[Show abstract][Hide abstract] ABSTRACT: The non-specific acute phase response in mice is associated with increased resistance to bacterial infection, which is critically mediated by granulocyte colony stimulating factor (G-CSF), but the behaviour of G-CSF in the human acute phase response is not known. Cardiothoracic surgery is a powerful acute phase stimulus and we show here that this procedure caused increased production of G-CSF, in addition to increases in the circulating concentrations of the proinflammatory cytokine interleukin (IL)-6 and the acute phase plasma proteins C-reactive protein (CRP) and serum amyloid A protein (SAA). Values of G-CSF correlated positively with IL-6 concentrations and circulating neutrophil counts, but not with CRP values. These results confirm that G-CSF is a physiological component of the acute phase response in humans that shares some of the same regulatory controls as IL-6, but its downstream effects are on neutrophils, not hepatic acute phase protein synthesis. Our observations are compatible with a protective role against bacterial infection for G-CSF in the human acute phase response, and support investigation of the prophylactic use of G-CSF in at-risk patients.
[Show abstract][Hide abstract] ABSTRACT: In this paper we review evidence in favor of a protective role for antibodies directed at the lipopolysaccharide (LPS) core region of Gram-negative bacilli. It will be shown that, given the right animal model, polyclonal antisera raised against O-antigen lacking, rough mutant strains, can be shown to protect animals against lethal sepsis due to a variety a bacterial species. Evidence for a protective role obtained from clinical studies will also be discussed. It will be stressed how difficult it is to prove that the antibodies directed against the LPS-core, and not other proteins present in the polyclonal anti-rough strain antisera, were responsible for the observed protective effects. The solution to some of these problems is the production of monoclonal antibodies (Mabs). We will discuss the findings that purified anti-core Mabs may give false-positive outcomes of protection studies, due to the induction of tolerance by small amounts of contaminating endotoxin, present in the antibody preparation. The need to administer the Mab therapeutically instead of prophylactically to the test animals, will be stressed. It will be shown how we succeeded in determining the epitope-specificities of several anti-core antibodies. It will be shown also that use of Elisa alone for characterization of anti-core Mabs may lead to false conclusions concerning epitope specificities. We will prove that synthetic KDO-(=ketodeoxycctanate) and lipid A-containing antigens are indispensable tools for thorough characterization of Mabs. Using this methodology we were able to discriminate between specific antigen-antibody interactions, and the ability of some Mabs to bind “non-specifically” to hydrophobic surfaces. The relationship between epitope specificity and protective effects of anti-core Mabs will be described. Finally, we will demonstrate that a KDO-specific Mabs is capable of protecting mice against lethal sepsis; in other words, it will be shown conclusively that Mabs to defined epitopes in LPS core region are cross-protective.
[Show abstract][Hide abstract] ABSTRACT: A case of an 18 year old woman is reported who presented with a pyrexia of unknown origin having returned from a trip to India. She initially had constitutional symptoms only, which rapidly progressed to a multisystem disorder. The difficulty in making the diagnosis of polyarteritis nodosa, especially with the possible differential diagnosis of infection after her recent travel, is discussed. The discussion reviews the condition of polyarteritis nodosa and analyses the diagnostic difficulties in this case.
Postgraduate Medical Journal 12/2002; 78(925):685-6. · 1.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The viridans streptococci (VS) are associated with the development of a rapidly fulminant shock syndrome in neutropenic patients. A panel of 52 VS strains isolated from the blood of neutropenic patients was used to demonstrate the ability of culture supernatants and cell walls of VS to induce release of the proinflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-8 from whole blood in a dose- and strain-dependent fashion. Intercellular adhesion molecule-1 was shown to be markedly up-regulated on endothelial cells after incubation with plasma from blood exposed to cell walls or culture supernatants. This up-regulation was blocked by IL-1 receptor antagonist but not neutralizing antibody against TNF-alpha. These data may help explain the pathogenesis of the viridans streptococcal shock syndrome in neutropenic patients.
The Journal of Infectious Diseases 03/2002; 185(3):357-67. · 5.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sepsis is the systemic response to infection, and is caused by gram positive and gram negative bacteria in approximately equal numbers. In recent years it has become increasingly clear that there are significant differences between sepsis caused by gram positive and gram negative infections, both in the microbial components that initiate the injury, and the host responses that follow it. These differences will assume greater importance in developing targeted therapeutic interventions to severe sepsis.
Journal of chemotherapy (Florence, Italy) 12/2001; 13 Spec No 1(1):153-8. · 0.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We used flow cytometry and confocal immunofluorescence microscopy to study the localization of Salmonella typhimurium in spleens of infected mice. Animals were inoculated intragastrically or intraperitoneally with S. typhimurium strains, constitutively expressing green fluorescent protein. Independently of the route of inoculation, most bacteria were found in intracellular locations 3 days after inoculation. Using a panel of antibodies that bound to cells of different lineages, including mononuclear phagocyte subsets, we have shown that the vast majority of S. typhimurium bacteria reside within macrophages. Bacteria were located in red pulp and marginal zone macrophages, but very few were found in the marginal metallophilic macrophage population. We have demonstrated that the Salmonella SPI-2 type III secretion system is required for replication within splenic macrophages, and that sifA(-) mutant bacteria are found within the cytosol of these cells. These results confirm that SifA and SPI-2 are involved in maintenance of the vacuolar membrane and intracellular replication in vivo.
[Show abstract][Hide abstract] ABSTRACT: We have shown previously that an isogenic SPEA-negative Streptococcus pyogenes strain did not attenuate virulence in a murine model of necrotizing fasciitis. The aim of this study was to confirm that streptococcal pyrogenic exotoxin A (SPEA) is not crucial for streptococcal invasiveness in murine invasive infection. The SPEA-negative S. pyogenes (H326) was complemented with speA extra-chromosomally to create strain H361 which produced 2.2-fold more SPEA compared with the parental speA(+)wild-type (H305). The growth phase-regulated expression of SPEA in vitro was unaffected in this strain. Complementation with speA resulted in reduced virulence and bacterial counts in invasive murine infection. SPEA production was quantitated from muscle tissue of infected mice. However, H361 did not produce more SPEA than H305 in vivo. We conclude that SPEA does not play a key role in invasive murine streptococcal infection.
[Show abstract][Hide abstract] ABSTRACT: Bacterial superantigens are believed to cause septic shock, although, because of the lack of superantigen-sensitive infection models, proof that superantigenicity underlies shock pathogenesis is lacking. This work demonstrates a clear superantigen effect in septic shock resulting from bacterial infection. Transgenic expression of human leukocyte antigen (HLA)-DQ, but not HLA-DR, specifically augments lymphocyte responses to streptococcal pyrogenic exotoxin A (SPEA). HLA-DQ transgenic mice had increased mortality after administration of SPEA or infection with Streptococcus pyogenes. Immune activation during infection was HLA-DQ transgene-dependent and was manifested by Vbeta-specific T cell repertoire changes and widespread lymphoblastic tissue infiltration. Unlike earlier models, which used toxin-induced shock, these T cell superantigen responses and lymphoblastoid changes were observed during invasive streptococcal sepsis. Lymphoid activation was undetectable in HLA-DQ mice infected with an isogenic SPEA(-) strain, which proves that a single superantigen can play a role in sepsis pathogenesis.
The Journal of Infectious Diseases 08/2001; 184(2):166-73. · 5.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The difficulty in identifying new treatment modalities that significantly reduce the mortality and morbidity rates associated with sepsis has highlighted the need to reevaluate the approach to clinical trial design. The United Kingdom Medical Research Council convened an International Working Party to address these issues.
The subject areas that were to be the focus of discussion were identified by the co-chairs, and group leaders were nominated. Preconference reading material was circulated to group members.
Small-group discussion fed into an iterative process of feedback from plenary sessions, followed by the formulation of recommendations. Finally, each working group prepared a summary of its recommendations and these are reported herein.
There were five key recommendations. First, investigators should no longer rely solely on the American College of Chest Physicians/Society of Critical Care Medicine definitions of sepsis or sepsis syndrome as the basis of trial entry. Entry criteria should be based on three principles: a) All patients should have infection; b) there should be evidence of a pathologic process that represents a biologically plausible target for the proposed intervention, for example, an abnormal circulating level of a biological marker pertinent to the study drug; and c) patients should fall into an appropriate category of severity (usually severe sepsis). Second, investigators should use a scoring system for organ dysfunctions that has been validated and that can be incorporated into all sepsis studies; agreement on the use of a single system would simplify comparisons between studies. Third, the primary outcome measure generally should be mortality rates, but under appropriate circumstances major morbidities could be considered as primary end points. Regardless of choice of the duration to primary end point, patients should be followed for > or =90 days. Fourth, sample size needs to be based on a realistic assessment of achievable effect size based on knowledge of the at-risk population. Fifth, subgroups should be few in number and should be defined a priori on the basis of variables present before randomization.
Important changes in several aspects of trial design may improve the quality of clinical studies in sepsis and maximize the chance of identifying effective therapeutic agents.
Critical Care Medicine 04/2001; 29(4):880-6. · 6.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The production of superantigenic exotoxins by Gram positive bacteria underlies the pathology of toxic shock syndrome. Future treatment strategies for superantigen-mediated diseases are likely to be directed at blocking the three-way interaction between superantigen, T cell receptor and major histocompatibility class II molecule, which inititates an excessive and disordered inflammatory response. In this article, we review the first published data to address one such strategy in the context of other recognised and experimental treatments.
Critical Care 02/2001; 5(2):53-5. · 4.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Serum amyloid P component (SAP) binds to Streptococcus pyogenes, and we show here that it also binds to Neisseria meningitidis, including a lipopolysaccharide (LPS)-negative mutant, and to rough variants of Escherichia coli. Surprisingly, this binding had a powerful antiopsonic effect both in vitro and in vivo, reducing phagocytosis and killing of bacteria. Furthermore, SAP knockout mice survived lethal infection with S. pyogenes and rough E. coli J5, organisms to which SAP binds. The susceptibility of SAP(-/-) mice was fully restored by injection of isolated human SAP. However, SAP(-/-) mice were more susceptible than wild-type animals to lethal infection with E. coli O111:B4, a smooth strain to which SAP does not bind, suggesting that SAP also has some host defense function. Although SAP binds to LPS in vitro, SAP(-/-) mice were only marginally more susceptible to lethal LPS challenge, and injection of large amounts of human SAP into wild-type mice did not affect sensitivity to LPS, indicating that SAP is not a significant modulator of LPS toxicity in vivo. In contrast, the binding of SAP to pathogenic bacteria enabled them to evade neutrophil phagocytosis and display enhanced virulence. Abrogation of this molecular camouflage is thus potentially a novel therapeutic approach, and we show here that administration to wild-type mice of (R)-1-[6-(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine -2- carboxylic acid, a drug that inhibits SAP binding, significantly prolonged survival during lethal infection with E. coli J5.
Proceedings of the National Academy of Sciences 01/2001; 97(26):14584-9. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to characterize abnormalities of coagulation in mice with experimental, invasive group A, streptococcal shock, in an attempt to explain the prolongation of the activated partial thromboplastin time identified in patients with streptococcal toxic shock syndrome.
A longitudinal descriptive animal model study of coagulation times and single coagulation factors in mice infected with Streptococcus pyogenes. This was followed by an experimental study to determine whether streptococci or streptococcal products could activate the human contact system in vitro.
University infectious diseases and hemostasis molecular biology laboratories.
CD1 outbred mice.
Coagulation times, single factor assays, and bradykinin assays were conducted on murine plasma at different times after streptococcal infection and compared with uninfected mice. In experiments in which streptococcal products were co-incubated with human plasma, we compared coagulation times, single factor assays, and activities against a range of chromogenic substrates with control plasma. In a murine model of streptococcal necrotizing fasciitis, the activated partial thromboplastin times were significantly prolonged in infected mice compared with controls, whereas prothrombin times were normal, suggesting an isolated abnormality of the intrinsic pathway. Bleeding was not seen. Prolongation of activated partial thromboplastin time was associated with reduced factor XII and prekallikrein, whereas levels of factors VIII, IX, XI, and high molecular weight kininogen were elevated. In vitro studies suggested that streptococcal supernatants can activate prekallikrein, in addition to causing plasminogen activation through the action of streptokinase.
Prolongation of activated partial thromboplastin time in streptococcal toxic shock syndrome is associated with activation of the contact system, possibly contributing to the profound shock associated with streptococcal toxic shock syndrome.
Critical Care Medicine 12/2000; 28(11):3684-91. · 6.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the role of mitogenic factor (MF) in streptococcal pathogenesis, the structural gene (mf) encoding this protein was disrupted in a clinical isolate of Streptococcus pyogenes H293, to yield the isogenic mutant H363. Growth in enriched broth and on blood agar was unaffected by disruption of mf. Cell-free broth supernatants from H293 and H363 demonstrated identical promitogenic activities when co-incubated with human peripheral blood mononuclear cells, even when diluted 100000-fold, showing that MF is not a major streptococcal mitogen compared with other secreted superantigens. Disruption of mf resulted in complete loss of DNase B production and detectable DNase activity in H363 compared with the parent strain, confirming that the single gene mf, which is present in all group A streptococcal M serotypes studied, encodes DNase B. Despite loss of DNase activity, the virulence of S. pyogenes in a mouse model of necrotizing fasciitis and myositis was unaffected.
[Show abstract][Hide abstract] ABSTRACT: A retrospective analysis of 7 patients with streptococcal toxic shock revealed isolated prolongation of the activated partial thromboplastin time, which returned to normal during recovery. Levels of factor XII were reduced in 2 patients who had single factor assays performed, consistent with activation of the kallikrein-kinin system. We speculate that bradykinin release following activation of the kallikrein-kinin system in streptococcal toxic shock may underlie the features of pain, capillary leaking, and severe hypotension characteristic of this syndrome.
[Show abstract][Hide abstract] ABSTRACT: A considerable body of evidence has accumulated that implicates endotoxin in the pathogenesis of the sepsis syndrome. This has raised interest in the possibility of measuring endotoxin as a surrogate marker of Gram negative infection, particularly since conventional microbiological tests have an inevitable delay. The Limulus amoebocyte lysate (LAL) assay has been used most widely to measure endotoxin in clinical samples. However, there are several important limitations that need to be borne in mind. These include the dangers of contamination, lack of precision and accuracy, and both false positive and false negative results. Endotoxaemia is present in the blood of about 30% of patients with bacteraemia, but endotoxaemia does not predict either Gram negative bacteraemia or Gram negative infection, nor does it predict survival from sepsis. There is some correlation with severity of sepsis, but the level of precision is poor. At the present time, there is no place for routine endotoxin testing in clinical practice. In particular, the positive predictive value of the test is insufficiently high to be of clinical use. It may be that the LAL assay, or one of the newer developments, may be more useful in excluding Gram negative infection, but that remains to be shown.
Intensive Care Medicine 02/2000; 26 Suppl 1:S51-6. · 5.26 Impact Factor