lactoferrin and Candidiasis

To discuss the potential clinical benefits of lactoferrin in preterm and term infants, as well as in young children and to review information on the burden of neonatal sepsis. Current evidence on the mechanisms that explain the role of human milk in the neonatal and infant anti-infective responses will be briefly reviewed and preclinical research data on the potential mechanisms of action by which lactoferrin may impact infant gut health, gut immune development and functions, including the lactoferrin effects on the neonatal microbiome, will be examined. Finally, updated translational research on lactoferrin will be presented and discussed and the current evidence from prospective randomized controlled trials in neonates, infants, and toddlers will be analyzed. These randomized controlled trials demonstrate that lactoferrin has a clinically significant impact on feeding, the microbiome, and clinical outcomes in neonates and infants.

Topics: Anti-Infective Agents; Candidiasis; Child, Preschool; Enterocolitis, Necrotizing; Gastrointestinal Tract; Humans; Infant; Infant Nutritional Physiological Phenomena; Infant, Newborn; Intensive Care, Neonatal; Lactoferrin; Microbiota; Milk, Human; Sepsis

Neonatal sepsis remains a feared cause of morbidity and mortality in the neonatal period. Maternal, neonatal, and environmental factors are associated with risk of infection, and a combination of prevention strategies, judicious neonatal evaluation, and early initiation of therapy are required to prevent adverse outcomes. This article reviews recent trends in epidemiology and provides an update on risk factors, diagnostic methods, and management of neonatal sepsis.

Topics: Adaptive Immunity; Anti-Infective Agents; Antibodies, Monoclonal; Antifungal Agents; Bacterial Infections; Biomarkers; Blood Cell Count; C-Reactive Protein; Candidiasis; Escherichia coli Infections; Fluconazole; Genomics; Humans; Immunity, Innate; Immunoglobulins, Intravenous; Infant, Newborn; Infant, Newborn, Diseases; Lactoferrin; Polymerase Chain Reaction; Predictive Value of Tests; Proteomics; Risk Factors; Sepsis; Streptococcal Infections; Streptococcus agalactiae

Invasive fungal infections are recognized as an important cause of morbidity and mortality in the immunocompromised host. Rapid initiation of adequate antifungal treatment is often hampered by the limitations of current diagnostic methods. This review encompasses the promises and limitations of newer tracers (believed to target the infectious agents), i.e., radiolabeled antimicrobial peptides, antifungals and chitin-specific agents, for fungal infection imaging by scintigraphy. In mice (99m)Tc-labeled peptides derived from human ubiquicidin (UBI29-41) and lactoferrin (hLF1-11) distinguished local Candida albicans and Aspergillus fumigatus infections from sterile inflammatory processes, but not from bacterial infections. Clinical trials showed that (99m)Tc-UBI29-41 can distinguish infections from inflammatory lesions with 80% specificity and 100% sensitivity. (99m)Tc-hLF1-11 was able to monitor the antifungal effects of fluconazole on C. albicans infections. Moreover, (99m)Tc-fluconazole proved to be an excellent tracer for C. albicans infections as it did not accumulate in bacterial infections and inflammatory processes. However this tracer poorly detected A. fumigatus infections. Furthermore, (123)I-chitinase and (99m)Tc-HYNIC-CBP21 accumulated in both C. albicans and A. fumigatus infections in mice at later time points. In conclusion, despite the recent advances in radiolabeled imaging techniques for invasive fungal infections, the search for better tracers for fungal infection imaging should be continued.

Topics: Animals; Antifungal Agents; Aspergillosis; Aspergillus fumigatus; Candida albicans; Candidiasis; Chitin; Diagnosis, Differential; Fluconazole; Humans; Lactoferrin; Mice; Peptide Fragments; Radioactive Tracers; Radionuclide Imaging; Radiopharmaceuticals; Sensitivity and Specificity; Technetium

Immunosuppressed children and adults have a higher prevalence of oropharyngeal candidiasis. In this patient population, anti-fungal therapy of this condition is often ineffective, and new approaches to treatment are needed. The use of bovine lactoferrin is considered a promising option in treating oropharyngeal candidiasis. Here we review the results of in vitro and in vivo studies that have examined the antimicrobial characteristics of bovine lactoferrin as an adjunctive therapy for oropharyngeal candidiasis.

Topics: Animals; Candidiasis; Cattle; Humans; Immunotherapy; Lactoferrin; Mucous Membrane

The increasing frequency of fungal infections in immunocompromised patients together with the emergence of strains resistant to currently used antifungal drugs point to an increased need for a new class of antimycotics. Antimicrobial peptides are promising candidates for the treatment of fungal infections since they have both mechanisms of action distinct from available antifungal agents and the ability to regulate the host immune defence systems as well. This review focuses on Candida albicans as a large amount of work on the mechanisms of action of classical antifungals as well as antimicrobial peptides, such as defensins, protegrins, histatins and lactoferrin (LF)-derived peptides, has been performed in this yeast. Analogues of these antimicrobial peptides and combinations of antimicrobial peptides with classical antimycotics are under investigation for treatment of candidiasis.

Topics: Anti-Bacterial Agents; Antifungal Agents; Candida albicans; Candidiasis; Defensins; Humans; Lactoferrin; Proteins

Recent advances in the research on host defense mechanisms against infections with Candida and Aspergillus were reviewed. Modes of the defense mechanisms were divided into three phases by thedifferent physiological circumstances surrounding the fungi: 1, the exocrine fluid in which fungi exist on the mucosal membranes; 2, the tissues invaded by fungi with the circulating blood; and 3, the limited lesions where fungi continue to be alive with a restricted blood flow. In each defense mechanism, theroles of the endogenous antifungal substances such as lactoferrin, defensins and calprotectin and leukocytes were discussed.

Topics: Aspergillosis; Candidiasis; Defensins; Humans; Lactoferrin; Leukocyte L1 Antigen Complex; Leukocytes; Membrane Glycoproteins; Neural Cell Adhesion Molecules; Proteins

The role of iron in certain clinical infections is revealed. In normal persons the antibacterial and antifungal properties of blood and other tissue fluids cannot be maintained unless there are exceptionally low levels of available iron. This is controlled by the presence of the unsaturated iron-binding proteins, transferrin and lactoferrin. In several clinical conditions an abnormal availability of iron is responsible for fatal septicaemia. This is because the phagocytic system is overwhelmed by rapidly growing organisms when iron is freely available.

Topics: Bacterial Infections; Candidiasis; Carrier Proteins; Disease Susceptibility; Humans; Iron; Iron-Binding Proteins; Lactoferrin; Leukemia; Transferrin; Transferrin-Binding Proteins; Virulence

Topics: Animals; Bacterial Adhesion; Candida albicans; Candidiasis; Cytokines; Humans; Kidney; Lactoferrin; Male; Mice; Mice, Inbred C57BL

Candida albicans is a commensal fungus in human mucosal surfaces, including the oral cavity. Lactoferrin (LF) and the lactoperoxidase (LPO) system, which are host protection components in exocrine secretions, each exhibit weak anti-candida activity. We herein examined the effects of the combination of LF and the LPO system on C. albicans. Morphological observations indicated that the combination of LF and the LPO system reduced the mycelial volume of C. albicans and changed the size and shape of cells more than each agent alone. The combination of LF and the LPO system also exerted strong inhibitory effects on the cellular metabolic activity and adhesive hyphal form of C. albicans. A checkerboard analysis revealed that the anti-candida activity of LF and the LPO system was synergistic. These results suggest that the combination of LF and the LPO system is useful for preventing candidiasis.

Topics: Animals; Antifungal Agents; Candida albicans; Candidiasis; Cattle; Dose-Response Relationship, Drug; Drug Synergism; Humans; Lactoferrin; Lactoperoxidase

Lysozyme and lactoferrin have anti-candidal activity. Candida dubliniensis is associated with oral candidiasis. Candida infections are managed with nystatin, amphotericin B, caspofungin, ketoconazole, fluconazole, and chlorhexidine. Candida species undergo a brief exposure to therapeutic agents in the mouth. There is no data on the influence of limited exposure to antimycotics on the sensitivity of C. dubliniensis to lactoferrin and lysozyme. Hence, this study observed the changes in the sensitivity of C. dubliniensis to anti-candidal action of lactoferrin and lysozyme after transitory exposure to sub-lethal concentrations of antifungals.. After determination of the minimum inhibitory concentration (MIC), 20 C. dubliniensis isolates were exposed to twice the concentration of MIC of nystatin, amphotericin B, caspofungin, ketoconazole, fluconazole, and chlorhexidine for 1 h. Drugs were removed by dilution and thereafter the susceptibility of these isolates to lysozyme and lactoferrin was determined by colony-forming unit quantification assay.. Exposure of C. dubliniensis to nystatin, amphotericin B, caspofungin, ketoconazole, fluconazole, and chlorhexidine resulted in an increase in susceptibility to lysozyme by 9.45, 30.82, 30.04, 50.64, 55.60, and 50.18%, respectively (p < 0.05 to p < 0.001). Exposure of C. dubliniensis to nystatin, amphotericin B, caspofungin, ketoconazole, fluconazole, and chlorhexidine resulted in an increase in susceptibility to lactoferrin by 13.54, 16.43, 17.58, 19.60, 21.32, and 18.73, respectively (p < 0.05 to p < 0.001).. Brief exposure to nystatin, amphotericin B, caspofungin, ketoconazole, fluconazole, and chlorhexidine enhances the antifungal effect of lysozyme and lactoferrin on C. dubliniensis isolates in vitro.

Topics: Anti-Infective Agents; Antifungal Agents; Candida; Candidiasis; Humans; Kuwait; Lactoferrin; Mouth Diseases; Muramidase

Topics: Adult; Body Mass Index; Breast Diseases; Breast Feeding; Calcium; Candidiasis; Female; Humans; Infant; Infant, Newborn; Lactoferrin; Male; Mastitis; Milk, Human; Mothers; Neuropeptides; Plasma; Postpartum Period; Prospective Studies; Surveys and Questionnaires; Sweden; Young Adult

To determine the role of human lactoferrin (hLF) in protecting the oral cavities of mice against Candida albicans infection in lactoferrin knockout (LFKO(-/-)) mice was compared to wild-type (WT) mice. We also aim to determine the protective role of hLF in LFKO(-/-) mice.. Antibiotic-treated immunosuppressed mice were inoculated with C. albicans (or sham infection) by oral swab and evaluated for the severity of infection after 7 days of infection. To determine the protective role of hLF, we added 0·3% solution of hLF to the drinking water given to some of the mice. CFU count, scoring of lesions and microscopic observations were carried out to determine the severity of infection. LFKO(-/-) I mice showed a 2 log (P = 0·001) higher CFUs of C. albicans in the oral cavity compared to the WT mice infected with C. albicans (WTI). LFKO(-/-) I mice given hLF had a 3 log (P = 0·001) reduction in CFUs in the oral cavity compared to untreated LFKO(-/-) I mice. The severity of infection, observed by light microscopy, revealed that the tongue of the LFKO(-/-) I mice showed more white patches compared to WTI and LFKO(-/-) I + hLF mice. Scanning electron microscopic observations revealed that more filiform papillae were destroyed in LFKO(-/-) I mice when compared to WTI or LFKO(-/-) I + hLF mice.. Human LF is important in protecting mice from oral C. albicans infection. Administered hLF may be used to prevent C. albicans infection.. Human LF, a multifunctional iron-binding glycoprotein can be used as a therapeutic active ingredient in oral healthcare products against C. albicans.

Topics: Administration, Oral; Animals; Anti-Bacterial Agents; Candida albicans; Candidiasis; Humans; Lactoferrin; Male; Mice; Mice, Knockout; Mouth Diseases; Tongue

Antimicrobial peptides are promising antibiotics as they possess strong antimicrobial activity and very broad spectra of activity. However, administration of an antibiotic with a very broad spectrum of activity disrupts normal microflora and increases the risks of other fatal infections. To solve the problem, we designed a novel antimicrobial peptide that is activated by virulent proteases of pathogenic organisms. We constructed a peptide composed of three domains, namely an antimicrobial peptide (lactoferricin) as the active center, a protective peptide (magainin intervening sequence) that suppresses antimicrobial activity, and a specific linker that joins these two components and is efficiently cleaved by virulent proteases. We utilized Candida albicans as a model organism that produces secreted aspartic proteases as a virulence attribute. We screened for a peptide sequence efficiently cleaved by secreted aspartic proteases isozymes and identified a GFIKAFPK peptide as the most favorable substrate. Subsequently, we chemically synthesized a peptide containing the GFIKAFPK sequence. The designed peptide possessed no antimicrobial activity until it was activated by secreted aspartic proteases isozymes. Furthermore, it demonstrated selective antimicrobial activity against C. albicans, but not against Saccharomyces cerevisiae. A designed peptide like the one described in this study may protect normal microflora, resulting in enhanced safety as a therapeutic.

Topics: Amino Acid Sequence; Antifungal Agents; Aspartic Acid Proteases; Candida albicans; Candidiasis; Drug Design; Humans; Lactoferrin; Molecular Sequence Data; Peptide Hydrolases

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.

Topics: Animals; Anti-Infective Agents; Candida; Candidiasis; Cryptococcus neoformans; Dermatomycoses; Disease Models, Animal; Humans; Lactoferrin; Mice; Microbial Viability; Treatment Outcome

Nosocomial infection with antibiotic-resistant strains is a major threat to critical care medicine. Selective decontamination of the digestive tract (SDD) is one of the strategies used to reduce ventilator-associated pneumonia and sepsis in critically ill patients. In the present study, we performed pathogenic challenges of the digestive tract in a transgenic milk-fed animal model to test whether porcine lactoferrin (pLF) is an effective SDD regimen.. Transgenic mice expressing recombinant pLF in their milk at a mean+/-SD concentration of 120+/-13.6 mg/L during the lactation stage fed normal CD-1 mice pups for 4 weeks. The pups were subsequently challenged with pathogenic Escherichia coli, Staphylococcus aureus, and Candida albicans.. Compared with the control groups fed wild-type (normal) milk, the groups fed pLF-enriched milk demonstrated statistically significant improvements in weight gain; lower bacterial numbers in intestinal fluid, blood, and liver; healthier microvilli in the small intestine; and alveoli in the lungs.. Our results showed that oral administration of pLF-enriched milk to mice led to broad-spectrum antimicrobial activity in the digestive tract and protected the mucosa of the small intestine from injury, implying that pLF can be used as an effective SDD regimen.

Topics: Animals; Animals, Newborn; Bacteremia; Bacterial Infections; Body Weight; Candidiasis; Cytokines; Escherichia coli Infections; Gastrointestinal Tract; Immunohistochemistry; Intestines; Lactation; Lactoferrin; Lung; Mice; Mice, Transgenic; Milk; Polymerase Chain Reaction; Recombinant Proteins; Staphylococcal Infections; Swine

The study aimed to describe the patterns and density of early tracheal colonization among intubated patients and to correlate colonization status with levels of antimicrobial peptides and inflammatory cytokines.. The was a prospective cohort study.. The study was conducted in medical and cardiovascular intensive care units of a tertiary referral hospital.. Seventy-four adult patients admitted between March 2003 and May 2006 were recruited for the study.. Tracheal aspirates were collected daily for the first 4 days of intubation using standardized, sterile technique and sent for quantitative culture and cytokines, lactoferrin and lysozyme measurements.. The mean acute physiology and chronic health evaluation (APACHE II) score in this cohort was 24 +/- 7. Proportion of subjects colonized by any microorganism increased over the first 4 days of intubation (47%, 60%, 70%, 70%, P = .08), but density of colonization for bacteria or yeast did not change significantly. No known risk factors predicted tracheal colonization on day 1 of intubation. Several patterns of colonization were observed (persistent, transient, new colonization, and clearance of initial colonization).The most common organisms cultured were Candida albicans and coagulase-negative Staphylococcus. Levels of cytokines, lactoferrin, or lysozyme did not change over time and were not correlated with tracheal colonization status. Four subjects (6%) had ventilator-associated pneumonia.. The density of tracheal colonization did not change significantly over the first 4 days of intubation in medical intensive care unit patients. There was no correlation between tracheal colonization and the levels of antimicrobial peptides or cytokines. Several different patterns of colonization may have to be considered while planning interventions to reduce airway colonization.

Topics: Adult; APACHE; Candidiasis; Case-Control Studies; Colony Count, Microbial; Cross Infection; Cytokines; Female; Humans; Inflammation; Intensive Care Units; Intubation, Intratracheal; Lactoferrin; Logistic Models; Male; Middle Aged; Multivariate Analysis; Muramidase; Pneumonia, Ventilator-Associated; Prospective Studies; Respiration, Artificial; Respiratory Mucosa; Risk Factors; Staphylococcal Infections; Statistics, Nonparametric; Suction; Time Factors; Trachea

Neonatal sepsis causes significant mortality and morbidity. Coagulase-negative staphylococci (CoNS) and Candida frequently cause neonatal sepsis at >72 h of age. Lactoferrin, which is present in human milk, is a component of innate immunity and has broad-spectrum antimicrobial activity. The synergistic effects of lactoferrin with antibiotics against neonatal isolates have not been systematically evaluated. Here, eight clinical strains (seven neonatal) of CoNS and three strains (two neonatal) of Candida albicans were studied. MIC50 and MIC90 values of human recombinant lactoferrin (talactoferrin; TLF), vancomycin (VAN) and nafcillin (NAF) against CoNS, and of TLF, amphotericin B (AMB) and fluconazole (FLC) against C. albicans, were evaluated according to established guidelines. Antimicrobial combinations of TLF with NAF or VAN against CoNS, and TLF with AMB or FLC against C. albicans, were evaluated by a checkerboard method with serial twofold dilutions. Synergy was evaluated by the median effects principle, and combination indices and dose reduction indices were reported at 50, 75 and 90% inhibitory effect at several drug-dose ratios. It was found that TLF acted synergistically with NAF and VAN against CoNS, and with AMB and FLC against C. albicans, at multiple dose effects and drug-dose ratios with few exceptions. In synergistic combinations, drug reduction indices indicated a significant reduction in doses of antibiotics, which may be clinically relevant. Thus TLF acts synergistically with anti-staphylococcal and anti-Candida agents commonly used in neonatal practice and is a promising agent that needs to be evaluated in clinical studies.

Topics: Anti-Bacterial Agents; Antifungal Agents; Candida albicans; Candidiasis; Coagulase; Drug Synergism; Humans; Infant, Newborn; Lactoferrin; Microbial Sensitivity Tests; Recombinant Proteins; Sepsis; Staphylococcal Infections; Staphylococcus

Because the human lactoferrin-derived peptide, hLF(1-11), exerts potent in vitro candidacidal activity, we investigated whether it displays antifungal activity against disseminated Candida albicans infections.. Neutropenic mice were intravenously infected with C. albicans and, 24 h later, were injected with hLF(1-11); 18 h later, the number of viable yeasts in the kidneys was determined microbiologically, the size and number of infectious foci were determined histologically, and serum cytokine levels were determined by immunoassays.. hLF(1-11) was effective (maximum reduction, 1.5 logs) against disseminated C. albicans infections, and its antifungal activity leveled off at a concentration of 0.4 ng of hLF(1-11)/kg of body weight. The antifungal activity of hLF(1-11) was increased in mice injected with interleukin (IL)-10 neutralizing antibodies, which suggests that IL-10 reduces the antifungal activity of hLF(1-11). In agreement with this result was the finding that injection of high doses of hLF(1-11) into infected mice was accompanied by increased levels of IL-10 in serum. Microscopic analysis revealed that infectious foci in kidneys of hLF(1-11)-treated mice contained mainly blastoconidia, whereas filamentous forms were abundant in untreated mice. The peptide inhibited the in vitro morphological transition of C. albicans, in a dose-dependent manner. : hLF(1-11) is effective against disseminated C. albicans infections; and its effects on C. albicans viability and virulence and on host cells may explain this antifungal activity.

Topics: Animals; Antifungal Agents; Candida albicans; Candidiasis; Carrier Proteins; Dose-Response Relationship, Drug; Drug Resistance, Fungal; Female; Fluconazole; Humans; Interleukin-10; Kidney Diseases; Lactoferrin; Mice; Neutropenia; Specific Pathogen-Free Organisms

To establish a novel strategy for the control of fungal infection, we examined the antifungal and neutrophil-activating activities of antimicrobial peptides. The duration of survival of 50% of mice injected with a lethal dose of Candida albicans (5 x 10(8) cells) or Aspergillus fumigatus (1 x 10(8) cells) was prolonged 3 to 5 days by the injection of 10 microg of peptide 2 (a lactoferrin peptide) and 10 microg of alpha-defensin 1 for five consecutive days and was prolonged 5 to 13 days by the injection of 0.1 microg of granulocyte-monocyte colony-stimulating factor (GM-CSF) and 0.5 microg of amphotericin B. When mice received a combined injection of peptide 2 (10 microg/day) with amphotericin B (0.5 microg/day) for 5 days after the lethal fungal inoculation, their survival was greatly prolonged and some mice continued to live for more than 5 weeks, although the effective doses of peptide 2 for 50 and 100% suppression of Candida or Aspergillus colony formation were about one-third and one-half those of amphotericin B, respectively. In vitro, peptide 2 as well as GM-CSF increased the Candida and Aspergillus killing activities of neutrophils, but peptides such as alpha-defensin 1, beta-defensin 2, and histatin 5 did not upregulate the killing activity. GM-CSF together with peptide 2 but not other peptides enhanced the production of superoxide (O2-) by neutrophils. The upregulation by peptide 2 was confirmed by the activation of the O2- -generating pathway, i.e., activation of large-molecule guanine binding protein, phosphatidyl-inositol 3-kinase, protein kinase C, and p47phox as well as p67phox. In conclusion, different from natural antimicrobial peptides, peptide 2 has a potent neutrophil-activating effect which could be advantageous for its clinical use in combination with antifungal drugs.

Topics: alpha-Defensins; Amphotericin B; Animals; Antifungal Agents; Aspergillosis; Aspergillus fumigatus; Candida albicans; Candidiasis; Cells, Cultured; Female; Lactoferrin; Mice; Neutrophil Activation; Peptides; Signal Transduction

Oral administration of lactoferrin (LF), an innate-defense protein present in exocrine secretions such as milk and in neutrophils, is reported to improve host-protection against infections with microorganisms including pathogenic fungi, possibly due to an immunomodulatory effect. This study aimed to evaluate the effect of bovine LF feeding on peritoneal macrophage activities in mice intraperitoneally injected with inactivated Candida albicans. Time course analysis during the 14 days following Candida-priming revealed that LF administration slightly increased the number of peritoneal exudate cells, and significantly enhanced the production of superoxide anion (O2(-)) and nitric oxide (NO) by peritoneal macrophages at day 7. LF administration facilitated NO production and Candida hyphal-growth inhibition by macrophages derived from Candida-primed mice but not non-primed mice, suggesting that the action of LF is dependent on the immune status of the host. LF administration altered the kinetics of cytokines in the peritoneal lavage fluid of Candida-primed mice. Enhancement of cytokine levels by LF was observed for IL-12 at day 5 and IFN-gamma at day 9, but not for TNF-alpha or IL-10. In conclusion, LF feeding augmented the activities of macrophages in a manner dependent on Candida-priming and these effects may be related to enhanced cytokine levels.

Topics: Animals; Candida albicans; Candidiasis; Cytokines; Female; Kinetics; Lactoferrin; Macrophages, Peritoneal; Mice; Mice, Inbred BALB C; Nitric Oxide; Specific Pathogen-Free Organisms; Superoxides

The aim of this study was to investigate whether technetium-99m labelled fluconazole can distinguish fungal from bacterial infections. Fluconazole was labelled with (99m)Tc and radiochemical analysis showed less than 5% impurities. The labelling solution was injected into animals with experimental infections. For comparison, we used two peptides for infection detection, i.e. UBI 29-41 and hLF 1-11, and human IgG, all labelled with (99m)Tc. Mice were infected with Candida albicans or injected with heat-killed C. albicans or lipopolysaccharides to induce sterile inflammation. Also, mice were infected with Staphylococcus aureus or Klebsiella pneumoniae. Next, accumulation of (99m)Tc-fluconazole and (99m)Tc-labelled peptides/IgG at affected sites was determined scintigraphically. (99m)Tc-fluconazole detected C. albicans infections (T/NT ratio=3.6+/-0.47) without visualising bacterial infections (T/NT ratio=1.3+/-0.04) or sterile inflammatory processes (heat-killed C. albicans: T/NT ratio=1.3+/-0.2; lipopolysaccharide: T/NT ratio=1.4+/-0.1). C. albicans infections were already seen within the first hour after injection of (99m)Tc-fluconazole (T/NT ratio=3.1+/-0.2). A good correlation (R(2)=0.864; P<0.05) between T/NT ratios for this tracer and the number of viable C. albicans was found. Although (99m)Tc-UBI 29-41 and (99m)Tc-hLF 1-11 were able to distinguish C. albicans infections from sterile inflammatory processes in mice, these (99m)Tc-labelled peptides did not distinguish these fungal infections from bacterial infections. It is concluded that (99m)Tc-fluconazole distinguishes infections with C. albicans from bacterial infections and sterile inflammations.

Topics: Animals; Aspergillosis; Aspergillus fumigatus; Candidiasis; Diagnosis, Differential; Fluconazole; Humans; Immunoglobulin G; Inflammation; Lactoferrin; Leukopenia; Lipopolysaccharides; Male; Mice; Myositis; Peptide Fragments; Radionuclide Imaging; Reproducibility of Results; Ribosomal Proteins; Sensitivity and Specificity; Technetium; Thigh; Tissue Distribution

This study compared the possibilities and limitations of 99mTc-labeled synthetic peptides derived from two human antimicrobial peptides, namely, ubiquicidin (UBI) and lactoferrin (hLF), for the scintigraphic detection of bacterial and fungal infections in mice and rabbits. The rationale of our approach was that selected peptides accumulate in infected areas but not in sterile inflammatory lesions, because they bind preferentially to microorganisms. 99mTc-labeled human neutrophil peptides (defensins), ciprofloxacin, and human polyclonal IgG were included as control agents.. 99mTc-labeled peptides and control agents were injected intravenously into animals that had been injected intramuscularly 18 h earlier with multidrug-resistant Staphylococcus aureus, Klebsiella pneumoniae, or fluconazole-resistant Candida albicans. Sterile inflammatory sites were induced by the injection of heat-killed microorganisms or lipopolysaccharide (LPS) into the thigh muscle. Up to 4 h after injection, the accumulation of 99mTc-labeled compounds in the infected/inflamed thigh muscles was determined using scintigraphic techniques and radioactivity counts in dissected tissues.. Scintigraphy revealed that 99mTc-labeled peptides UBI 29-41, UBI 18-35, UBI 31-38, hLF 1-11, and defensins, which showed preferential in vitro binding to microorganisms in a former study, accumulated at a significantly higher rate (P < 0.01) in bacterial and C. albicans infections in mice and rabbits than in inflamed tissues induced by heat-killed microorganisms or by LPS. No significant difference in the accumulation of 99mTc-labeled ciprofloxacin was observed between infected and sterile inflamed thigh muscles in mice.. 99mTc-labeled antimicrobial peptides UBI 29-41, UBI 18-35, UBI 31-38, hLF 1-11, and defensins accumulate significantly in tissues infected with gram-positive and gram-negative bacteria and C. albicans. Significantly lower (P < 0.01) accumulation of these peptides occurs in sterile inflamed tissues. These data indicate that the peptides preferentially tag microorganisms at the site of infection, which is in agreement with their preferential binding to the microorganisms in vitro and in vivo. 99mTc-labeled ciprofloxacin does not distinguish between infections and sterile inflammatory lesions, which implies that its specificity for the detection of bacterial infections is not warranted.

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacterial Infections; Candidiasis; Ciprofloxacin; Defensins; Drug Resistance, Multiple; Immunoglobulin G; Inflammation; Klebsiella Infections; Lactoferrin; Male; Mice; Rabbits; Radionuclide Imaging; Radiopharmaceuticals; Ribosomal Proteins; Staphylococcal Infections; Staphylococcus aureus; Technetium

To develop a new strategy to control candidiasis, we examined in vivo the anticandidal effects of a synthetic lactoferrin peptide, FKCRRWQWRM (peptide 2) and the peptide that mimics it, FKARRWQWRM (peptide 2'). Although all mice that underwent intraperitoneal injection of 5 x 10(8) Candida cells with or without peptide 2' died within 8 or 7 days, respectively, the survival times of mice treated with 5 to 100 microg of intravenous peptide 2 per day for 5 days after the candidal inoculation were prolonged between 8.4 +/- 2.9 and 22.4 +/- 3.6 days, depending on the dose of peptide 2. The prolongation of survival by peptide 2 was also observed in mice that were infected with 1.0 x 10(9) Candida albicans cells (3.2 +/- 1.3 days in control mice versus 8.2 +/- 2.4 days in the mice injected with 10 microg of peptide 2 per day). In the high-dose inoculation, a combination of peptide 2 (10 microg/day) with amphotericin B (0.1 microg/day) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (0.1 microg/day) brought prolonged survival. With a combination of these agents, 60% of the mice were alive for more than 22 days. Correspondingly, peptide 2 activated phagocytes inducing inducible NO synthase and the expression of p47(phox) and p67(phox), and peptide 2 increased phagocyte Candida-killing activities up to 1.5-fold of the control levels upregulating the generation of superoxide, lactoferrin, and defensin from neutrophils and macrophages. These findings indicated that the anticandidal effects of peptide 2 depend not only on the direct Candida cell growth-inhibitory activity, but also on the phagocytes' upregulatory activity, and that combinations of peptide 2 with GM-CSF and antifungal drugs will help in the development of new strategies for control of candidiasis.

Topics: Amphotericin B; Animals; Antifungal Agents; Candida albicans; Candidiasis; Drug Therapy, Combination; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Lactoferrin; Macrophages; Mice; Mice, Inbred CBA; Neutrophils; Peptides; Up-Regulation

The antimicrobial protein lactoferrin (Lf) is present in plasma and in mucosal secretions. Using ELISA we analysed plasma and saliva of HIV-infected patients, patients with AIDS, and healthy controls for the presence of secreted Lf. The plasma Lf levels of AIDS patients (classification C3) were significantly lower (p < 0.001) as compared to asymptomatic and symptomatic HIV infected patients, or controls. In addition, plasma Lf levels closely correlated with neutrophilic granulocyte counts in the HIV-infected patients. Thus, basal plasma Lf levels are likely the result of Lf release by neutrophilic granulocytes. The Candida titres present in the oral cavity were determined in a part of the HIV-infected patient group. As it appeared, the presence of this opportunistic pathogen always coincided with low levels of salivary Lf levels. We conclude that Lf, as part of the nonspecific immune system, might play an important role in the first line of defense against opportunistic microbial infections in AIDS patients.

Topics: Acquired Immunodeficiency Syndrome; AIDS-Related Opportunistic Infections; Candidiasis; Humans; Lactoferrin; Leukocyte Count; Neutrophils; Saliva

Because of the rising incidence of failures in the treatment of oropharyngeal candidosis in the case of severely immunosuppressed patients (mostly human immunodeficiency virus [HIV]-infected patients), there is need for the development of new, more effective agents and/or compounds that support the activity of the common antifungal agents. Since lactoferrin is one of the nonspecific host defense factors present in saliva that exhibit antifungal activity, we studied the antifungal effects of human, bovine, and iron-depleted lactoferrin in combination with fluconazole, amphotericin B, and 5-fluorocytosine in vitro against clinical isolates of Candida species. Distinct antifungal activities of lactoferrin were observed against clinical isolates of Candida. The MICs generally were determined to be in the range of 0.5 to 100 mg. ml(-1). Interestingly, in the combination experiments we observed pronounced cooperative activity against the growth of Candida by using lactoferrin and the three antifungals tested. Only in a limited concentration range was minor antagonism detected. The use of lactoferrin and fluconazole appeared to be the most successful combination. Significant reductions in the minimal effective concentrations of fluconazole were found when it was combined with a relatively low lactoferrin concentration (1 mg/ml). Such combinations still resulted in complete growth inhibition, while synergy of up to 50% against several Candida species was observed. It is concluded that the combined use of lactoferrin and antifungals against severe infections with Candida is an attractive therapeutic option. Since fluconazole-resistant Candida species have frequently been reported, especially in HIV-infected patients, the addition of lactoferrin to the existing fluconazole therapy could postpone the occurrence of species resistance against fluconazole. Clinical studies to further elucidate the potential utility of this combination therapy have been initiated.

Topics: Amphotericin B; Antifungal Agents; Candida; Candidiasis; Culture Media; Drug Synergism; Fluconazole; Flucytosine; Humans; Lactoferrin; Microbial Sensitivity Tests

Preparations of human cervical mucus were obtained from fifteen female volunteers with or without vaginal candidiasis. Their content of lactoferrin and inhibitory effects on the growth of Candida albicans were estimated. The concentration of lactoferrin in cervical mucus was measured by enzyme linked immunosorbent assay (ELISA). Lactoferrin concentration of the eleven preparations among them was more than 0.2 mg/ml. The effects of these cervical mucus preparations on Candida growth were examined in vitro. Candida growth was not inhibited by the addition of 1/200 diluted cervical mucus alone, however, anti-Candida activity of murine neutrophils was augmented by such an addition. These results suggest that the combination of neutrophils and cervical mucus may have an important role in defense against Candida infection in the vaginal mucosa.

Topics: Adult; Animals; Candida albicans; Candidiasis; Cervix Mucus; Female; Humans; Lactoferrin; Mice; Mice, Inbred C3H; Middle Aged; Neutrophils

We investigated the effects of lactoferrin (Lf)-related compounds on growth inhibition of Candida albicans by neutrophils or antifungal agents in vitro. Human neutrophils partially inhibited the growth of C.albicans. The growth inhibition caused by human neutrophils was augmented by the addition of human Lf at concentrations which did not show any inhibitory effect in the absence of neutrophils. Similar observations were obtained also with the following combinations: human neutrophils + bovine Lf, murine neutrophils + bovine Lf, and murine neutrophils + iron saturated bovine Lf, but not in the case of murine neutrophils + human transferrin. The minimum inhibitory concentration (MIC) of azole antifungal agents, clotrimazole, ketoconazole, fluconazole, and itraconazole was reduced by 1/4 to 1/16 in the presence of a sub-MIC level of each of bovine Lf, bovine Lf pepsin hydrolysate, and the antimicrobial peptide "lactoferricin B" (Lfcin B). Other types of antifungal agents, amphotericin B, nystatin, and flucytosine did not show such combined effects with these Lf-related compounds. The anti-Candida activity of bovine Lf or Lfcin B in combination with clotrimazole was shown to be synergistic by checkerboard analysis. Clinically isolated azole-resistant C. albicans strains were more susceptible to bovine Lf or Lfcin B than azole-susceptible strains. Trailing growth of an azole-resistant strain in the presence of fluconazole was reduced by the addition of sub-MIC levels of bovine Lf or Lfcin B. These results suggest that Lf-related compounds even at relatively low concentrations may function as an antifungal effector in combination with neutrophils thereby modulating azole antifungal efficacies in vivo.

Topics: Animals; Antifungal Agents; Candida albicans; Candidiasis; Cattle; Clotrimazole; Drug Synergism; Humans; Lactoferrin; Mice; Neutrophil Activation

Lactoferrin and lysozyme (muramidase) are non-immune defence factors present in various exocrine secretions, including saliva. Previous studies have shown that both proteins, either singly or in combination, are bactericidal in nature and their combined activity is synergistic. As little is known of their interactions with Candida species, 20 oral isolates of C. krusei and 5 isolates of C. albicans were studied for their susceptibility to human apo-lactoferrin and lysozyme, either singly or in combination, using an in vitro assay system. The two species exhibited significant interspecies differences in susceptibility to lactoferrin (p < 0.05), but not for lysozyme; C. krusei being more sensitive to lactoferrin (c 1.4 times) than C. albicans. Both species revealed significant intraspecies differences in their susceptibility to lysozyme (p < 0.05), but not for lactoferrin. No synergistic antifungal activity of the two proteins on either Candida species was noted. The results imply that the variable expression of the fungicidal activity of lactoferrin and lysozyme on Candida species may modulate the oral carriage of yeasts in a complex manner.

Topics: Antifungal Agents; Apoproteins; Candida; Candida albicans; Candidiasis; Humans; Lactoferrin; Mouth Mucosa; Muramidase; Regression Analysis

Lipopolysaccharides (LPSs) from Escherichia coli, Serratia marcescens, and Salmonella typhimurium, at doses from 1 to 100 ng/ml, strongly enhanced growth inhibition of Candida albicans by human polymorphonuclear leukocytes (PMN) in vitro. Flow cytometry analysis demonstrated that LPS markedly augmented phagocytosis of Candida cells by increasing the number of yeasts ingested per neutrophil as well as the number of neutrophils capable of ingesting fungal cells. LPS activation caused augmented release of lactoferrin, an iron-binding protein which itself could inhibit the growth of C. albicans in vitro. Antibodies against lactoferrin effectively and specifically reduced the anti-C. albicans activity of both LPS-stimulated and unstimulated PMN. Northern (RNA blot) analysis showed enhanced production of mRNAs for interleukin-1 beta, tumor necrosis factor alpha, and interleukin-6 and in neutrophils within 1 h of stimulation with LPS. The cytokines were also detected in the supernatant of the activated PMN, and their synthesis was prevented by pretreatment of LPS-stimulated PMN with protein synthesis inhibitors, such as emetine and cycloheximide. These inhibitors, however, did not block either lactoferrin release or the anti-Candida activity of LPS-stimulated PMN. These results demonstrate the ability of various bacterial LPSs to augment neutrophil function against C. albicans and suggest that the release of a candidastatic, iron-binding protein, lactoferrin, may contribute to the antifungal effect of PMN. Moreover, the ability to produce cytokines upon stimulation by ubiquitous microbial products such as the endotoxins points to an extraphagocytic, immunomodulatory role of PMN during infection.

Topics: Candida albicans; Candidiasis; Cycloheximide; Emetine; Gene Expression; Humans; Immunity, Cellular; In Vitro Techniques; Interleukin-1; Interleukin-6; Lactoferrin; Lipopolysaccharides; Neutrophils; Phagocytosis; RNA, Messenger; Tumor Necrosis Factor-alpha