Endotoxin-like Activity Research Articles

Inhibitory effect of BMAP-28 on Leptospiral Lipopolysaccharide-Induced TLR2-Dependent Immune Response in Bovine Cells.

BackgroundBovine leptospirosis is a widespread zoonotic disease, leading to serious economic losses in animal production and causing potential hazards to human health. Leptospiral lipopolysaccharide (L-LPS) plays an important role in leptospirosis pathogenicity.ObjectivesWith respect to L-LPS endotoxin-like activity, we examined bovine immune response to L-LPS and the inhibitory ability of bovine myeloid antimicrobial peptide-28 (BMAP-28) against L-LPS-induced immune activation in bovine cells.Materials and MethodsIn this study, L-LPS-induced proinflammatory cytokine production in bovine cells was quantitatively measured with real-time PCR and ELISA, and we determined which cell membrane receptors (toll-like receptor [TLR]2 and TLR4) played a major role. In addition, the ability of BMAP-28 to inhibit L-LPS-induced endotoxin-like immune activation in bovine cells was determined by the decrease in cytokine secretion.ResultsL-LPS showed the ability to induce cytokine production in bovine cells, and its induction was TLR2-dependent. BMAP-28 was used to inhibit L-LPS-induced endotoxin-like activity. The function of BMAP-28 was to inhibit LPS-induced TLR2 expression and cytokine production.ConclusionsIn this study, the L-LPS immune response of bovine cells was significant, indicating that TLR2 is the predominant receptor for L-LPS. Due to L-LPS endotoxin-like activity, we found a strategy through using BMAP-28 to prevent L-LPS-induced TLR2-dependent immune activation in bovine cells.

Glycolaldehyde‐modified β‐lactoglobulin AGEs are unable to stimulate inflammatory signaling pathways in RAGE‐expressing human cell lines

Advanced glycation endproducts (AGEs) are suspected to stimulate inflammatory signaling pathways in target tissues via activation of the receptor for AGEs. Endotoxins are generally recognized as potential contamination of AGE preparations and stimulate biological actions that are very similar as or identical to those induced by AGEs. In our study, we used glycolaldehyde-modified β-lactoglobulin preparations as model AGEs and employed two methods to remove endotoxin using either affinity columns or extraction with Triton X-114 (TX-114). Affinity column-purified AGEs retained their ability to stimulate inflammatory signaling as measured by mRNA expression of inflammatory cytokines in the human lung epithelial cell line Beas2b. However, glycolaldehyde-modified AGEs purified by extraction with TX-114 did not show any stimulation of mRNA expression of inflammatory cytokines. The presence of a cell stimulating endotoxin-like activity was demonstrated in the detergent phase after extraction with TX-114, thus indicating that not AGEs but a lipophilic contamination was responsible for the stimulation of inflammatory signaling. Our results demonstrate that glycolaldehyde-modified AGEs are unable to induce inflammatory signaling in receptor for AGE-expressing cells. The observed cell-activating activity can be ascribed to an endotoxin-like lipophilic contamination present in AGE preparations and affinity column purification was insufficient to remove this contamination.

Endotoxin-like effects in acute phase response to Trypanosoma brucei brucei infection are not due to gastrointestinal leakage

Trypanosomosis is mainly an immunological and inflammatory response mediated by increased levels of pro-inflammatory cytokines. Evidence suggests that pathological changes produced during infection with trypanosomes could be initiated by nonspecific endotoxin-like substances in trypanosomes and/or Gram-negative secondary bacterial infection. Studies in trypanosome-infected rats indicate damage to the gastrointestinal tract (GIT) accompanied by increased leakage of the GIT mucosa. The current study was carried out to determine the in vivo response to endotoxin-like substances of Trypanosomabrucei brucei. To this purpose we neutralized the entrance of endotoxin through the GIT using polymyxin-B treatment and monitored the plasma concentration of the acute phase proteins SAP and Hp. The results in this study, where infection was performed in the presence of oral antibiotic that is not absorbed from GIT and which binds to and inactivates endotoxin, show that the elevated plasma levels of endotoxin-like activity and the resulting acute phase response indicated by an increase in levels of Hp and SAP, are due to trypanosome infection. Results obtained in the present study indicate that GIT is not the major source of elevated plasma endotoxin-like activity levels and the observed acute phase response was due to an increase in the levels of acute phase proteins SAP and haptoglobin.Therefore trypanosomes are responsible for the elevated plasma endotoxin-like activity levels and the subsequent systemic acute phase response in the host.

Secondary bacterial infection in plasma endotoxin levels and the acute‐phase response of mice infected withTrypanosoma brucei brucei

Murine Trypanosoma brucei brucei infection leads to elevated plasma endotoxin-like activity levels not related to parasitaemia levels accompanied by the development of acute-phase response and increased plasma levels of serum amyloid P (SAP) and haptoglobin (Hp). To determine the source of the endotoxin-like activity and role of secondary bacterial infection in the pathogenesis of trypanosomosis, infected mice were treated with the antibiotic ciprofloxacin. Plasma endotoxin-like activity levels, irrespective of treatment, were elevated three- to fourfold, beginning 7 days after infection. Plasma protein concentrations increased markedly following infection from 7 days after infection (DAI). Peak Hp and SAP concentrations in ciprofloxacin-treated and -untreated infected mice were attained 7 and 14 DAI, respectively. Thereafter, both protein levels gradually declined until the end of the experiment, but Hp levels for non-treated mice declined up to 21 DAI and thereafter significantly increased on 28 and 35 DAI. Whole-trypanosome lysate and the membrane-enriched fraction demonstrated endotoxin-like activity, with the former having higher levels. The results suggest that the endotoxin-like activity in trypanosome fractions and plasma of infected mice is due to the trypanosome. Further elevation of haptoglobin during the late stages of infection in non-treated mice suggests the involvement of secondary bacterial infection.

Lipopolysaccharide binding protein in the acute phase response of experimental murine Trypanosoma brucei brucei infection

Cellular responses to lipopolysaccharide (LPS) are enhanced by LPS-binding protein (LBP). The present study investigated the acute phase response of LBP during Trypanosoma brucei brucei infection in mice. Mean plasma concentrations of LBP increased two-fold by the seventh day following infection, but decreased to intermediate levels by the 14th day. There were no significant differences in LBP concentrations of infected/antibiotic-treated and infected/untreated mice. At 35 days post-infection, the infected mice were treated with the anti-trypanosomal diminazine aceturate (Berenil ®). LBP levels of the mice then decreased to pre-infection levels within one-week. This demonstrated that LBP is an acute phase protein during murine trypanosomosis. Furthermore, opportunistic secondary bacterial infection during trypanosomosis did not seem to play an important role in the changes in plasma LBP levels. We speculate that the marked concomitant increases in plasma LBP and endotoxin-like activity following murine trypanosome infection might play an important role in the pathogenesis of trypanosomosis.

Wolbachia in the inflammatory pathogenesis of human filariasis.

Filarial nematodes cause some of the most debilitating diseases in tropical medicine. Recent studies, however, have implicated the parasites' endosymbiotic Wolbachia bacteria, rather than the nematode, as the cause of inflammatory-mediated filarial disease. Soluble extracts of a variety of filarial species stimulate innate inflammatory responses, which are absent or reduced when using extracts derived from species either devoid of bacteria, or those cleared of bacteria by antibiotics. Characterization of the molecular nature of the bacterial derived inflammatory stimulus points toward an endotoxin-like activity that is dependent on the pattern recognition receptors CD14 and TLR4 and can be inhibited by lipid A antagonists. TLR4 dependent inflammation has been shown to occur in the systemic inflammatory adverse reaction to Brugia malayi following anti-filarial chemotherapy and in the development of neutrophil-mediated ocular inflammation in a mouse model of river blindness. The development of acute and severe inflammatory responses in people infected with Brugia malayi and Onchocerca volvulus is associated with the release of Wolbachia into the blood following death or damage of the worms after anti-filarial chemotherapy. Together these studies suggest that Wolbachia are the principal cause of acute inflammatory filarial disease. Accumulated exposure to acute episodes of inflammation may also underlie the development of chronic filarial pathology. The use of antibiotic therapy to target Wolbachia of filarial parasites may therefore provide a means to prevent the development of filarial pathology.

Wolbachia Endosymbiotic Bacteria of Filarial Nematodes. A New Insight into Disease Pathogenesis and Control

Filarial nematodes are parasitic worms that cause some of the most devastating of all tropical diseases such as elephantiasis and river blindness. Studies on the inflammatory pathogenesis of filarial disease have shown that endotoxin-like activity derived from endosymbiotic Wolbachia bacteria is the major inflammatory stimulus of filarial nematodes. Wolbachia appear to have evolved as essential symbionts of their filarial nematode hosts. Antibiotic depletion of bacteria shows that they are required for normal fertility and development of the worm and may even protect the parasites from host immunity. In addition to the uncovering of a fascinating symbiotic relationship, this discovery means we can now consider using antibiotics as a new approach to the treatment of filarial diseases.

MALP-2, a Mycoplasma lipopeptide with classical endotoxic properties: end of an era of LPS monopoly?

Although some activities of LPS are shared by other bacterial components, for half a century LPS has been regarded as unique in displaying many pathophysiological activities. Here we report on a synthetic lipopeptide, MALP-2 from Mycoplasma fermentans, which expresses potent endotoxin-like activity and whose lethal toxicity is comparable to that of LPS. With the exception of the Limulus lysate gelation test, in which MALP-2 was approximately 1000-fold less active than LPS, the synthetic lipopeptide induced all activities tested for, and in most cases to an extent comparable to that of LPS. Unlike LPS, the biological activities of MALP-2 were expressed both in LPS-responder and in LPS-non-responder mice (BALB/c/l, C57BL10/ScCr), indicating that MALP-2 signaling, unlike that of LPS, is not transduced via the Toll-like receptor (Tlr) 4 protein.MALP-2 expressed no toxicity in normal or sensitized Tlr2 knockout (Tlr2(-/-)) mice indicating that its toxic activity is induced via Tlr2 signaling. The phenomenology of the lethal shock induced by MALP-2 in normal or sensitized mice, i.e. the kinetics of its development and symptoms of illness exhibited by the treated animals, was very reminiscent of the lethal shock induced by LPS.

Environmental modulation of oral treponeme virulence in a murine model.

This investigation examined the effects of environmental alteration on the virulence of the oral treponemes Treponema denticola and Treponema pectinovorum. The environmental effects were assessed by using a model of localized inflammatory abscesses in mice. In vitro growth of T. denticola and T. pectinovorum as a function of modification of the cysteine concentration significantly enhanced abscess formation and size. In contrast, growth of T. denticola or T. pectinovorum under iron-limiting conditions (e.g., dipyridyl chelation) had no effect on abscess induction in comparison to that when the strains were grown under normal iron conditions. In vivo modulation of the microenvironment at the focus of infection with Cytodex beads demonstrated that increasing the local inflammation had no effect on lesion induction or size. In vivo studies involved the determination of the effects of increased systemic iron availability (e.g., iron dextran or phenylhydrazine) on the induction, kinetics, and size of lesions. T. denticola induced significantly larger lesions in mice with iron pretreatment and demonstrated systemic manifestations of the infectious challenge and an accompanying spreading lesion with phenylhydrazine pretreatment (e.g., increases in circulating free hemoglobin). In contrast, T. pectinovorum virulence was minimally affected by this in vivo treatment to increase iron availability. T. denticola virulence, as evaluated by lesion size, was increased additively by in vivo iron availability, and cysteine modified growth of the microorganism. Additionally, galactosamine sensitized mice to a lethal outcome following infection with both T. denticola and T. pectinovorum, suggesting an endotoxin-like activity in these treponemes. These findings demonstrated the ability to modify the virulence capacity of T. denticola and T. pectinovorum by environmental conditions which can be evaluated by using in vivo murine models.

Transient alteration in intestinal permeability to technetium Tc99m diethylenetriaminopentaacetate during the prodromal stages of alimentary laminitis in ponies

Abstract Objectives To determine whether mucosal permeability is altered during the prodromal stages of alimentary laminitis. Animals 15 healthy adult ponies. Procedures Intestinal permeability was evaluated for control ponies (n = 5) and for ponies 4 to 12 (n = 5) and 20 to 28 (n = 5) hours after administration of carbohydrate overload. Mucosal permeability was determined by measuring the percentage of orally administered technetium Tc99m diethylenetriaminopentaacetate (99mTc-DTPA) excreted in urine during an 8-hour period, then measuring blood radioactivity at hourly intervals. Plasma endotoxin-like activity was measured by use of a chromogenic Limulus amebocyte assay. Results Urinary excretion of 99mTc-DTPA was 2.45% of administered dose for control ponies, and was 16.67% of administered dose 4 to 12 hours and 3.57% of administered dose 20 to 28 hours after administration of carbohydrate. Conclusions A marked but transient increase in intestinal permeability was observed early in the prodromal stages of alimentary laminitis. Clinical Relevance Absorption of substances from the intestine may be an initiating event in alimentary laminitis. (Am J Vet Res 1998;59:1431–1434)

Endotoxemia Following Enteral Refeeding in Children

Plasma endotoxin-like activity, tumor necrosis factor alpha (TNFalpha) concentrations, core body temperature, and liver functions were measured before and after enteral feeding in children who had been deprived of enteral feeding for 5 days because of their illness. Transient endotoxemia and elevations in plasma TNFalpha concentrations occurred. Core body temperature, aspartate aminotransferase, alamine aminotransferase, and bilirubin concentrations were normal in patients who had elevated plasma endotoxin-like activity. Transient endotoxemia following enteral feeding may be due to the translocation from the gastrointestinal (GI) tract as a result of increased mesenteric circulation and peristalsis. No clinical consequences were noted despite transient endotoxemia. The transient endotoxemia is not due to the immature GI tract; instead, it results from enteral feeding following the deprivation of enteral feeds.

A sublethal dose of LPS to pregnant rats induces TNF-alpha tolerance in their 0-day-old offspring.

The newborn has high mortality in septic shock. Induction of endotoxin tolerance may prevent endotoxic shock in the newborn. The present study showed that a small dose of Salmonella enteritidis lipopolysaccharide (S. ent. LPS), Rc mutant Escherichia coli lipopolysaccharide (J5 LPS), or tumor necrosis factor-alpha (TNF-alpha) given to pregnant rats on the 19th day of gestation induced endotoxin tolerance in their 0-day-old offspring. S. ent. LPS or J5 LPS injected into pregnant rats increased plasma endotoxin-like activity in dams, although not in their fetuses, and increased plasma TNF-alpha concentration in both dams and their fetuses. The endotoxin-tolerant newborn rats were also resistant to TNF-alpha. In those newborn rats, an LPS injection increased plasma TNF-alpha concentration and liver TNF-alpha mRNA abundance. These experiments showed that the endotoxin tolerance could be due to TNF-alpha tolerance. In conclusion, prenatal treatment of dams with a small dose of S. ent. LPS, J5 LPS, or TNF-alpha was beneficial in preventing endotoxic shock in the newborn.

Role of endotoxin-like contaminants in the apparent anti-inflammatory activity of bovine superoxide dismutase.

Bovine CuZn superoxide dismutase (SOD: 1, 3 and 10 mg/kg) dose-dependently reduced carrageenan-induced paw edema in rats when administered intravenously 30 min before irritant injection. However, heat-treated SOD (10 mg/kg) was as effective as native SOD (10 mg/kg) although the enzymic activity was reduced to 9.7%. Examination of the contaminants of the native SOD revealed a fairly large amount of endotoxin-like activity, 47 ng as Escherichia coli endotoxin per mg, and 59.6% of this activity remained after heat treatment. Bovine CuZn SOD (1, 3 and 10 mg/kg), which contained negligible endotoxin-like materials and 1.5 times more enzyme units, had no effect on edema under the same conditions. Furthermore, Escherichia coli endotoxin (10, 100 and 1000 ng/kg) reduced edema dose-dependently. These results suggest that contamination by endotoxin-like materials is responsible for the anti-inflammatory action of the SOD preparation we observed. Hence, the anti-inflammatory action of contaminating endotoxin-like materials may lead to misinterpretation as a protective effect of SOD unless stringent precautions are taken against endotoxin-like contaminants in the SOD under examination.

Prothrombotic events in the prodromal stages of acute laminitis in horse

SUMMARY Prothrombotic changes occurring in the prodromal stages of carbohydrate-induced laminitis were investigated. Hemostatic alterations were evaluated by determining platelet counts, platelet survival, activated partial thromboplastin time, one-stage prothrombin time, and monocyte procoagulant activity. Thrombosis of vessels in the hoof wall was evaluated by contrast arteriography and histologic examination. Of 5 horses, 4 became lame between 28 and 52 hours after carbohydrate administration. Mean platelet count in laminitis-affected horses was lower throughout the prodromal stages of laminitis, compared with that in control horses, but differences were not statistically significant. However, survival of indium-111-labeled platelets was less than the value in control horses by 6 hours after carbohydrate administration. Arteriography of disarticulated feet revealed marked reduction in blood supply to hooves in laminitis-affected horses. Histologic examination of the laminar dermis disclosed microthrombi in venules of the laminar dermis in 2 of 4 affected horses. Statistically significant changes in prothrombin time were not observed, and changes in activated partial thromboplastin time were slight and occurred only at the onset of lameness. Statistically significant changes in monocyte procoagulant activity were not observed. Plasma endotoxin-like activity was not detected in laminitis-affected horses. These data indicate that platelet survival was decreased within the first 6 hours after induction of carbohydrate-induced laminitis, but systemic activation of the coagulation system was not detected.

Elimination of trace endotoxin protein from rough chemotype LPS

The phenol-chloroform-petroleum ether (PCP) extraction is the method of choice for isolating LPS chemotypes from rough mutant bacteria. However, we have observed that a high percentage of PCP-purified LPS preparations contain trace levels of protein contaminants, and these protein contaminants may exhibit endotoxin-like activity. To obtain protein-free rough LPS, a modified phenol-water extraction procedure was developed for use as a final step to follow PCP extraction. Using this procedure, Salmonella minnesota Ra, Rc, Rd and Re LPS were recovered in yields of 82-100% from the original PCP-extracted preparations. Yields were determined by recovery of KDO and supported by analysis of silver stained SDS-PAGE gels. Although the original PCP-purified chemotypes were contaminated by major 41, 38.5, 29.5, 23, 17 and 14 kDa proteins (detected by Western blotting and gold staining), the repurified LPS contained no detectable protein. The repurified LPS retained all of its original bioactivity as defined by the ability to stimulate normal C3H/OuJ macrophages to secrete TNF. The importance of utilizing repurified LPS in biologic studies was illustrated by the loss of > 99% of activity when repurified LPS was used to stimulate TNF secretion by LPS 'hyporesponsive' C3H/HeJ macrophages.

Suppression by methylprednisolone of augmented plasma endotoxin-like activity and interleukin-6 during cardiopulmonary bypass

It has been reported that plasma endotoxin, measured by Limulus amoebocyte lysate assays, markedly increased during extracorporeal circulation (ECC). Therefore, this study was undertaken to see if pretreatment with methylprednisolone 30 mg kg-1 modifies the endotoxaemia and associated increases in plasma cytokines in 17 patients undergoing cardiac surgery requiring ECC. We found that methylprednisolone suppressed significantly the ECC-induced increases in plasma endotoxin, measured by a conventional Limulus amoebocyte lysate assay (Toxicolor), and interleukin-6. Plasma concentrations of endotoxin, measured by a highly specific chromogenic Limulus test (Endospecy test), tumour necrosis factor-alpha and interleukin-1 beta did not increase significantly in either the control or methylprednisolone groups.

Biochemical properties of the outer membrane of Treponema denticola

The outer membranes (OMs) from serovars a, b, and c of Treponema denticola, originally isolated from periodontal patients, were prepared. Dialysis of the OMs against 20 mM MgCl2 yielded the aggregable (A) and the nonaggregable (NA) moieties of the OMs. The absence of muramic acid, adenosine triphosphatase, hexokinase, and nucleic acid as well as electron microscopy indicated that the OM preparations were homogeneous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the A and NA moieties of the OMs showed approximately 25 Coomassie brilliant blue R-250 stain-positive bands or 47 silver-stained polypeptides. The relative molecular masses ranged between 14 and 97 kDa. The electrophoretic polypeptide profiles of the A and NA moieties shared many similarities among serovars a, b, and c. However, they exhibited variation in the overall pattern, intensity, or location of the polypeptide stained zones. This was especially true for serovar b. Two-dimensional electrophoretic studies showed an excess of 100 silver-stained spots with isoelectric points of 4.6 to 7.0 and relative molecular masses in the 14- to 97-kDa range. The OMs contained simple proteins, glycoproteins, and lipoproteins. The NA moieties of the OMs contained 4 to 6, 10 to 12, and 4 to 6 glycopeptides as well as two, seven, and two lipoprotein bands for serovars a, b, and c, respectively. The A moieties of the OMs showed 7 to 9, 11 to 13 and 5 to 6 glycopeptides as well as four, five, and three lipoprotein bands for serovars a, b, and c, respectively. Lipopolysaccharide was detected in the OMs of the three serovars following removal of proteins with proteinase K, pronase and silver staining of sodium dodecyl sulfate-polyacrylamide gels, or removal of lipopolysaccharide from the OMs by hot phenol extraction. The 66- and 53-kDa bands were present in serovars b and c, while a band with a relative molecular mass of 45 kDa was present only in serovar c. Endotoxin-like activity was also shown in the OMs of the three serovars by the Limulus amebocyte clotting assay and the chick embryo lethality test. This is the first report on selected biochemical properties of the OM macromolecules of three known serovars of T. denticola.

Biological Activity of Endotoxin and Cytokine.

Bacterial endotoxin, which is thought to be a direct cause of shock or tissue damage of multi-organs in Gram-negative septis, has been studied on their biological activities for a long time. In the last several years, the advance in endotoxin research has been very rapid. Macrophage activation is the first and essential step in the course of endotoxin action and is followed by the release of many kinds of mediators including cytokines such as tumor necrosis factor α (TNFα)/cachectin, interleukin-1 (IL-1), interleukin-6 (IL-6), interferons, and colony-stimulating factors, and lipid derivatives such as prostaglandines, leukotrienes, and platelet-activating factor. Of these mediators, TNF α/cachectin and IL-1 play a central role in the multiple activities of endotoxin. TNF α and IL-1 have many similar activities and exert alone or cooperatively the endotoxin-like activities such as lethal toxicity, pyrogenesis, promotion of coagulation and leukocyte adherence to endothelial cells, and induction of acute phase proteins. Endotoxin tolerance and Shwartzman reaction are also mediated through these inflammatory cytokines. On the basis of these findings, some remedies using anti-TNF α monoclonal antibody or antagonist of IL-1 are under research for septic shock as well as clinical use of TNF α for cancer therapy.

Endotoxin-like activity associated with lyme disease Borrelia

The newly recognized spirochete, Borrelia burgdorferi, the causative agent of Lyme Disease, has been examined for endotoxin-like activities as measured by the standard Farmacopea Ufficiale della Republica Italiana rabbit fever test and the Limulus amoebocyte lysate assay. The suspension of heat-killed microorganism caused a febrile response at a dose of 1 X 10(8) bacteria pro kilo. Similar results were obtained in the Limulus assay where the heat-killed spirochetes stimulated formation of solid clot until the concentration of 1 X 10(5) per ml. Both in pyrogen test and in Limulus assay heat-killed Escherichia coli exhibited a higher degree of potency. These results show that LD-Borrelia possess endotoxin-like activities which could help in understanding the pathogenesis of the clinical symptomatology of the disease.

1088 CLINICAL CORRELATIONS OF CSF ENDOTOXIN-LIKE ACTIVITY IN GRAM-NEGATIVE MENINGITIS

Detection of endotoxin-like activity (ELA) in cerebrospinal fluid (CSF) by limulus amebocyte lysate (LAL) gelation has been suggested to be a useful technique for diagnosis of gram-negative meningitis. We prospectively screened 1503 CSF specimens with a microassay utilizing 20 μl of CSF added to 20 μl of limulus reagent on an endotoxin-free slide. Specimens and controls were observed for gel formation after 60 minutes incubation at 37°C in a moisture chamber. Serial dilutions of CSF were used to quantitate ELA. The limit of sensitivity of the assay was 0.01 ng/ml. All ELA positive (+) specimens were subjected to confirmatory retesting, after which 49 (3.3%) were ELA (+). Comparison with the 38 available culture results revealed that 33 specimens (87%) were culture (+), that 3 of the 5 culture negative (-) specimens were from patients on therapy for gram-negative meningitis and a fourth was from a neonate. Overall specificity of the test was 99.6% with a (+) predictive value of 86.8%. There was one false (-) specimen giving an overall sensitivity of 97.4% but a (-) predictive value of 99.9%. ELA values > 150 ng/ml correlated with the occurrence of seizures (p 3000 ng/ml with peripheral leukopenia and hypoglycorrhachia (p 5×104 ng/ml with death (p<0.005). We conclude that LAL micromethod gelation test is a sensitive, specific, simple screening test for gram-negative meningitis that exhibits prognostic value.