.8 respectively. From these findings, it is suggested that the residual DNA damage and not the initial damage is reflected in the cellular inactivation. It is inferred that the difference in repair of the various lesions is a reflection of the differences in the complexity of the clustered damage produced by these radiations.

Alpha Particles↗
PubMed1993

Role of theta toxin, a sulfhydryl-activated cytolysin, in the pathogenesis of clostridial gas gangrene.

Clostridial infections cause a wide variety of dramatic infections and intoxications. In each case the major virulence factors are extracellular toxins. Clostridium perfringens produces potent exotoxins, which are its major virulence factors. theta Toxin, a thiol-activated cytolysin, causes the clear zone of hemolysis around colonies on blood-agar plates, suppresses myocardial contractility ex vivo, and induces shock within 1 to 2 hours in vivo. Low concentrations of theta toxin induce priming and degranulation of polymorphonuclear leukocytes (PMNs) and functional up-regulation of PMN-dependent adherence molecules such as the integrin CD11/CD18, whereas higher concentrations are cytotoxic. Similarly, theta toxin causes concentration- and time-dependent induction of endothelial cell synthesis of platelet-activating factor, a potent proinflammatory lipid autocoid that mediates endothelial cell-dependent adherence of PMNs. These data suggest that theta toxin in high concentrations is a potent cytolysin and promotes direct vascular injury at the site of infection. At lower concentrations theta toxin activates PMNs and endothelial cells, and in so doing promotes vascular injury distally by activating adherence mechanisms. The rapid tissue necrosis associated with C. perfringens infection may be related to progressive vascular compromise orchestrated by dysregulated host-cell responses induced by theta toxin.

Animals↗
PubMed1993

Evaluation of therapy with hyperbaric oxygen for experimental infection with Clostridium perfringens.

The effects of inoculum size and treatment delays on the efficacy of hyperbaric oxygen (HBO) were evaluated in a murine model of Clostridium perfringens myositis in which the infection was treated with an HBO regimen identical to that used for humans. The efficacies of treatment with penicillin, metronidazole, or clindamycin--alone or in combination with HBO--were also assessed. Survival was inversely related to the size of the bacterial inoculum used for challenge, and delays in treatment markedly reduced the efficacies of all single and combination regimens. When animals were challenged with > 10(8) colony-forming units, survival was significantly higher among those treated with clindamycin or metronidazole than among those treated with penicillin. HBO alone did not improve survival at any inoculum tested. However, when administered early, HBO plus metronidazole or penicillin demonstrated significant additive efficacies in animals challenged with > or = 10(9) organisms. Clindamycin was more effective at the higher inocula than penicillin, metronidazole, or HBO, and its superior efficacy was not further enhanced by adjunctive therapy with HBO.

Animals↗
PubMed1993

Penicillin-binding protein expression at different growth stages determines penicillin efficacy in vitro and in vivo: an explanation for the inoculum effect.

Mechanisms to explain the "inoculum effect" have not been elucidated in gram-positive infections. A mouse model of group A streptococcal myositis was used to compare the efficacies of two beta-lactams, penicillin and ceftriaxone, and a protein synthesis inhibitor, clindamycin, at three different inoculum sizes. beta-lactams were more susceptible to inoculum effects than was clindamycin both in vivo and in vitro (P < .05). The large inocula were hypothesized to reach stationary phase of growth sooner than smaller inocula both in vitro and in vivo. The penicillin-binding protein (PBP) patterns from membrane proteins isolated from mid-log-phase and stationary-phase cultures of Streptococcus pyogenes were compared. Binding of radiolabeled penicillin by all PBPs was decreased in stationary cells; however, PBPs 1 and 4 were undetectable at 36 h. Thus, the loss of certain PBPs during stationary-phase growth in vitro may be responsible for the inoculum effect observed in vivo and may account for the failure of penicillin in both experimental and human cases of severe streptococcal infection.

Animals↗
PubMed1993

Superantigens associated with staphylococcal and streptococcal toxic shock syndrome are potent inducers of tumor necrosis factor-beta synthesis.

The role of tumor necrosis factor-alpha (TNF alpha) in the pathogenesis of severe bacterial infections has been studied extensively. However, the role of TNF beta, a lymphokine with biologic activities similar to those of TNF alpha, has received little attention. Therefore, the purpose of this study was to examine the production of TNF beta by peripheral blood mononuclear cells in response to lipopolysaccharide (LPS) and the superantigens staphylococcal toxic shock syndrome toxin 1 (TSST-1) and streptococcal pyrogenic exotoxin A (SPEA). Though LPS was a more potent inducer of TNF alpha than was TSST-1 or SPEA, TSST-1 and SPEA were both more potent inducers of TNF beta. The superantigens TSST-1 and SPEA were more potent inducers of total TNF (TNF alpha and TNF beta) than was LPS. These data suggest that the induction of TNF beta synthesis may be a unique pathway by which superantigens associated with severe streptococcal and staphylococcal infections mediate shock and multiorgan failure characteristic of toxic shock syndrome.

Antigens, Bacterial↗
PubMed1993

Clostridium perfringens invasiveness is enhanced by effects of theta toxin upon PMNL structure and function: the roles of leukocytotoxicity and expression of CD11/CD18 adherence glycoprotein.

Clostridium perfringens infections are characterized by the lack of an inflammatory response at the site of infection and rapidly progressive margins of tissue necrosis. Studies presented here investigated the role of theta toxin from C. perfringens in the pathophysiology of these events. Mice passively immunized with neutralizing monoclonal antibody against theta toxin and challenged with an LD100 of log phase C. perfringens had significantly less mortality than untreated controls. Intramuscular injection of killed, washed C. perfringens in mice induced a massive time-dependent influx of polymorphonuclear leukocytes (PMNL) into tissue; injection of either viable, washed C. perfringens or killed organisms plus theta toxin dramatically attenuated PMNL influx although PMNL accumulated in adjacent vessels. The anti-inflammatory effects could not be attributed to an absence of chemoattractants since C. perfringens proteins had chemotactic factor activity, and killed bacilli generated serum-derived chemotactic factors. Scanning and transmission electron microscopy demonstrated the dramatic leukocidal effects of high doses of theta toxin on PMNL. In contrast, sublethal concentrations of theta toxin primed PMNL chemiluminescence, disrupted PMNL cytoskeletal actin polymerization/disassembly, and stimulated functional upregulation of CD11b/CD18 adherence glycoprotein. In summary, these results demonstrate that theta toxin is an important virulence factor in C. perfringens infection. In a concentration-dependent fashion, theta toxin contributes to the pathogenesis of clostridial gangrene by direct destruction of host inflammatory cells and tissues, and by promoting dysregulated PMNL/endothelial cell adhesive interactions.

Actins↗
PubMed1992

Rapid screening for deletion mutations in the hprt gene using the polymerase chain reaction: X-ray and alpha-particle mutant spectra.

Conditions were devised for the isolation of DNA from single-mutant colonies on dishes, to give reproducible results in the polymerase chain reaction (PCR). Primers for 3 exons of the hamster hprt gene were used in a multiplex reaction to show rapidly whether the mutants carried deletions at these sites. 138 independent mutants were screened in total, some spontaneous and others induced by X-rays or by alpha-particles from plutonium-238. Few deletions were found among the spontaneous set, while 'total' gene deletions formed about half the mutants found after irradiation. At equitoxic doses, little difference in mutant spectrum was found for the X-ray set compared to the alpha-particle set. This rapid technique should be applicable to many instances of comparative mutagenesis.

Alpha Particles↗
PubMed1992

Direct comparison between protons and alpha-particles of the same LET: I. Irradiation methods and inactivation of asynchronous V79, HeLa and C3H 10T1/2 cells.

A direct comparison was carried out of the biological effectiveness of protons and alpha-particles of the same linear energy transfer (LET) under identical conditions with a variety of in vitro biological systems. Monolayers of mammalian cells were irradiated with accelerated beams of protons (1.2 and 1.4 MeV) and alpha-particles (30 and 35 MeV) corresponding to LETs of 23 and 20 keV microns-1 for each particle type. For V79-4 cells it was observed that the linear term of the dose-response for cell inactivation by protons was significantly greater than that for alpha-particles of the same LET. For HeLa and HeLa S3 cells, also, the linear term appeared to be greater for protons, but this was not observed with more limited data for C3H 10T1/2 cells. The result for V79 cells is in agreement with the report of Belli et al. (1989) who observed that the biological effectiveness of protons rose sharply between 17 and 30 keV microns-1 in strong contrast to alpha-particles which reached a peak effectiveness at greater than 100 keV microns-1. These results place new constraints on the biologically relevant features of the microscopic structure of radiation tracks, and have implications for the mechanistic and practical comparison between radiations.

Alpha Particles↗