In vitro susceptibility of filamentous fungi to itraconazole, voriconazole and posaconazole by Clinical and Laboratory Standards Institute reference method and E-test.

The use of anti-fungal agents has increased dramatically in recent years and new drugs have been developed. Several methods are available for determinations of their specific biological activities, i.e. the standard method for minimum inhibitory concentration-determination is described in M-38 [Clinical and Laboratory Standards Institute document M-38 (CLSI M-38)]. However, alternative methods, such as the E-test, are currently available in Mycology laboratories. The susceptibilities of clinical isolates of Aspergillus spp. (n = 29), Fusarium spp. (n = 5), zygomycetes (n = 21) and Schizophyllum (n = 1) were determined for itraconazole, voriconazole and posaconazole, using the CLSI M-38-A broth dilution method and also by the E-test. A good overall agreement (83.7%) between the two methods for all drugs and organisms was observed. Analyses of voriconazole showed a better agreement (93%) between the methods than posaconazole and itraconazole (85% and 74% respectively). Aspergillus spp. were the most susceptible fungi to the anti-fungal agents tested in this study. Posaconazole was the most active drug against filamentous fungi in vitro, followed by itraconazole and voriconazole. The latter (voriconazole) demonstrated no significant in vitro activity against zygomycetes.

Structure-microbicidal activity relationship of synthetic fragments derived from the antibacterial alpha-helix of human lactoferrin.

There is a need for new microbicidal agents with therapeutic potential due to antibiotic resistance in bacteria and fungi. In this study, the structure-microbicidal activity relationship of amino acid residues 14 to 31 (sequence 14-31) from the N-terminal end, corresponding to the antibacterial alpha-helix of human lactoferrin (LF), was investigated by downsizing, alanine scanning, and substitution of amino acids. Microbicidal analysis (99% killing) was performed by a microplate assay using Escherichia coli, Staphylococcus aureus, and Candida albicans as test organisms. Starting from the N-terminal end, downsizing of peptide sequence 14-31 showed that the peptide sequence 19-31 (KCFQWQRNMRKVR, HL9) was the optimal length for antimicrobial activity. Furthermore, HL9 bound to lipid A/lipopolysaccharide, as shown by neutralizing endotoxic activity in a Limulus assay. Alanine scanning of peptide sequence 20-31 showed that Cys20, Trp23, Arg28, Lys29, or Arg31 was important for expressing full killing activity, particularly against C. albicans. Substituting the neutral hydrophilic amino acids Gln24 and Asn26 for Lys and Ala (HLopt2), respectively, enhanced microbicidal activity significantly against all test organisms compared to the amino acids natural counterpart, also, in comparison with HL9, HLopt2 had more than 10-fold-stronger fungicidal activity. Furthermore, HLopt2 was less affected by metallic salts than HL9. The microbicidal activity of HLopt2 was slightly reduced only at pH 7.0, as tested in the pH range of 4.5 to 7.5. The results showed that the microbicidal activity of synthetic peptide sequences, based on the antimicrobial alpha-helix region of LF, can be significantly enhanced by optimizing the length and substitution of neutral amino acids at specific positions, thus suggesting a sequence lead with therapeutic potential.

Bacterial vaginosis--a microbiological and immunological enigma.

The development of bacterial vaginosis (BV) among women of childbearing age and the resulting quantitative and qualitative shift from normally occurring lactobacilli in the vagina to a mixture of mainly anaerobic bacteria is a microbiological and immunological enigma that so far has precluded the formulation of a unifying generally accepted theory on the aetiology and clinical course of BV. This critical review highlights some of the more important aspects of BV research that could help in formulating new basic ideas respecting the biology of BV, not least the importance of the interleukin mediators of local inflammatory responses and the bacterial shift from the normally occurring lactobacilli species: L. crispatus, L. gasseri, L. jensenii, and L. iners to a mixed flora dominated by anaerobic bacteria.

The role of the Lps gene in experimental ulcerative colitis in mice.

The effects of the Lps gene on the development of experimental ulcerative colitis were studied in two genetically different mouse strains: C57B1 and C3H. Acute colitis was induced by adding 3% dextran sulfate sodium (DSS) to the drinking water for a 7-day (C57B1 and C3H) or a 10-day (C57B1) experimental period. Although the DSS treatment initiated the same type of morphological changes in the colon in all groups of mice, an earlier onset and persistent intestinal bleeding occurred in the Lpsn mice (sensitive to lipopolysaccharide, LPS) in comparison with the Lpsd mice (hyporesponsive to LPS). Rectal bleeding appeared on day 7 in 90% of the Lpsn compared to 13% of the Lpsd mice (p < 0.0001). In C57B1 mice, followed for three additional days, 50% of the Lpsn mice died and the surviving animals showed as well as rectal bleeding a large number of Gram-negative bacteria in the liver and spleen. In contrast, the Lpsd mice of the C57B1 strain appeared unaffected by the treatment, although a transient rectal bleeding occurred in 90% on day 8. Also, significantly fewer Gram-negative bacteria were found in the liver and spleen. Even though significantly increased serum endotoxin levels were seen in all DSS-treated groups compared to controls on day 7, the serum levels of TNF alpha were significantly increased only in the Lpsn mice. In DSS-induced colitis the Lpsn genotype conferred on the mice an increased LPS susceptibility, resulting in an augmentation of the inflammatory response to Gram-negative bacteria and their endotoxins. The results suggest that LPS-induced host effector mechanisms significantly enhanced the intestinal bleeding, systemic inflammatory response, and mortality in mice with DSS-induced colitis. In addition, the host defense against the invading and systemically spread bacteria most probably involved additional genes.

The mammary gland-infant intestine immunologic dyad.

The human infant has a very small immune system and needs the support of the mother with the transplacentally arrived IgG antibodies to protect tissues with inflammatogenic and energy-consuming defense. The mucous membranes, where most infections occur, need support via the specialized secretory IgA antibodies and the many other mucosal defense mechanisms provided via the mother's milk. This defense is not inflammatogenic and energy-consuming. We learn about additional defense factors in the milk, like the anti-secretory factor, which seems to protect against diarrhoea. The milk contains numerous growth factors and cytokines, like leptin, which may promote the development of the intestine as well as the immune system. Results are appearing giving interesting evidence for enhanced protection against infection also after the termination of breastfeeding. This may occur via the priming of the infant's immune system after uptake of anti-idiotypic antibodies and lymphocytes from the milk. A breastfeeding motivation study in a large Pakistani village resulted in a 50% decrease of diarrhoea and infant mortality. Deep interviews with the mothers and the traditional birth attendants suggested that even better results may be obtained.

[Increased procalcitonin level in bacteriogenic metabolic disturbances. A new possibility for diagnosis and treatment monitoring in sepsis]. / Prokalcitoninökning vid bakteriellt betingade metabola störningar. Ny möjlighet vid diagnostik och behandlingsuppföljning av sepsis.

Earlier observations of increased plasma concentrations of immunoreactive calcitonin (32 amino acids) in sepsis and other non-tumorous conditions may be explained by increased secretion of procalcitonin, the 116-amino acid prohormone. At present, the site(s) of origin of procalcitonin in sepsis, the factors regulating its biosynthesis and release, the route(s) of its elimination from blood as well as its biological function(s) are unknown. The rapid increase in procalcitonin concentration in sepsis--in some patients earlier than that of C-reactive protein--and decrease upon successful chemotherapy makes procalcitonin a potentially important biomarker in monitoring patients with suspected or confirmed sepsis.

Fungicidal activity of human lactoferrin-derived peptides based on the antimicrobial αß region.

Owing to the increasing number of infections in hospitalised patients caused by resistant strains of fungi, there is a need to develop new therapeutic agents for these infections. Naturally occurring antimicrobial peptides may constitute models for developing such agents. A modified peptide sequence (CFQWKRAMRKVR; HLopt2) based on amino acid residues 20-31 of the N-terminal end of human lactoferrin (hLF) as well as a double-sized human lactoferricin-like peptide (amino acid residues 16-40; HLBD1) were investigated for their antifungal activities in vitro and in vivo. By in vitro assay, HLopt2 was fungicidal at concentrations of 12.5-25 µg/mL against Cryptococcus neoformans, Candida albicans, Candida krusei, Candida kefyr and Candida parapsilosis, but not against Candida glabrata. HLopt2 was demonstrated to have ≥ 16-fold greater killing activity than HLBD1. By inducing some helical formation caused by lactam bridges or by extending the assay time (from 2h to 20 h), HLBD1 became almost comparable with HLopt2 in its fungicidal activity. Killing of C. albicans yeast cells by HLopt2 was rapid and was accompanied by cytoplasmic and mitochondrial membrane permeabilisation as well as formation of deep pits on the yeast cell surface. In a murine C. albicans skin infection model, atopic treatment with the peptides resulted in significantly reduced yields of Candida from the infected skin areas. The antifungal activities of HLopt2 in vitro and in vivo suggest possible potential as a therapeutic agent against most Candida spp. and C. neoformans. The greatly improved antifungal effect of the lactam-modified HLBD1 indicates the importance of amphipathic helix formation for lethal activity.

A novel monoclonal antibody recognizing beta(1-3) glucans in intact cells of Candida and Cryptococcus.

The cell walls of all medically important fungi contain a unique polyglucose compound, beta(1-3) glucan. In the present study, murine monoclonal antibodies were produced against linear and beta(1-6) branched beta(1-3) glucans, and their specificities were characterized for reactivity to other beta glucans, fungal cell wall fragments, and fungal cells. Their reactivity was also compared with that of rabbit polyclonal antibodies raised against the same immunogens. Two mouse monoclonal antibodies (AG and BG) recognized immunoreactive epitopes in beta(1-3)(1-6) glucan by ELISA. In an inhibition assay of the anti-beta(1-3)(1-6) activity of the monoclonals, the homologous antigen effectively inhibited the activity as expected, while beta(1-3) also inhibited the assay but to a much lesser extent. No inhibition was obtained by beta(1-3)(1-4) or beta(1-6), while a cell wall extract of Candida albicans (PPM) effectively inhibited both monoclonals. Cell wall fragments of C. albicans (CaCW) and Cryptococcus neoformans (CnCW) inhibited the anti-beta(1-3)(1-6) activity of AG, while BG was much less or not inhibited at all. Immunofluorescence confirmed the unique antibody specificity of AG by its recognition of a beta(1-3)(1-6)-associated epitope on the cell surfaces of C. albicans,C. krusei, C. glabrata, and nonencapsulated C. neoformans. The epitope for the AG antibody is suggested to be present in the branching point of beta(1-3)(1-6), or in the randomly coiled beta(1-3) polyglucan due to the presence of branches. Thus, monoclonal antibodies to beta(1-3)(1-6) glucans may have potential as tools in the laboratory diagnosis of invasive yeast infections.

Heterogeneity of lipid A: structural determination by 13C and 31P NMR of lipid A fractions from lipopolysaccharide of Escherichia coli 0111.

Purified lipid A from Escherichia coli 0111 was fractionated by thin-layer chromatography, and seven major bands were studied by 13C and 31P NMR. All lipid A fractions except one had fatty acids, 3-hydroxytetradecanoic acid, 3-(acyloxy)tetradecanoic acid, and phosphate groups bonded to the diglucosamine backbone. The remaining fraction was shown to be phosphatidylethanolamine. The number of substituents found showed that in all fractions all sites available for C-acylation (C-3, C-4, and C-3') and N-acylation (C-2 and C-2') carried acylic substituents. The number, ranging from four to six, and type of ester-bound carboxylic acid residues as well as the number of phosphate groups differed among the fractions. The three fastest moving bands all had three unsubstituted hydroxy fatty acids and one phosphate group (C-4'), while the slower moving bands had four hydroxy fatty acids and two phosphate groups. Unsubstituted 3-hydroxytetradecanoic acid residues were amide-bound to the disaccharide in all but one of the fractions. In summary, the heterogeneity of E. coli 0111 lipid A is found to be a consequence of a variation of the number and composition of carboxylic acid residues and of varying phosphate content.

Lipid A in anaerobic bacteria.

The ability of lipid A preparations from strains of Bacteroides, Fusobacterium, and Veillonella to inhibit the lipid A-anti-lipid A reaction in an enzyme-linked immunosorbent assay was tested. Anti-lipid A serum was prepared with lipid A from Salmonella minnesota R595, and lipid A from Escherichia coli EH100 was used as control antigen. Preparations from three of four different species of Bacteroides were unable to inhibit the anti-lipid A activity, whereas lipid A preparations from Fusobacterium and Veillonella strains inhibited 50% of the activity at 1 to 141 micrograms. One of the Bacteroides strains, Bacteroides oralis, showed a very weak inhibiting activity at the highest concentration used. The results confirm that Bacteroides species have a unique lipopolysaccharide structure, in contrast to other anaerobic genera which have a lipopolysaccharide structure similar to that of the Enterobacteriaceae.

Lipid A and anti-lipid A.

Lipid A in free form, in crude antigen preparations, and on Formalin-treated Escherichia coli and Salmonella minnesota R595 was employed in studies of its antigenic composition, immunogenicity, and availability on gram-negative bacteria. Analyses with immunodiffusion and crossed immunoelectrophoresis of isolated lipid A preparations revealed three components. Inhibition experiments with enzyme-linked immunosorbent assay showed that the lipid A structure was not exposed on the tested smooth or rough E. coli strains or on S. minnesota R595. In crude O antigen preparations from some of the strains, however, lipid A was available for reaction with antibodies. The inaccessibility of lipid A on the bacterial surface may explain the poor protective capacity of anti-lipid A antibodies against bacterial infections. An enzyme-linked immunosorbent assay was more sensitive for measuring anti-lipid A antibody activity than indirect hemolysis or indirect hemagglutination. With an enzyme-linked immunosorbent assay it was shown that in rabbits the immunogenicity of lipid A was approximately the same when coated on erythrocytes or, as is more commonly done, when lipid A-coated hydrolyzed bacteria were used. Some antisera from rabbits immunized with E. coli of different serotypes showed activity against lipid A, with a higher frequency for antisera from rabbits immunized with R mutants.

Antibodies to lipid A: occurrence in humans.

Lipid A, the toxic part of the bacterial endotoxin, is a common antigen for many gram-negative bacteria. Antibodies to lipid A occur naturally in humans; they have been found in 10%-34%, and even up to 73%, of individuals tested, as detected by indirect hemolysis and enzyme-linked immunosorbent assay (ELISA), respectively. Inflammatory bowel diseases (Crohn's disease or ulcerative colitis) cause changes in the level of antibodies to lipid A, as compared with that found in healthy control subjects. Increased levels of antibodies to lipid A are seen in both children and adults with infections due to gram-negative bacteria, such as urinary tract infections (UTI). The highest titers of IgG in serum, as detected by ELISA, have been recorded in patients with development or progression of renal scarring associated with UTI. Since lipid A may play a role in the pathogenesis of renal impairment, the determination of the level of antibodies to lipid A may help in the diagnosis of certain forms of UTI. Possible beneficial roles of antibodies to lipid A during septicemia caused by gram-negative bacteria in humans are still unclear.

Lipid A fractions analyzed by a technique involving thin-layer chromatography and enzyme-linked immunosorbent assay.

A modified enzyme-linked immunosorbent assay (ELISA), with alkaline phosphatase as enzyme, was used for the study of antigenicity of lipid A fractions directly on thin-layer chromatographic (TLC) plates. For visualization a gel slab containing the enzyme substrate was placed on the plate containing enzyme-conjugated antibodies. The plate was read by a thin-layer chromatogram spectrophotometer. The immunoassay was both highly specific and quite sensitive. Sensitivity was superior to levels obtained by staining the plate with molybdenum blue (for phosphate) or orcinol (for carbohydrate). Fractions of lipid A from Escherichia coli 0111, Shigella flexneri or Salmonella minnesota R595, after being separated by thin-layer chromatography, were analyzed using rabbit anti-(lipid A) serum. Patterns obtained by scanning the same plates for phosphate staining and for the TLC-ELISA corresponded well. For comparison with TLC-ELISA, an inhibition assay was run using a tube ELISA. The tube ELISA, run in aqueous medium, showed that fractions 6-8 (those having the highest RF values) had the least activities. In contrast, TLC-ELISA did not detect large differences between fractions 2-7. This discrepancy probably reflected limited aqueous solubility of fractions 6 and 7. We conclude that TLC-ELISA might reveal antigenic activities of lipids that could be missed by other methods. The data suggested that all fractions, except for fraction 8, were similar in their antigenicity by TLC-ELISA. Differences in antigenicity between the fractions occurred when the fractions were tested in free form in an aqueous environment and these differences possibly could have been due to different solubilities of individual fractions.

Endotoxin shedding by enterobacteria: free and cell-bound endotoxin differ in Limulus activity.

The endotoxin activities of gram-negative bacteria and their lipopolysaccharides (LPS) have been quantitated by a chromogenic Limulus amocbocyte lysate (CLAL) assay. When bacterial cell exposing various cell surface structures were compared, the highest Limulus activities were found in R strains of Escherichia coli and Salmonella typhimurium mutants. E. coli with K antigens did not differ from K-negative strains. By measuring beta-hydroxymyristic acid (3-OH tetradecanoic acid, beta-OHC14:0), it was possible to compare the CLAL activities of LPS bound to bacterial cells, LPS shed into the culture medium, and purified LPS. After 16 h of growth, the cell-free culture supernatants of three E. coli O1K1 strains and S. typhimurium showed CLAL activities 14.3 to 20.3 times higher than did the corresponding bacterial cell suspensions in relation to their beta-OHC14:0 contents. Four other E. coli strains (O serotypes O14, O24, and O75) and the S. typhimurium 395 R mutants MR5 and MR6 showed CLAL values 2.8 to 7.9 times higher in their culture supernatants. LPS of E. coli O1K1 and S. typhimurium had lower CLAL activities than the culture supernatants (1/10 and 1/4, respectively). Although the beta-OHC14:0 concentrations of the culture supernatants were approximately half those of the corresponding bacterial cells, all had CLAL values that were 2 to 21 times higher. The bacterial cell suspension, culture supernatant, and purified LPS of S. typhimurium MS were compared by CLAL assay and a quantitative enzyme-linked immunosorbent assay based on monoclonal antibodies to the O5 antigen. Endotoxin shed into the culture medium was the most CLAL-active form of LPS, while purified LPS was the most antigen-active form. The results emphasize the importance of appropriate standards when quantifying endotoxin in various states. In conclusion, E. coli and S. typhimurium bacteria shed significant amounts of endotoxin into the surrounding medium during growth. This form of LPS is more CLAL active than the cell-bound or purified LPS.

Circulating beta (1-3) glucan and immunoglobulin G subclass antibodies to Candida albicans cell wall antigens in patients with systemic candidiasis.

Invasive candidiasis in patients who are immunocompromised or in intensive care units (ICUs) presents both diagnostic and therapeutic problems. We previously described antibodies that were directed against Candida albicans cell wall fragments (CW), periodate-treated CW (CW(IO4)), phosphopeptidomannan (PPM), and beta(1-3) glucan. In this study, circulating fungal antigens [mannan and beta(1-3) glucan] and immunoglobulin G (IgG) subclass antibodies to these cell wall antigens (anti-CW) were analyzed in patients with systemic candidiasis. Sera were collected from 14 patients on two or three consecutive occasions, starting on the day when candidiasis was culture proven. The sera were analyzed by enzyme-linked immunosorbent assay. The control groups consisted of lactating mothers (n = 9) (group I) who had breast milk that was positive for C. albicans and also had acute inflammation of the nipples, and age-matched blood donors (n = 10) (group II). Within the first 3 weeks of Candida infection all of the patients were positive for beta(1-3) glucan by the Gluspecy test, but no patients were positive for mannan in the less-sensitive Pastorex Candida test. The controls were negative for both beta(1-3) glucan (<20 pg/ml) and mannan (<2.5 ng/ml). IgG1 anti-CW and IgG2 anti-PPM antibodies were the most discriminatory antibodies. The ratio of IgG1 anti-CW to IgG2 anti-PPM was significantly lower in nonsurviving patients than in the other patients within the first week of candidiasis (P = 0.019). The IgG2 levels of anti-CW(IO4) and antiglucan antibodies correlated strongly (r = 0.681; P < 0.0001), and the absence of these antibodies was associated with increased levels of beta(1-3) glucan. Increased levels of IgG1 anti-CW or IgG2 anti-PPM antibodies (titer of > or = 3 logs) or of a combination of the two antibodies (log sum, > or = 5) showed 92% sensitivity, 100% specificity, and positive predictive values. In conclusion, beta(1-3) glucan and the two subclass antibodies appear to be early specific markers for the laboratory diagnosis of candidiasis. Furthermore, the kinetics of beta(1-3) glucan appearance in serum may assist in evaluating the therapeutic efficacy of antifungal treatments.

Endotoxin is angiogenic.

The formation of new blood vessels, angiogenesis, is an important event in inflammation, wound healing and tumour growth. Mediators produced by various cells when exposed to endotoxin include cytokines (tumour necrosis factor, interleukins 1 and 6, and basic fibroblast growth factor) which, it has been suggested, stimulate angiogenesis. The angiogenic effect of endotoxin (lipopolysaccharide, LPS) was studied in rats using the quantitative mesenteric window assay. Adult rats were injected intraperitoneally with Escherichia coli LPS (5 pg/ml-20,000 ng/ml) twice daily for 4.5 consecutive days and were sacrificed 14 days after the start of this treatment. An angiogenic response was observed at concentrations of > 2 ng/ml in a dose-dependent manner. No inflammatory cellular exudate was seen in the test tissue at the time of angiogenesis analysis. Suppressed body-weight gain, a marker of the systemic effect of LPS in the rat, was significant only at the highest dose tested. The data suggest that endotoxin-mediated neovascularization could be a component of inflammation and wound healing.

Candida albicans cell wall antigens for serological diagnosis of candidemia.

Serological tests for diagnosis of disseminated fungal infections in the immunocompromised host are used with varying results. In the present study, the relative ability of antibodies to specifically recognize Candida albicans cell wall components was evaluated in order to find antigenic markers for serological diagnosis of candidemia. Native C. albicans cell wall fragments (CW), periodate- (CWIO4) and proteinase-K- (CWP) treated CW, a mildly extracted phosphopeptidomannan (PPM), and beta(1-3)(1-6)-glucan were used as antigens in ELISA with sera from rabbits immunized with C. albicans (n = 10), patients with culture proven candidemia (n = 8) and healthy individuals (n = 8). The antibody response in rabbits consisted predominantly of anti-PPM antibodies, a finding that was substantiated by inhibition-ELISA. Consistently, periodate treatment (CW104) destroyed a major proportion of the antigenic epitopes. Low rabbit antibody levels were found against glucan, the major Candida cell wall component. These results supported the conclusion that glucan is localized mainly in the inner part of the C. albicans cell wall. In contrast to rabbits' serum IgG antibody response against PPM, which was at least tenfold higher than that raised against CW, patients with candidemia had similar IgG antibody levels against both antigens. These levels were significantly higher than those seen in healthy controls (CW, P = 0.0005 and PPM, P < 0.0001). Although the human anti-glucan and anti-CWIO4 IgG antibody levels were low overall, they were nonetheless significantly increased in the patient group (P = 0.0159 for antiglucan and P = 0.0491 for anti-CWIO4). In addition, a correlation was noticed between levels of these antibodies. No significant differences were found between patients and controls for IgM antibodies when CW, CWIO4, PPM and Glu were used as antigens. In conclusion, IgG antibodies to PPM and native cell wall fragments (CW) were highly discriminatory for recognition of candidemia and these antigens are thus promising candidates for use in serodiagnosis.

Orally administered bovine lactoferrin systemically inhibits VEGF(165)-mediated angiogenesis in the rat.

Lactoferrin (Lf) systemically suppresses tumor growth and metastasis by unknown mechanisms. We have studied the effect of orally administered iron-unsaturated bovine Lf on angiogenesis induced by VEGF(165) and IL-1-alpha in adult rats using the mesenteric-window angiogenesis assay. VEGF(165) is a major angiogenic factor in most, if not all, tumors and other angiogenesis diseases of clinical relevance. A number of objective angiogenesis variables were analyzed using microscopic morphometry and image analysis. Lf treatment significantly inhibited the VEGF(165)-mediated response in terms of microvessel spatial extension, overall vascularity and incidence of crossover. The response to IL-1-alpha decreased significantly only in terms of microvessel crossover. In vitro, Lf exerted an antiproliferative effect on endothelial cells. To our knowledge, Lf is the first endogenous protein that has been shown to be antiangiogenic following oral administration. The oral administration of Lf thus appears to be of potential interest as an antiangiogenesis treatment modality in the clinical setting. Since tumor growth is angiogenesis dependent, the extensive therapeutic potential warrants further study to elucidate the mechanisms responsible for the angiostatic effect of Lf.