Innate immune recognition of, and response to, Clostridium sordellii.

Clostridium sordellii, an anaerobic pathogen, has recently been associated with rapidly fatal infections following medically induced abortions and injecting drug use. Patients with C. sordellii infection display few signs of inflammation such as fever, or redness and pain at the site of infection. We hypothesized that this could be due to reduced recognition of the organism by Toll-like receptors (TLRs) of the innate immune system. An ELAM-NF-kappaB luciferase reporter system in TLR-transfected HEK cells was used to measure TLR-dependent recognition of washed, heat-killed C. sordellii and other pathogenic clostridial species. Results demonstrated that all clostridia were well recognized by TLR2 alone and that responses were greatest when TLR2 was co-expressed with TLR6. Further, isolated human monocytes produced the pro-inflammatory cytokine TNFalpha and the immunoregulator IL-10 in response to C. sordellii. In addition, C. sordellii-stimulated monocytes produced 30% less TNFalpha following treatment with an anti-TLR2 blocking antibody. These data demonstrate that innate immune recognition of, and response to, cell-associated components of C. sordellii and other clostridial pathogens are mediated by TLR2 in combination with TLR6. We conclude that the characteristic absence of inflammatory signs and symptoms in C. sordellii infection is not related to inadequate immune detection of the organism, but rather is attributable to a species-specific immune system dysfunction that remains to be elucidated.

Clostridium sordellii infection: epidemiology, clinical findings, and current perspectives on diagnosis and treatment.

Clostridium sordellii infections pose difficult clinical challenges and are usually fatal. Most commonly, these infections occur after trauma, childbirth, and routine gynecological procedures, but they have recently been associated with medically induced abortions and injection drug use. We report 2 fatal cases, one of which was associated with minor trauma, and the other of which was associated with normal childbirth, and we summarize the clinical features of 43 additional cases of reported C. sordellii infection. Of these 45 cases, 8 (18%) were associated with normal childbirth, 5 (11%) were associated with medically induced abortion, and 2 (0.4%) were associated with spontaneous abortion. The case-fatality rate was 100% in these groups. Ten (22%) of the C. sordellii infections occurred in injection drug users, and 50% of these patients died. Other cases of C. sordellii infection (in 19 patients [43%]) occurred after trauma or surgery, mostly in healthy persons, and 53% these patients died. Overall, the mortality rate was 69% (31 of 45 patients). Eighty-five percent of all patients with fatal cases died within 2-6 days of initial infection, and nearly 80% of fatal cases developed leukemoid reactions. Rapid diagnostic tests and improved treatments are needed to reduced the morbidity and mortality associated with this devastating infection.

Clostridium perfringens phospholipase C-induced platelet/leukocyte interactions impede neutrophil diapedesis.

Clostridium perfringens gas gangrene is a fulminant necrotizing infection in which inflammatory cells are notably absent from infected tissues but are often massed within adjacent vessels. It has been shown that C. perfringens phospholipase C (PLC) stimulates formation of large intravascular platelet/leukocyte complexes and that PLC-induced activation of platelet gpIIbIIIa plays a major role. In vivo, such aggregates contribute to microvascular thrombosis and ischaemic necrosis of tissue. However, the effects of adherent platelets on neutrophil diapedesis have not been established. The present work investigated (1) the contribution of platelet P-selectin (CD62P) to PLC-induced cellular complex formation and (2) the effects of platelet adhesion on neutrophil diapedesis. The effects of anti-gpIIbIIIa and anti-CD62P strategies on PLC-induced complex formation were measured by flow cytometry and followed by light microscopy. Both platelet gpIIbIIIa and CD62P contributed to the formation of platelet/leukocyte complexes. Specifically, gpIIbIIIa mediated the formation of large platelet/platelet aggregates that were tethered to the leukocyte principally via CD62P. Neutrophil diapedesis, quantified by a transendothelial cell migration assay and visualized by electron microscopy, was significantly reduced (>60%) by the adherence of large platelet aggregates. It was concluded that the absence of a tissue inflammatory response in C. perfringens gas gangrene is due, in part, to impaired neutrophil mobility caused by large aggregates of adherent platelets induced by PLC. Further, an adjunctive immunotherapeutic strategy targeting both gpIIbIIIa and CD62P may improve the tissue inflammatory response, prevent vascular occlusion, maintain tissue viability, and reduce the need for radical amputation in patients with clostridial gas gangrene.

The hormonal and inflammatory responses to pelvic reconstructive surgery following major trauma.

Patients undergoing trauma sustain an initial injury followed by further physiological challenges during surgery. Plasma osteocalcin (OC), a marker of osteoblastic activity, declines after major surgery. Increased cortisol secretion, and other components of the perioperative stress response, may play a role in mediating this response. We have examined the osteocalcin, hormonal and cytokine responses in twenty patients undergoing post-traumatic pelvic reconstruction surgery. We measured plasma osteocalcin, serum cortisol, bone specific alkaline phosphatase (BSAP), IL-6, IL-8, IL-10, plasma epinephrine and norepinephrine concentrations for up to 3 days after surgery. We recorded an increase in IL-6, IL-10 and epinephrine concentrations perioperatively and a fall in OC and BSAP concentrations. There were no significant changes in cortisol or IL-8 concentrations. Patients undergoing pelvic reconstruction surgery following trauma have a preserved inflammatory and catecholamine response but the cortisol response may be obtunded. Osteocalcin concentrations are affected by factors other than glucocorticoids.

M type 1 and 3 group A streptococci stimulate tissue factor-mediated procoagulant activity in human monocytes and endothelial cells.

Streptococcal toxic shock syndrome (StrepTSS) is an invasive infection characterized by marked coagulopathy, multiple organ failure, and rapid tissue destruction and is strongly associated with M type 1 and 3 group A streptococci (GAS). Initiation of the coagulation cascade with formation of microvascular thrombi contributes to multiple organ failure in human cases of gram-negative bacteremia; however, little is known regarding the mechanism of coagulopathy in StrepTSS. Thus, we investigated the abilities of several strains of M type 1 and 3 GAS isolated from human cases of StrepTSS to stimulate production of tissue factor (TF), the principal initiator of coagulation in vivo. Washed, killed M type 1 and 3 GAS, but not M type 6 GAS, elicited high-level TF-mediated procoagulant activity from both isolated human monocytes and cultured human umbilical vein endothelial cells. M type 1 GAS consistently elicited higher levels of TF from monocytes than did M type 3 GAS. GAS-induced TF synthesis in monocytes did not correlate with production of tumor necrosis factor alpha or interleukin-8. Conversely, M type 3 GAS were consistently more potent than M type 1 GAS in stimulating endothelial cell TF synthesis. These results demonstrate that (i) M type 1 and 3 strains of GAS are potent inducers of TF synthesis, (ii) GAS-induced TF synthesis is not simply an epiphenomenon of cytokine generation, and (iii) induction of TF in endothelial cells and monocytes may be M type specific. In total, these findings suggest that a novel interaction between GAS and host cells contributes to the observed coagulopathy in StrepTSS.

Osteocalcin and the hormonal, inflammatory and metabolic response to major orthopaedic surgery.

Plasma osteocalcin, a marker of osteoblastic activity, decreases after major abdominal and gynaecological surgery. Increased cortisol secretion and other hormonal and inflammatory components of the peri-operative stress response may play a role in mediating this response. We assessed the effects of three different anaesthetic techniques on peri-operative osteocalcin concentrations. Thirty-six female patients undergoing elective total hip replacement were randomly assigned to receive propofol, propofol plus 'three-in-one' block or etomidate as part of a general anaesthetic technique. We measured plasma osteocalcin and serum cortisol, bone specific alkaline phosphatase, interleukin-6, plasma epinephrine, norepinephrine, plasma glucose and cystatin C concentrations for up to 3 days after surgery. Etomidate successfully inhibited the cortisol response to surgery but plasma osteocalcin declined in all patients. This was accompanied by increased plasma catecholamines, interleukin-6 and glucose concentrations, and decreased cystatin C-values. Inhibition of the cortisol response to surgery failed to prevent a decrease in plasma osteocalcin concentrations after surgery, suggesting that other factors such as cytokines or catecholamines may play a significant role.

Efficacy of varying concentrations of hyaluronidase in peribulbar anaesthesia.

We have compared the efficacy of adding varying concentrations of hyaluronidase to a standard mixture of 2% lidocaine and 1% ropivacaine to provide peribulbar anaesthesia for cataract surgery. We used (i) the time to adequate anaesthesia for surgery and (ii) ocular and eyelid movement scores at 8 min after block as clinical endpoints. Ninety patients were randomly allocated to receive 7-10 ml of equal volumes of 2% lidocaine and 1% ropivacaine without hyaluronidase or with hyaluronidase 15 IU ml(-1) or 150 IU ml(-1). Median time at which the block was adequate for surgery was 6 min in all groups (interquartile range 4-12 min). Median eyelid movement scores were similar in all groups, but the ocular movement scores at 8 min were significantly lower in the group which received hyaluronidase 150 IU ml(-1) than in the group not given hyaluronidase (P<0.03). There were no differences between groups in the incidence of minor complications. A high concentration of hyaluronidase resulted in a statistically significantly lower ocular movement score at 8 min; the clinical relevance of this finding is uncertain.

Epidural analgesia and arterial reconstructive surgery to the leg: effects on fibrinolysis and platelet degranulation.

It has been suggested that the incidence of early graft occlusion after arterial reconstructive surgery to the leg may be decreased by epidural analgesia. This effect may be mediated by the suppression of the usual cortisol response to surgery, which results in increased circulating plasminogen activator inhibitor-1 with consequent adverse effects on fibrinolysis. To investigate this and other potential mechanisms, 30 patients undergoing arterial reconstructive surgery to the leg were randomized to receive either general anaesthesia or general anaesthesia plus epidural analgesia. Post-operative analgesia was provided by morphine infusion or epidural analgesia, respectively. Blood samples were collected at 0, 2, 4, 6, 12 and 24 h, and 2, 3 and 5 days and analysed for cortisol, plasminogen activator inhibitor-1 antigen, interleukin-6 and beta thromboglobulin. The incidence of graft-related and systemic complications was recorded for 30 days. Only one patient developed early graft occlusion that required embolectomy and eventually amputation. There were no significant changes from control values in either group of patients in circulating cortisol, plasminogen activator inhibitor-1 and beta thrombogobulin (a marker for platelet degranulation). Interleukin-6 values increased significantly in both groups after 4 h and remained elevated until day 3. There were no significant differences between the groups in any variable measured. We conclude that any effect of epidural analgesia on early graft patency is unlikely to be mediated by fibrinolysis or platetlet degranulation.

Molecular epidemiology of nga and NAD glycohydrolase/ADP-ribosyltransferase activity among Streptococcus pyogenes causing streptococcal toxic shock syndrome.

Severe invasive group A streptococcal (GAS) infections emerged in the late 1980s, yet no single virulence factor has been common to all isolates from infected patients. A strong association was recently found between isolates of such cases (regardless of M type) and the production of NAD glycohydrolase (NADase). Of interest, all M-1 strains isolated after 1988 were positive for NADase, whereas virtually all M-1 GAS were previously negative for NADase. Genetic analysis demonstrated that GAS isolates were >96% identical in nga and >99% identical in their upstream regulatory sequences. Furthermore, because NADase-negative strains did not produce immunoreactive NADase, we concluded that additional regulatory element(s) control NADase production. NADase purified from GAS altered neutrophil-directed migration and chemiluminescence responses and had potent ADP-ribosyltransferase activity. In summary, the temporal relationship of NADase expression, alone or with other streptococcal virulence factors, may contribute to the pathogenesis of invasive GAS infections.

Clostridial gas gangrene. I. Cellular and molecular mechanisms of microvascular dysfunction induced by exotoxins of Clostridium perfringens.

Mechanisms responsible for the rapid tissue destruction in gas gangrene are not well understood. To examine the early effects of Clostridium perfringens exotoxins on tissue perfusion, a rat model of muscle blood flow was developed. Intramuscular injection of a clostridial toxin preparation containing both phospholipase C (PLC) and theta-toxin caused a rapid (1-2 min) and irreversible decrease in blood flow that paralleled formation of activated platelet aggregates in venules and arterioles. Later (20-40 min), aggregates contained fibrin and leukocytes, and neutrophils accumulated along vascular walls. Flow cytometry confirmed that these clostridial toxins or recombinant PLC induced formation of P-selectin-positive platelet aggregates. Neutralization of PLC activity in the clostridial toxin preparation completely abrogated human platelet responses and reduced perfusion deficits. It is concluded that tissue destruction in gas gangrene is related to profound attenuation of blood flow initiated by activation of platelet responses by PLC.

Clostridial gas gangrene. II. Phospholipase C-induced activation of platelet gpIIbIIIa mediates vascular occlusion and myonecrosis in Clostridium perfringens gas gangrene.

Clostridium perfringens gas gangrene is a fulminant infection, and radical amputation remains the single best treatment. It has been hypothesized that rapid tissue destruction is related to tissue hypoxia secondary to toxin-induced vascular obstruction, and previous studies demonstrated that phospholipase C (PLC) caused a rapid and irreversible decrease in skeletal muscle blood flow that paralleled the formation of intravascular aggregates of activated platelets, fibrin, and leukocytes. In this study, flow cytometry demonstrated that PLC stimulated platelet/neutrophil aggregation in a gpIIbIIIa-dependent fashion. Pretreatment of animals with heparin or depletion of leukocytes reduced blood-flow deficits, and aggregate formation caused by PLC. It is concluded that fulminant tissue destruction in gas gangrene results from profound attenuation of blood flow caused by PLC-induced, gpIIbIIIa-mediated formation of heterotypic platelet/polymorphonuclear leukocyte aggregates. Therapeutic strategies that target gpIIbIIIa may prevent vascular occlusion, maintain tissue viability, and provide an alternative to radical amputation for patients with this infection.

Construction and virulence testing of a collagenase mutant of Clostridium perfringens.

Clostridium perfringens produces several extracellular toxins and enzymes, including an extracellular collagenase or kappa toxin that is encoded by the colA gene. To determine if the ability to produce collagenase was a significant virulence factor in cases of gas gangrene or clostridial myonecrosis that are caused by C. perfringens, a chromosomal colA mutant was constructed by homologous recombination and subsequently virulence tested in the mouse myonecrosis model. The results clearly indicate that loss of the ability to produce collagenase does not alter the ability of the mutant to establish a virulent infection. By contrast, infection with a mutant unable to produce alpha-toxin led to a marked decrease in virulence. These results indicate that collagenase is not a major determinant of virulence in C. perfringens -mediated clostridial myonecrosis.

Staging of the baboon response to group A streptococci administered intramuscularly: a descriptive study of the clinical symptoms and clinical chemical response patterns.

Group A streptococcal infections, ranging from necrotizing fasciitis and myositis to toxic shock syndrome, have increased over the last 10 years. We developed the first primate model of necrotizing fasciitis and myositis. Thirteen baboons were inoculated intramuscularly with group A streptococci (GAS). Eleven animals survived for > or = 11 days before sacrifice, and two animals died within 2 days. The site of inoculation of the survivors exhibited an intense neutrophilic influx (stage I), followed by a lymphoplasmacytic influx (stages II and III). This was accompanied by the appearance of markers of an acute and then a chronic systemic inflammatory response. In contrast, the site of inoculation of the two nonsurvivors exhibited intravascular aggregates of neutrophils at its margin with no influx of neutrophils and with extensive bacterial colonization. We conclude that GAS inoculation induces a local and systemic acute neutrophilia followed by a chronic lymphoplasmacytic response; failure, initially, of neutrophilic influx into the site of inoculation predisposes to systemic GAS sepsis and death; and this three-stage primate model approximates the human disease.

Type-specific opsonophagocytosis of group A Streptococcus by use of a rapid chemiluminescence assay.

A whole-blood chemiluminescence (CL) assay was developed to determine the presence of type-specific opsonic antibodies against group A streptococcus (GAS). Convalescent sera with high bactericidal activities against an M-1 serotype were used to opsonize different M-types of GAS. CL responses were monitored for 20 min, and results were expressed as integral counts/minute per phagocyte. CL responses of phagocytes incubated with M-1 GAS opsonized with homologous (M-1) serum were significantly higher than responses of phagocytes incubated with heterologous (M-3) GAS. Adsorption of convalescent serum against the homologous, but not the heterologous, strain markedly reduced the CL response, demonstrating type specificity. The CL assay showed a high correlation with the indirect bactericidal test (r=0.90). In conclusion, this CL assay is a rapid, highly sensitive, specific, and reproducible method for quantifying type-specific opsonic antibodies against GAS and will be a useful tool for future clinical, basic science, and epidemiological studies.

Etomidate and the osteocalcin response to gynaecological surgery.

Circulating osteocalcin is a good marker of osteoblastic activity and decreases significantly after stressful physiological states such as major surgery. Glucocorticoids are known to inhibit osteoblastic activity and result in a decline in circulating osteocalcin. We used etomidate to inhibit the cortisol response to routine gynaecological surgery to determine if this would prevent the postoperative decline in osteocalcin. Twenty-four patients were allocated randomly to receive either thiopental or etomidate for induction of anaesthesia; all other aspects of anaesthesia and perioperative management were standardized. In the thiopental group, circulating cortisol increased significantly at 2 and 6 h after the start of surgery and plasma osteocalcin concentrations decreased significantly to almost 50% of baseline values at 48 h. Etomidate abolished the cortisol response to surgery, and circulating osteocalcin concentrations did not change after operation. There was a significant difference in osteocalcin concentration between the groups at 48 h. We conclude that the cortisol response to surgery is associated with a postoperative decrease in circulating osteocalcin.

Maternal cotinine level during pregnancy and birthweight for gestational age.

BACKGROUND: Recent studies have found that cotinine is a better predictor of birthweight than the number of cigarettes smoked in pregnancy. In this paper we test this hypothesis and use cotinine to explore the effect of environmental tobacco smoke (ETS) on birthweight. METHODS: In all, 1254 white women were interviewed at booking, 28 and 36 weeks about the number and brand of cigarette smoked. Cotinine was assayed from blood samples taken on the day of interview. The outcome was birthweight for gestational age. RESULTS: There was good agreement between self-reported smoker/non-smoker status and maternal cotinine with 1.3% women mis-reported as non-smokers at booking, 0.6% and 1.8% mis-reported at 28 and 36 weeks respectively. Among smokers, cotinine was more closely related to birthweight than the number of cigarettes smoked at all three time points (r = -0.25 versus r = -0.16 at booking). A reduction in cotinine between booking and 28 weeks was associated with increased birthweight but the effect was not statistically significant. Among non-smokers the association between birthweight and cotinine was not statistically significant after adjusting for maternal height, parity, sex and gestational age. Difference in mean birthweight between non-smokers in the lower and upper quintiles of cotinine was 0.2% (95% CI: -2.4, 2.8). Pooling the results of 10 studies plus our own gave an estimated difference in mean birthweight between women unexposed and exposed to passive smoke of 31 g (95% CI: 19, 44). CONCLUSIONS: Cotinine is a better predictor of birthweight than the reported number of cigarettes smoked. If biochemical analysis is impossible, then self-reported smoking habit should be obtained prospectively using a structured approach. Any effect on birthweight of maternal passive smoking during pregnancy is small compared with the effects of maternal active smoking.

Alpha toxin from Clostridium perfringens induces proinflammatory changes in endothelial cells.

Alpha toxin from Clostridium perfringens type A, a phospholipase C, has been implicated in many of the localized and systemic features of gas gangrene. We demonstrated that human endothelial cells synthesize two vasoactive lipids, platelet-activating factor (PAF) and prostacyclin, in response to alpha toxin treatment. The stimulated synthesis of PAF required the enzymatic activity of the toxin and subsequent protein kinase C activation. Alpha toxin-treated endothelial cells accumulated the products of the phospholipase C reaction, diacylglycerol and ceramide, and exhibited a decrease in the enzymatic precursors phosphatidylcholine and sphingomyelin. Furthermore, the temporal accumulation of PAF depended on the concentration of the toxin in the overlying medium and was blocked in the presence of a neutralizing antibody. The cultured endothelial cells also exhibited enhanced neutrophil adhesion in response to alpha toxin which was mediated through the PAF receptor and P-selectin. P-selectin expression by endothelial cells and extravascular neutrophil accumulation were also observed in tissue sections from alpha toxin-injected Sprague-Dawley rats. These endothelial cell-mediated processes are important in maintaining vascular homeostasis and, when activated in a dysregulated manner by C. perfringens alpha toxin, may contribute to localized and systemic manifestations of gas gangrene including enhanced vascular permeability, localized neutrophil accumulation, and myocardial dysfunction.

Clostridial gas gangrene: evidence that alpha and theta toxins differentially modulate the immune response and induce acute tissue necrosis.

The rapid extension of necrosis and an absence of polymorphonuclear leukocytes (PMNL) at the site of infection are two hallmarks of Clostridium perfringens gas gangrene. While both alpha and theta toxins profoundly affect PMNL function and viability in vitro, their roles in muscle destruction and impairment of the inflammatory response in vivo have not been investigated. Comparative histopathologic examinations were performed on animals infected with either wild-type C. perfringens, or isogenic, toxin-deficient mutants of C. perfringens. Tissue destruction was modest in animals infected with the alpha toxin-deficient mutant; destruction was more pronounced in tissues infected with the theta toxin-deficient mutant or the wild-type strain. alpha and theta toxins also displayed differing abilities to modulate the inflammatory response. Histopathologic studies in which recombinant toxins were injected together with killed, washed C. perfringens further substantiated these tissue-destructive and differential antiinflammatory effects.

The effect of cortisol suppression on interleukin-6 and white blood cell responses to surgery.

BACKGROUND: The endocrine and immune changes associated with surgery are well documented, but the interaction between them has not been fully evaluated. Cortisol production during surgery can be suppressed by etomidate and we have used this to investigate the relationship between the cortisol response and immune changes in the perioperative period. METHODS: We have measured the cortisol, interleukin-6 and white cell responses to elective abdominal hysterectomy in 8 healthy female patients, who received etomidate 0.3 mg kg-1 for induction of anaesthesia. A control group of 8 subjects received thiopentone. Both groups of patients received vecuronium and fentanyl 2 micrograms kg-1 and anaesthesia was maintained with nitrous oxide in oxygen and isoflurane 0.5-1.0%. Venous blood samples were collected before and during surgery and up to 24 h in the postoperative period. RESULTS: Serum interleukin-6 values were significantly greater at 6 and 12 h (P < 0.05) in those patients who received etomidate. Inhibition of the serum cortisol response to surgery in the etomidate group was also associated with less marked lymphopenia at 4 h (P < 0.05). There was no significant difference in neutrophil granulocyte counts between the two groups. CONCLUSION: In conclusion, endogenous corticosteroids modulate the interleukin-6 response to surgery.