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PubMed2005

Functional and morphological changes of the gut barrier during the restitution process after hemorrhagic shock.

AIM: To investigate the functional, morphological changes of the gut barrier during the restitution process after hemorrhagic shock, and the regional differences of the large intestine and small intestine in response to ischemia/reperfusion injury. METHODS: Forty-seven Sprague-Dawley rats with body weight of 250-300 g were divided into two groups: control group (sham shock n = 5) and experimental group (n = 42). Experimental group was further divided into six groups (n = 7 each) according to different time points after the hemorrhagic shock, including 0(th) h group, 1st h group, 3rd h group, 6th h group, 12th h group and 24th h group. All the rats were gavaged with 2 mL of suspension of lactulose (L) (100 mg/2 mL) and mannitol (M) (50 mg/each) at the beginning and then an experimental rat model of hemorrhagic shock was set up. The specimens from jejunum, ileum and colon tissues and the blood samples from the portal vein were taken at 0, 1, 3, 6, 12 and 24 h after shock resuscitation, respectively. The morphological changes of the intestinal mucosa, including the histology of intestinal mucosa, the thickness of mucosa, the height of villi, the index of mucosal damage and the numbers of goblet cells, were determined by light microscope and/or electron microscope. The concentrations of the bacterial endotoxin lipopolysaccharides (LPS) from the portal vein blood, which reflected the gut barrier function, were examined by using Limulus test. At the same time point, to evaluate intestinal permeability, all urine was collected and the concentrations of the metabolically inactive markers such as L and M in urine were measured by using GC-9A gas chromatographic instrument. RESULTS: After the hemorrhagic shock, the mucosal epithelial injury was obvious in small intestine even at the 0(th) h, and it became more serious at the 1st and the 3rd h. The tissue restitution was also found after 3 h, though the injury was still serious. Most of the injured mucosal restitution was established after 6 h and completed in 24 h. Two distinct models of cell death-apoptosis and necrosis-were involved in the destruction of rat intestinal epithelial cells. The number of goblet cells on intestinal mucosa was reduced significantly from 0 to 24 h (the number from 243+/-13 to 157+/-9 for ileum, 310+/-19 to 248+/-18 for colon; r = -0.910 and -0.437 respectively, all P<0.001), which was the same with the large intestine, but the grade of injury was lighter with the values of mucosal damage index in 3 h for jejunum, ileum, and colon being 2.8, 2.6, 1.2, respectively. The mucosal thickness and the height of villi in jejunum and ileum diminished in 1 h (the average height decreased from 309+/-24 to 204+/-23 microm and 271+/-31 to 231+/-28 microm, r = -0.758 and -0.659, all P<0.001; the thickness from 547+/-23 to 418+/-28 microm and 483+/-45 to 364+/-35 microm, r = -0.898 and -0.829, all P<0.001), but there was no statistical difference in the colon (F = 0.296, P = 0.934). Compared with control group, the urine L/M ratio and the blood LPS concentration in the experimental groups raised significantly, reaching the peak in 3-6 h (L/M: control vs 3 h vs 6 h was 0.029+/-0.09 vs 0.063+/-0.012 vs 0.078+/-0.021, r = -0.786, P<0.001; LPS: control vs 3 h vs 6 h was 0.09+/-0.021 vs 0.063+/-0.012 vs 0.25+/-0.023, r = -0.623, P<0.001), and it kept increasing in 24 h. CONCLUSION: The gut barrier of the rats was seriously damaged at the early phase of ischemic reperfusion injury after hemorrhagic shock, which included the injury and atrophy in intestinal mucosa and the increasing of intestinal permeability. Simultaneously, the intestinal mucosa also showed its great repairing potentiality, such as the improvement of the intestinal permeability and the recovery of the morphology at different phases after ischemic reperfusion injury. The restitution of gut barrier function was obviously slower than that of the morphology and there was no direct correlation between them. Compared with the small intestine, the large intestine had stronger potentiality against injury. The reduction of the amount of intestinal goblet cells by injury did not influence the ability of intestinal mucosal restitution at a certain extent and it appeared to be intimately involved in the restitution of the epithelium.

Animals↗
PubMed1998

[The Clinical Significance of Plasma]

(1 --> 3)- beta - D -Glucan ( beta -glucan), a fungal cell wall component existing in plasma was measured by the kinetic turbidimetric limulus test. Here 3 reported cases have proven the clinical usefulness of plasma beta -glucan. Case 1: A 46-year-old female with non-Hodgkin's lymphoma was admitted for severe hepatitis type B in June 1997. During hospitalization, the fever rose with no response to antibiotics and with a negative blood culture. Although the plasma beta -glucan concentration was high, the patient was administered with antimycotics (fluconazole; FLCZ) and she showed some signs of improvement. Case 2: A 52-year-old male with jaundice was hospitalized in November 1997. The third day following the operation on the pancreas head carcinoma, the body temperature rose higher with a negative blood culture. The fourth day, the plasma beta -glucan concentration was positive with negative endotoxin. As some improvements were obserbed from taking FLCZ, he was discharged in January 1998. Case 3: A 19-year-old male with epilepsy was hospitalized for ARDS in August 1997. A butterfly-like shadow was observed in the chest roentgenogram (suspected malignant lymphoma). The high titer of beta -glucan has continued and endotoxin was detected. The symptoms showed some signs of improvement and the titer of beta -glucan reduced with FLCZ. Although a high level of beta -glucan still remained, the patient was discharged, but has to under go regular follow-up examinations. The measurment of beta -glucan proved very useful not only as a diagnosis for the screening of deep mycosis but also as monitoring for therapies.

Journal Article↗
PubMed1975

Properties of free endotoxin from Pasteurella multocida.

Free endotoxin (FET) from virulent encapsulated Pasteurella multocida or from an avirulent nonencapsulated mutant is capable of inducing active immunity, but the lipopolysaccharide (LPS) moiety of the endotoxin is not. These results suggest that a protein of P multocida is involved in the stimulation of active immunity. The serologic specificity of the FET is associated with the LPS moiety, which is related to a heat extracted antigen that is used for serotyping P multocida. The FET is capable of producing widespread vascular alteration and death. It is present in the vascular system of turkeys with acute fowl cholera, and it can be detected with the "Limulus" test for endotoxins and with the gel diffusion precipitin test.

Animals↗
PubMed2000

Occupational biohazards in agricultural dusts from India.

Sixteen samples of settled dusts deposited during handling of various granular plant materials (green gram, red gram, amaranth, rice, pearl millet, sorghum, wheat, maize) in small food storing and processing facilities (godowns) were collected in the region of Aurangabad (Southern India). The samples were examined by the dilution plating method for the concentration and species composition of Gram-positive mesophilic bacteria, Gram-negative mesophilic bacteria, thermophilic actinomycetes and fungi. They were also examined by Limulus test for the concentration of bacterial endotoxin. The total concentration of microorganisms (bacteria + fungi) in examined samples varied within a wide range of 1.4 x 10(5) - 8.45 x 10(8) cfu/g (median 8.36 x 10(6) cfu/g). On average, the most common were Gram-positive bacteria (87.84% of all isolates) followed by Gram-negative bacteria (11.12%). Less common were fungi (1.24%) and thermophilic actinomycetes (0.01%). Among isolated bacteria and fungi, there were many species known as causative agents of allergic alveolitis, asthma and organic dust toxic syndrome. The concentration of bacterial endotoxin in the examined samples ranged between 12.5 - 62500 microg/g (median 781.25 microg/g), being particularly large in the samples of dust from maize (6250 microg/g and 62500 microg/g) and pearl millet (6250 microg/g and 12500 microg/g). The results of the present work indicate that the agricultural dusts from India represent a potential hazard for the workers because of high concentrations of allergenic microorganisms and bacterial endotoxin. The particular risk is associated with handing of maize and pearl millet. Further studies on this subject with the use of aerobiological methods are highly desirable

Agricultural Workers' Diseases↗
PubMed2002

Abnormal accumulation in lipopolysaccharide in biliary epithelial cells of rats with self-filling blind loop.

Primary sclerosing cholangitis (PSC) is known to be frequently associated with inflammatory bowel diseases. In a rat with self-filling blind loop (SFBL), a proposed animal model for PSC, hepatobiliary inflammation has previously been demonstrated. In this study, we assessed the involvement of lipopolysaccharide (LPS), a bacterial endotoxin, in the pathogenesis of hepatobiliary inflammation of the SFBL model. The hepatic localization of LPS was examined by immunohistochemistry using an anti-lipid A antibody. The portal blood concentration of LPS was measured by an endotoxin-specific chromogenic Limulus test (Endospecy test). LPS was localized in the biliary epithelial cells (BECs) of rats with SFBL, and the portal blood concentration of LPS was significantly higher than that of sham-operated rats. Development of hepatobiliary inflammation, peribiliary fibrosis, and injury to the intestinal mucosa were histologically confirmed. Constriction in the biliary trees was radiologically demonstrated. These findings suggested that abnormal accumulation of LPS, which may be derived from portal blood, in BECs was involved in the pathogenesis of hepatobiliary inflammation with intestinal injury.

Animals↗
PubMed1975

[Enterobacteria and endotoxins in abdominal surgical pathology. Critical review and clinico-experimental studies].

A detailed critical review on the role of endotoxins from gram-negative bacteria in the pathogenesis of most of the intestinal experimental models of surgical interest is presented. Data of an investigation on 10 cases of acute appendicitis and 2 cases of intestinal occlusion associated with toxaemia are then presented: in all these cases the Limulus test significatively revealed the presence of endotoxin in the blood and in the peritoneal fluid, also in absence of bacteria in the blood. The implications of these results and the use of the test in clinical practice are discussed.

Animals↗
PubMed2002

[Determination of lipopolysaccharide by capillary zone electrophoresis].

OBJECTIVE: To detect lipopolysaccharides (LPS) in frangibility bacilli, control the procedure of LPS preparation, examine the ally of the product by high performance capillary zone electrophoresis (CZE), and obtain rapidly high alloy of LPS for scientific research. METHODS: Theoptyline was added to the sample as an internal standard. The LPS was separated and detected by CZE in sodium tetraborate-borate buffer. The concentration of the buffer was 30 mmol.L-1 (pH 8.0); the diameter of the capillary column was 75 microns, and the length was 57 cm; the CZE operative voltage was 25 kV, the electric current was 112 microA, and the detecting wavelength was 230 nm. RESULTS: CZE was first internally used to separate and detecte LPS in bacillus. The LPS had a good linearity in the range of 2-30 mg.L-1 (r = 0.996). The average recovery rate was 99%-101%, the in-day and day-to-day coefficients of variation (CV) were less than 5%, and the minimum detectable quantity was 1.0 mg.L-1. CONCLUSION: Compared with the limulus test (LT) and gel electrophoresis, this method can resist heat source interference and is more simple, rapid and accurate. Moreover, it can control the preparative process of LPS immediately, and may be an advanced method to determine LPS.

Electrophoresis, Capillary↗
PubMed2000

[Preliminary screen of anti-endotoxin activity of Chinese materia medica].

OBJECTIVE: To screen Chinese materia medica against E. coli. O111B4 endotoxin. METHOD: 70% alcohol extracts from 134 kinds of heat-clearing Chinese materia medica were demonstrated by using limulus test in vitro. The extracts which had anti-endotoxin effects were diluted to demonstrate their minimal anti-endotoxin concertnations. RESULTS: 75 kinds of Chinese materia medica possessed anti-endotoxin activity in various degrees in vitro. CONCLUSION: Most of heat-clearing Chinese materia medica possess anti-endotoxin effects in vitro.

Agglutination↗
PubMed2001

[Relationship between endotoxemia and APACHE-III scoring and integrated therapy of Chinese and Western medicine].

OBJECTIVE: To explore the relationship between endotoxemia and the severity of disease condition and therapeutic effect. METHODS: According to randomized controlled principle, the 153 acute infectious patients were divided into simple antibiotics treated group and antibiotics plus Chinese drugs combined treated group, and patients in each group were subdivided into 3 types according to acute physiology and chronic health evaluation (APACHE-III) scoring: type A (APACHE-III scoring < or = 20 points), type B (APACHE-III scoring 21-40 points) and type C (APACHE-III scoring > 40 points). The 77 cases in the simple treated group were 40 males and 37 females, aging 18-76 years, mean 46.5 +/- 27.5 years, 41 cases of type A, 28 of type B and 8 of type C, treatment course 10-14 days, mean 11.5 +/- 2.5 days. The 76 cases in the combined treated group were 39 males and 37 females, aging 18-70 years, mean 44.5 +/- 25.5 years, 37 of type A, 30 of type B and 9 of type C, treatment course 10-14 days, mean 10.5 +/- 2.5 days. Limulus test was used to determine the endotoxin content in peripheral blood of patients, and further analysis on the relationship between endotoxemia and APACHE-III scoring was conducted. RESULTS: Acute severe infectious patients whose APACHE-III scoring > 20 points occurred endotoxemia (P < 0.05), and the condition of disease was positively related to the APACHE-III scoring (r = 0.718, P < 0.05). Chinese drugs plus antibiotics can obviously alleviate endotoxemia (P < 0.05) and improve the prognosis of patients. CONCLUSION: Endotoxemia can serve as a referential parameter for predicting the severity of disease. Integrated therapy of Chinese and western medicine in treating bacterial infection revealed better results than that of antibiotics solely.

APACHE↗
PubMed1992

[Percutaneous transhepatic intraportal administration of amphotericin B to a patient with multiple liver abscesses due to Candida albicans: with monitoring by fungal index as a parameter of Candida volume].

A 59-year-old female with acute promyelocytic leukemia in remission was admitted to our center because of an episode of incidental high fever with general fatigue. She was found to have hepatomegaly. Abdominal CT revealed multiple liver abscesses and a positive culture was obtained for candida albicans from an aspirated abscess. She was treated with percutaneous transhepatic intraportal administration of amphotericin B in addition to oral and intravenous administration. We confirmed the remission of these abscesses by means of the fungal index which is the difference between the values of the limulus test and endotoxin specific test. The fungal index appears to be useful for early diagnosis and treatment of fungal infection.

Amphotericin B↗
PubMed2005

[Experimental study on the antagonistic activity of cationic multi-peptide mastoparan-1 against lipopolysaccharide].

OBJECTIVE: To explore the mechanism of cationic multi-peptide mastoparan-1 (MP-1) on the protection of mice from lipopolysaccharide (LPS) challenge. METHODS: Thirty Kunming mice were divided randomly into MP-1, injury, protection groups with 10 mice in each group. The mice in MP-1 group were injected with 3 mg/kg MP-1 by tail vein, while those in injury group were injected with 20 mg/kg LPS by tail vein, and those in protection group 3 mg/kg MP-1 within 20 seconds after 20 mg/kg LPS injection were injected. The effects of MP-1 on the protection of mice from LPS challenge were observed. In vitro, the affinity of MP-1 and PMB to LPS was compared by biosensor and FAST fit construct and expressed as Kd. And the neutralizing activity of MP-1 and PMB in dose of 5, 10, 20, 40 micromol/L on LPS (2 microg/L) was detected by dynamic turbidimetric limulus test with LPS neutralizing 0 micromol/L MP-1 and PMB as control. The mRNA expression levels of TLR4, TNF-alpha and IL-6 in murine peritoneal macrophages (PM phi) after exposure to LPS (100 ng/ml) were assayed by RT-PCR. RESULTS: MP-1 could significantly protect mice from LPS challenging with protection rate of 90%. In vitro, MP-1 had a high affinity (Kd value: 484.0 nmol/L) and neutralizing ability with LPS, but it was lower than that of PMB (Kd value: 18.9 nmol/L). The neutralizing effect of 20 and 40 micromol/L MP-1 was obviously stronger than that in 0 micromol/L (P < 0.01). MP could obviously inhibit the expression of TLR4, TNF-alpha and IL-6 mRNA in LPS-stimulated murine PM phi. CONCLUSION: MP-1 can evidently protect mice from lethal LPS challenge, and the mechanism might be related to the activity of MP-1 which binding and neutralizing LPS, blocking the combination LPS with its receptors. So the murine macrophage activation induced by LPS was inhibited.

Animals↗
PubMed1990

Ultrafiltration to remove endotoxins and other cytokine-inducing materials from tissue culture media and parenteral fluids.

The presence of small amounts of endotoxins are often undesirable when investigating cytokines such as interleukin-1 and tumor necrosis factor alpha. Polymyxin B, widely used to block endotoxins, does not block several forms of endotoxins, and at high concentrations, polymyxin B itself stimulates interleukin-1 production. Human peripheral blood mononuclear cells are highly sensitive to endotoxins; they respond with cytokine production to endotoxins at concentrations of 10-50 pg/ml and detect pyrogenic materials nonreactive in the Limulus test. In the present study, ultrafiltration using polysulfone filters was found to remove all interleukin-1- and tumor necrosis factor alpha-inducing substances produced in E. coli cultures. Interleukin-1- and tumor necrosis factor alpha-inducing substances derived from Pseudomonas aeruginosa cultures were also rejected by the filters. Ultrafiltration is therefore a convenient and effective procedure to remove interleukin-1 and tumor necrosis factor alpha-inducing substances from parenteral fluids and solutions that come in contact with blood such as fluids used in hemodialysis. This technique is also applicable for the large-scale production of culture media for mammalian cell expression of recombinant, pyrogen-free proteins intended for use in humans.

Biological Factors↗
PubMed1990

[Rapid diagnosis in microbiology, present and future (lecture)].

The authors analyze present-day rapid laboratory methods that are helpful in the diagnosis of various critical states in infectious diseases. Currently the immunologic and other than immunologic methods for visual detection of corpuscular antigens, soluble antigens in clinical material are the best rapid methods easy to perform. Methods with DNA probes are a promising alternative of these methods.

Bacterial Infections↗
PubMed1991

[Studying the levels of endotoxemia in meningococcal sepsis. Its relations to pregnancy and antibiotic treatment].

The most frequent cause of toxic shock in our area is meningococcal sepsis. It is currently assumed that endotoxin produce by this bacteria, a lipopolysaccharide with toxic properties, is able to trigger shock and DIC by stimulating both arachidonic acid pathways, among other actions. Previous studies in our laboratory demonstrated significant differences (p +/- 0.001) in the amounts of endotoxins released in vitro by strains from patients and healthy carriers and statistically related criteria of severity with mortality in 256 patients in our center over the last 10 years. In the present study we attempted to establish whether plasma levels of endotoxin were correlated with the severity of the disease. We studied 32 patients with meningococcal sepsis, dividing the subjects into two groups: those in whom six or more criteria of severity were present, and those in whom less than six criteria were found. Blood levels of endotoxin were determined upon admission and after the administration of antibiotics (penicillin and chloramphenicol) using the limulus test with a chromogenic substrate (Coatest, Endotoxin, Kabivitrum, Sweden). Levels of endotoxins were significantly higher in patients with more than six criteria of severity both upon admission (0.6 +/- 0.03) ng/ml) and 4 h. afterward (0.74 +/- 0.006 ng/ml) in comparison to children in whom the clinical picture was less serious (0.27 +/- 0.18 ng/ml and 0.27 +/- 0.18 ng/ml and 0.27 +/- 0.16 ng/ml7 t = 5.8 y t = 5.6 respectively. Endotoxin levels were highest in patients presenting shock, disseminated intravascular coagulation in the hypocoagulability phase and more than 8 criteria.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-Bacterial Agents↗
PubMed1979

[Fever and liver cirrhosis].

Occurrence of fever in a patient with liver cirrhosis should suggest the following: 1. Endotoxemia. Endotoxins are normally present in portal blood; in hepatic cirrhosis they are insufficiently cleared by the liver and their presence can be demonstrated in the systemic circulation by the "limulus test". Fever is one of the many consequences ascribed to the presence of endotoxins in the blood. 2. Infections. Cirrhosis and alcoholism (which often accompanies it) impair host defenses against bacteria and other organisms. Thus, infections are actually more frequent in hepatic cirrhosis as is shown by the example of bacterial endocarditis. Spontaneous bacterial peritonitis must be searched for carefully when ascites is present. 3. Alcoholic hepatitis. This diagnosis is established histologically. The usual symptoms, occurring with variable incidence, include anorexia, nausea and vomiting, abdominal pain, fever and jaundice in the presence of hepatomegaly, leukocytosis and an elevated SGOT. Differential diagnosis from obstructive jaundice and a severe prognosis without alcohol abstinence make early diagnosis mandatory. Its evolution in cirrhosis can be astonishingly rapid. In the absence of hepatic encephalopathy, corticosteroids do not appear to be recommended. 4. Hepatoma.

Ascites↗
PubMed1989

[Infection as a cause of multiple organ failure. Definition, pathophysiology and diagnostic parameters].

DEFINITION: Several clinical observations support the hypothesis that bacterial sepsis is the main aetiological factor in at least half of the patients developing multiple organ failure. Sepsis is defined as the pathophysiological alterations and life-threatening clinical consequences of the action of microorganisms or their products invading the blood stream from a focus of infection. The clinical course of sepsis is highlighted by initial multiorgan insufficiency progressing to severe multiple system organ failure. PATHOPHYSIOLOGY: Pathogenetic bacteria and bacterial toxins arise from the septic focus, overcome the defence mechanisms of the body, continuously invade the blood stream, activate the biological cascade systems and initiate release of mediators from blood and tissue cells. Endotoxin and activated mediators cause endothelial and organ cell dysfunction and cell damage by at least three mechanisms: maldistribution of blood flow; cytotoxia; direct inhibition of oxygen-utilising cell enzymes. CLINICAL PICTURE: The septic disease begins with unspecific signs caused by invasion of bacteria, followed by alterations of the circulatory system, the blood clotting system, the metabolism, initiating vital organ dysfunctions and finally acute respiratory, renal, gastrointestinal, hepatic failure and septic encephalopathy. DIAGNOSIS: The clinical diagnosis of sepsis is based on the finding of an obvious septic focus with the presence of at least four of the following 5 criteria: (I) fever above 38.8 degrees C or hypothermia below 35.5 degrees C; (II) tachypnoea (greater than 24/min) or hypocapnia (PaCO2 less than 32 mmHg); (III) tachycardia (greater than 100 Bpm), (IV) leucocytosis (greater than or equal to 15.000/mm3) or leucopenia (greater than 5.000/mm3); (V) presence of at least one indicator for inadequate organ perfusion like mental alterations, hypoxaemia (PaCO2 less than 75 mmHg while breathing room air), hyperlactataemia (greater than 1,6 mmol/l), diuresis below 30 ml/h, drop in systolic blood pressure below 100 mmHg. A positive blood culture or a positive limulus test are frequent but are not considered to be obligatory for the diagnosis of sepsis.

Bacterial Infections↗
PubMed1987

Bile salts, endotoxin and renal function in obstructive jaundice.

Surgical treatment for the relief of obstructive jaundice is still complicated by postoperative acute renal failure in almost 10 per cent of patients. Renal failure in the patient with jaundice is associated with the presence of bacterial endotoxin in the peripheral blood, and enteric endotoxin absorption is facilitated by the absence of bile salts from the intestine. Oral replacement of bile salts should prevent endotoxemia and renal failure. Forty-six patients with jaundice were studied. Twelve patients received sodium deoxycholate preoperatively, 12 received chenodeoxycholic acid and 22 acted as controls. Endotoxemia was measured by the limulus test and renal function assessed by 24 hour creatinine clearance. No patient given deoxycholate preoperatively had systemic endotoxemia or postoperative impairment of renal function. Endotoxemia was reduced in the chenodeoxycholic acid group, but not significantly, and renal function was not protected. Sodium deoxycholate is undergoing further evaluation in a multicenter randomized prospective study.

Acute Kidney Injury↗
PubMed1977

[Microbiological diagnosis of septicemia (author's transl)].

To detect microorganisms in the blood it is necessary not only that the microbiologist uses reliable methods, but also that the clinician takes a sufficient number of blood samples at the right point in time using a correct method for drawing the blood. The best results are obtained if the blood sample is transferred to the culture media at the bedside. The media should contain anticoagulants, osmosis stabilizers and preparations to neutralize the microbial action of the blood (caused by intrinsic and extrinsic factors). Up until now sodium polyanetholsulfonate ("Liquoid") has proved to be the most suitable additive. The procedure used for blood culturing must enable growth of aerobes, anaerobes and microbes with cell-wall damage. Today, modern methods such as radiometry, impedance measurement and microcalorimetry are used or are in the process of being developed which facilitate screening for positive cultures. Antigens, cell-wall constituents and metabolites of bacteria and fungi present in the blood stream can be detected by means of counter-immunoelectrophoresis, the Limulus test and gas chromatography, without culturing being necessary. Concentration techniques such as filtration and centrifugation are also being refined to enable a more reliable and earlier detection of septicemia.

Adult↗