Low dose of Concanavalin-A enhances innate immune response and prevents liver injury in mice infected with Candida albicans.

The mechanisms through which Candida albicans is recognized by immune cells and how it triggers host defence are not completely understood. In this study, we evaluated the effect of Concanavalin-A on the clearance of C. albicans by infected mice and their production of proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). Subgroups of 5 animals were pretreated with Con-A (250 mug mL(-1) PBS) and after 96 h were infected intraperitoneally with 10(7) cells of C. albicans CR15 (an isolate from a HIV+ person); 30 min, 2, 6, 24 or 72 h after infection the mice were sacrificed. Phagocytosis of C. albicans by peritoneal macrophages increased 30 min after infection in mice pretreated with Con-A. The liver presented the greatest number of CFUs, and this number was reduced by pretreatment with Con-A. Control animals infected with C. albicans presented a significant increase in plasmatic alanine aminotransferase, which was not observed in mice treated with Con-A. Two hours after infection the production of TNF-alpha in the liver of mice pretreated with Con-A was significantly increased. These results suggest that a single dose of Con-A caused a beneficial modulating action of the inflammatory response during infection with C. albicans.

Early membrane exposure of phosphatidylserine followed by late necrosis in murine macrophages induced by Candida albicans from an HIV-infected individual.

The hypothesis that Candida albicans isolate (CR1) from an HIV-infected individual induced apoptosis of macrophages was examined by optical microscopy, binding of annexin V-FITC and analyses of DNA degradation (TUNEL tests and agarose gel electrophoresis). Resident murine peritoneal macrophages co-incubated for 5-15 min with C. albicans CR1 bound annexin V, whereas macrophages incubated with either heat-inactivated strain CR1, C. albicans 577 (isolated from a patient with mucocutaneous candidiasis) or C. albicans FCF14 (a mutant that did not produce proteases and phospholipases) did not bind annexin for up to 2 h of observation. However, macrophages exposed to C. albicans CR1 did not present the pattern of DNA degradation typical of apoptosis. Macrophages became increasingly permeable to propidium iodide from 30 min to 2 h after their exposure to C. albicans CR1. Most of the phagocytosed C. albicans CR1 yeast cells switched to germ-tubes inside the macrophages after incubation for 1-2 h. These results show that macrophages exposed to C. albicans CR1 presented early signs of apoptosis but progressed to necrosis, and suggest that Candida strains that readily switch to germ-tubes inside those apoptotic cells might have a competitive advantage in vivo because released germ-tubes resist further attack by macrophages.

Phagocytic and candidacidal activities of macrophages from suckling and adult mice pretreated with concanavalin-A.

In this study, we investigated the effect of concanavalin-A (Con-A) on the activation of phagocytosis and killing of Candida albicans by peritoneal macrophages from suckling and adult mice. Pretreatment of adult mice with Con-A dose-dependently increased the percentage of macrophages phagocytosing C. albicans in vitro from 3.8 +/- 0.9 to 24.2 +/- 2.4 in the absence of serum opsonins. Addition of mannan (50 microg) and mannose (50 mM) to the incubation medium reduced phagocytosis from 21.5 +/- 1.3 to 4.7 +/- 1.9, suggesting that treatment with Con-A increased phagocytosis mediated by mannose receptors. Killing of C. albicans was also increased by increasing the dose of Con-A. Pretreatment of suckling mice with Con-A increased the macrophages' phagocytic and candidacidal activities by an amount similar to that observed in adult mice. Furthermore, suckling mice pretreated with Con-A survived an intraperitoneal inoculum of 5 x 10(7) C. albicans, whereas all control mice died within 24-48 h of infection. This suggested that increased phagocytosis and killing of C. albicans stimulated by the action of Con-A conferred early protection upon suckling mice experimentally infected with C. albicans.

Effects of medium composition, calcium, iron and oxygen on haemolysin production by Plesiomonas shigelloides isolated from water.

AIMS: The effects of medium composition, calcium, iron and oxygen tension on the haemolytic activity of Plesiomonas shigelloides were investigated. METHODS AND RESULTS: The haemolytic activity of seven strains of Ple. shigelloides was tested on the surface of Luria Agar (LA), Brain Heart Infusion Agar (BHIA) and Trypitic Soy Agar (TSA) containing 5% (v/v) sheep blood, and in the Agar Overlay (AO) assay. All strains produced beta-haemolysis in the AO assay in three media, and on the surface of LA. The kinetics of growth and haemolytic activity of Ple. shigelloides 9P3-1 were evaluated in six different media, and the highest production of haemolysin occurred in Luria Broth (LB). The haemolytic activity of 9P3-1 was stimulated by Ca2+ and inhibited by EDTA. Addition of iron to the culture medium did not affect bacterial growth, although it reduced bacterial haemolytic activity. In the presence of an iron chelator, growth of the 9P3-1 was inhibited, but its haemolytic activity was enhanced. CONCLUSION: The haemolytic activity of Ple. shigelloides depends on medium composition, and that it is regulated by iron and is calcium-dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: These results show the importance of optimization of media composition and oxygen tension for detection of Ple. shigelloides haemolytic activity.

Apoptosis of murine peritoneal macrophages induced by an avian pathogenic strain of Escherichia coli.

The mechanisms used by avian strains of Escherichia coli to invade the respiratory epithelia, leading to septicemia in poultry, are not well-established. In this work, we show that resident murine peritoneal macrophages infected in vitro with an avian strain of E. coli underwent apoptosis 4 h after infection (55.6% of apoptosis in infected cells versus 3.5% in non-infected cells). Heat-inactivated bacteria did not induce apoptosis and the inhibition of phagocytosis by pretreatment of cells with cytochalasin D reduced the number of apoptotic cells from 55.6 to 13.9% (P<0.05), showing that the bacteria must be intracellularly located and viable to induce apoptosis. Therefore, these data suggest that induction of macrophage apoptosis may be a pathogenic mechanism employed by avian E. coli to circumvent the host defences and invade the respiratory epithelia.

Phagocytosis of Candida albicans by concanavalin-A activated peritoneal macrophages.

We evaluated the effect of treatment of mice with concanavalin-A (Con-A) on the phagocytosis of glutaraldehyde-fixed Candida albicans by peritoneal macrophages. The mean number of unopsonized C. albicans blastoconidia phagocytosed in vitro by peritoneal macrophages was doubled (from 1.3+/-0.1 to 2.7+/-0.14) by pre-treatment of the donor mice with Con-A. The percent of peritoneal cells phagocytosing the blastoconidia in vitro was increased about four times (from 22.3+/-8.6 to 80.3+/-3.2) by Con-A. This increase in phagocytosis was about 50% inhibited by addition of mannan (50 microg) plus mannose (50 mM) to the assay medium, suggesting that it was mediated by mannose receptors (MR). Phagocytosis in vitro in the presence of fresh non-immune serum (5%) was also increased, from 84.3+/-5.0 for untreated macrophages to 100% for Con-A activated peritoneal macrophages and the mean number of opsonized C. albicans blastoconidia increased from 2.3+/-0.1 to 4. 6+/-0.1. These results suggest that treatment of mice with Con-A increased both the phagocytosis of C. albicans blastoconidia mediated by mannose receptors and by complement receptors.

Modulation of lectinophagocytosis of Escherichia coli by variation of pH and temperature.

The effect of variation of pH and temperature on the lectinophagocytosis of enteropathogenic Escherichia coli by polymorphonuclear leukocytes and macrophages elicited by thioglycolate medium was evaluated. The phagocytosis of enteropathogenic E. coli is dependent on pH, being maximal at pH 7.0 and reduced at pH 5.5 or 6.0. Mannan and mannose (as representative sugars that bind to phagocyte lectin receptors), are recognized by mannose receptors and reduced the phagocytic index at pH 7.0 (from 41.6 +/- 8.5 to 17.0 +/- 6.1) and at pH 6.0 (from 24.1 +/- 5.1 to 14.5 +/- 5.0), suggesting that mannose receptors, despite their reduced affinity for ligand at pH 6.0, also participate in phagocytosis of enteropathogenic E. coli. The inhibition of phagocytosis by anti-substance A antibody was also examined at pH 7.0 and at pH 6.0, decreasing (from 41.6 +/- 8.5 to 21.1 +/- 3.4) and (from 24.1 +/- 5.1 to 12.0 +/- 3.5), respectively. This antibody reduced the phagocytosis of enteropathogenic E. coli in phagocytic assays at 37 or 41 degrees C. These results suggest that the acidic pH decreased the affinity of mannose receptors to ligands on the surface of E. coli and also affected the binding of lectin from E. coli to N-acetylgalactosamine on phagocytes.

Pre-treatment with Concanavalin-A increases resistance of mice to peritoneal infection by Serratia marcescens.

Mice pre-treated with Concanavalin-A largely survived an intra-peritoneal inoculum of 2 x 10(7) Serratia marcescens, whereas all control mice died within 15 h of inoculation. A subpopulation of peritoneal macrophages from Con-A pre-treated mice was able to phagocytose the bacteria in vitro (6.7 SEM 1.2% phagocytosing cells) and in vivo (16.9 SEM 2.1%), whereas control phagocytes did not phagocytose S. marcescens. The survival of Con-A pre-treated mice allowed their immunisation with living bacteria, and the antiserum thus produced increased the phagocytosis of S. marcescens in vitro. Control mice largely survived an inoculum of S. marcescens suspended in 50% immune serum, although the bacteria were resistant to the bactericidal activity of that serum. These results suggest that, in contrast to the delayed humoral protection afforded by immunisation, phagocytosis by phagocytes activated by Con-A conferred early protection to mice against experimental infection by S. marcescens.

Effect of chemical stimulation of the dorsomedial hypothalamic nucleus on blood plasma glucose, triglycerides and free fatty acids in rats.

The effects of chemical stimulation of the dorsomedial hypothalamic nucleus (DMH) on blood plasma concentration of glucose, triglycerides, insulin, and free fatty acids (FFA) were investigated in anesthetized adult Wistar rats. Microinjection of 12.5 nmol of norepinephrine into the DMH increased blood plasma concentration of glucose and FFA, decreased triglycerides, and did not change plasma insulin within 5 min; after 20 min, blood glucose and FFA reached control values. Microinjection of epinephrine (12.5 nmol) into the DMH also increased blood plasma glucose concentration and decreased triglycerides after 5 min. These effects are probably mediated by beta-adrenergic mechanisms, because they were prevented by beta-adrenergic antagonist propranolol, but not by alpha-adrenergic antagonist prazosin. Microinjection into the DMH of glutamate, dopamine, or acetylcholine failed to cause any change in those metabolic parameters, corroborating the hypothesis that the DMH is part of a beta-adrenergic pathway involved in short-term modulation of the availability of glucose and FFA.

Adherence pili of pathogenic strains of avian Escherichia coli.

Fifty strains of Escherichia coli isolated from colisepticemic chickens in Londrina, Brazil, were examined for presence of gene sequences for pil and pap, hemagglutination, and adherence to chicken tracheal cells. Forty-one strains were pil+ and 22 of these showed mannose sensitive (MS) hemagglutination (MSHA) of guinea-pig erythrocytes, indicating that they possessed only type 1 pili. Seven strains were pap+ and 6 of these caused mannose resistant (MR) hemagglutination (MRHA) of human erythrocytes. Twenty-four strains (17 of which caused MSHA) showed MS-adherence to chicken tracheal cells and the remaining 26 showed MR-adherence. The former typically adhered to the mucus layer whereas the latter usually adhered to the mucosal epithelium. It is concluded that MS adherence to chicken tracheal cells is correlated with expression of type 1 fimbriae and that MR-adherence to chicken tracheal cells cannot always be attributed to P pili.

Cloned genes of the aerobactin system of virulent avian Escherichia coli do not confer virulence to recombinant strains.

1. We cloned the aerobactin region and its receptor from pMV14, a large nonconjugative plasmid isolated from the virulent strain UEL14, to assess the importance of the aerobactin iron uptake system as a virulence determinant in septicemic avian Escherichia coli. 2. The physical map of the region of the recombinant plasmid (pGMV1) containing the genes for synthesis of aerobactin and its receptor was very similar to the corresponding region in pABN1 containing the genetic determinants for the aerobactin system of pColV-K30. 3. The 74-kDa outer-membrane protein encoded by pGMV1 cross-reacted immunologically with the 74-kDa aerobactin receptor protein encoded by pABN1. 4. Various avirulent E. coli strains carrying the recombinant plasmid, which contains only the aerobactin system, were assayed for virulence and were found to be avirulent for chickens. Only the wild-type aerobactin-producing strain was virulent in a pathogenicity test for chickens. 5. These results show that the aerobactin system by itself does not confer virulence, and that other factors are necessary for virulence of avian strains of E. coli.

Cloned genes of the aerobactin system of virulent avian Escherichia coli do not confer virulence to recombinant strains

1. We cloned the aerobactin region and its receptor from pMV14, a large nonconjugative plasmid isolated from the virulent strain UEL14, to assess the importance of the aerobactin iron uptake system as a virulence determinant in septicemic avian Escherichia coli. 2. The physical map of the region of the recombinant plasmid (pGMV1) containing the genes for synthesis of aerobactin and its receptor was very similar to the corresponding region in pABN1 containing the genetic determinants for the aerobactin system of pColV-K30. 3. The 74-kDa outer-membrane protein encoded by pGMV1 cross-reacted immunologically with the 74-kDa aerobactin receptor protein encoded by pABN1. 4. Various avirulent E. coli strains carrying the recombinant plasmid, which contains only the aerobactin system, were assayed for virulence and were found to be avirulent for chickens. Only the wild-type aerobactin-producing strain was virulent in a pathogenicity test for chickens. 5. These results show that the aerobactin system by itself does not confer virulence, and that other factors are necessary for virulence of avian strains of E. coli

Spectrophotometric determination of total proteins in blood plasma with p-benzoquinone.

1. In the present study we have documented the use of the reagent, p-benzoquinone (PBQ) for the spectrophotometric determination of total proteins in blood plasma. 2. Since the products of reaction are stable for several hours at room temperature after the 20-min boiling step, the time at which absorbance is measured is not a critical factor. 3. Common anticoagulants such as EDTA, citrate, or heparin do not interfere with the PBQ method at concentrations used in clinical laboratories. 4. The products of the reaction between PBQ and either plasma (specific absorbance 2.33 x 10(-3) +/- 0.20 x 10(-3) micrograms cm-2) or purified proteins (specific absorbance 2.61 x 10(-3) +/- 0.31 x 10(-3) micrograms cm-2) show an absorption band at 350 nm, which follows Beer's law, and therefore can be used for analytical purposes. 5. The PBQ method has a lower limit of detection (4 micrograms/ml) than that of the biuret method (45 micrograms/ml) for a final reaction mixture of 5.0 and 4.2 ml, respectively.

Phagocytosis of enteropathogenic Escherichia coli and Candida albicans by lectin-like receptors.

1. Ingestion of enteropathogenic Escherichia coli or Candida albicans by thioglycollate-elicited macrophages and polymorphonuclear leukocytes was investigated in vitro, 2. Goat antiserum against mannose receptors caused about 50% inhibition of E. coli phagocytosis and about 90% inhibition of C. albicans phagocytosis. 3. E. coli and C. albicans uptake was inhibited by about 60% and 98%, respectively, by plating the macrophages onto substrates coated with poly-L-lysine-mannan. Further addition of 50 mM mannose to the medium significantly increased the inhibition of phagocytosis of E. coli by macrophages from 60.7 +/- 1.5 to 79.8 +/- 13.1 and by polymorphonuclear cells from 58.9 +/- 3.7 to 88.7 +/- 4.9. 4. Preincubation of phagocytic cells with antiserum against substance A of human erythrocytes reduced E. coli ingestion by 95%, but this inhibition was not observed when the antiserum was incubated with N-acetylgalactosamine (50 mM) before being added to the phagocytes. The phagocytosis of C. albicans was not inhibited by anti-substance A antiserum. 5. The phagocytosis of E. coli was inhibited by about 25% by the addition of 7.8 micrograms/ml soluble mannan to the medium, and by about 50% by the addition of 50 mMN-acetylgalactosamine; when both substances were added to the medium, an additive inhibition of about 75% was observed. 6. These results indicate that mannose receptors on the surface of phagocytic cells mediate E. coli or Candida albicans uptake and that the binding of bacteria to N-acetylgalactosamine residues from the membrane of phagocytes is also involved in the phagocytosis of E. coli.

Spectrophotometric determination of total proteins in blood plasma with p-benzoquinone

In the present study we have documented the use of the reagent p-benzoquinone (PBQ) for the spectrophotometric determination of total protein in blood plasma. Since the products of reaction are stable for several hours at room temperature after the 20-min boiling step, the time at which absorbance is measured is not a critical factor. Common anticoagulants such as EDTYA, citrate, or heparin do not interfere with the PBQ method at concentrations used in clinical laboratories. The products of the reaction between PBQ and either plasma (specific absorbance 2.33 x 10-3 ñ 0.20 x 10-3 ug cm -2) or purified proteins (specific absorbance 2.61 x 10-3 ñ 0.31 x 10-3 ug cm-2) show an absorption band at 350 nm, which follows Beer's law, and therefore can be used for analytical purposes. The PBQ method has a lower limit of detection (4 ug/ml) than that of biuret method (45 yg/ml) for a final reaction mixture of 5.0 and 4.2 ml, respectively

Phagocytosis of enteropathogenic Escherichia coli and Candida albicans by lectin-like receptors

1. Ingestion of enteropathogenic Escherichia coli or Candida albicans by thioglycollate-elicited macrophages and polymorphonuclear leukocytes was investigated in vitro, 2. Goat antiserum against mannose receptors caused about 50% inhibition of E. coli phagocytosis and about 90% inhibition of C. albicans phagocytosis. 3. E. coli and C. albicans uptake was inhibited by about 60% and 98%, respectively, by plating the macrophages onto substrates coated with poly-L-lysine-mannan. Further addition of 50 mM mannose to the medium significantly increased the inhibition of phagocytosis of E. coli by macrophages from 60.7 +/- 1.5 to 79.8 +/- 13.1 and by polymorphonuclear cells from 58.9 +/- 3.7 to 88.7 +/- 4.9. 4. Preincubation of phagocytic cells with antiserum against substance A of human erythrocytes reduced E. coli ingestion by 95%, but this inhibition was not observed when the antiserum was incubated with N-acetylgalactosamine (50 mM) before being added to the phagocytes. The phagocytosis of C. albicans was not inhibited by anti-substance A antiserum. 5. The phagocytosis of E. coli was inhibited by about 25% by the addition of 7.8 micrograms/ml soluble mannan to the medium, and by about 50% by the addition of 50 mMN-acetylgalactosamine; when both substances were added to the medium, an additive inhibition of about 75% was observed. 6. These results indicate that mannose receptors on the surface of phagocytic cells mediate E. coli or Candida albicans uptake and that the binding of bacteria to N-acetylgalactosamine residues from the membrane of phagocytes is also involved in the phagocytosis of E. coli