Host defense against oropharyngeal and vaginal candidiasis: Site-specific differences

Mucosal candidiasis is extremely common in immunocompromised patients. However, the prevalence of site-specific infection (i.e., oropharyngeal, vaginal, and esophageal candidiasis) can be quite variable depending on the immune status of the host. While vulvovaginal candidiasis is common in normal healthy women, oropharyngeal and esophageal candidiasis are more frequently encountered under immunocompromised states. Candida albicans, the causative agent in most cases of candidiasis, is a commensal organism of the gastrointestinal and lower female reproductive tracts. Thus, most healthy individuals have demonstrable Candida-specific immunity in the peripheral circulation. The pathogenic state is often precipitated by a deficiency or dysfunction in this immunity. Studies from animal models, women with recurrent vulvovaginal candidiasis, and HIV-infected individuals, however, suggest that distinct host defense mechanisms may function against oropharyngeal and vulvovaginal candidiasis. While cell-mediated immunity (CMI) appears important for protection against oropharyngeal candidiasis (OPC), there is little evidence to indicate that T cell-mediated immunity is protective against vulvovaginal candidiasis (VVC). Furthermore, whereas both local and systemically derived immune defenses appear important for protection against OPC, host defenses that protect against VVC appear limited to the local tissue and possibly restricted to innate mechanisms. Thus, current evidence suggests that VVC, unlike OPC, may not represent a strict opportunistic infection.     

Growth promotion and cell binding ability of bovine lactoferrin to Bifidobacterium longum

Lactoferrin, a major whey protein of human milk, is considered as growth promoter for bifidobacteria, the predominant microorganisms of human intestine. In the present study, in vitro growth promotion and cell binding ability of bovine lactoferrin to several strains of Bifidobacterium longum have been demonstrated. A dose-dependent as well as strain-dependent growth promotion effect by lactoferrin was observed. Cell binding ability of lactoferrin was inspected under an inverted confocal laser scanning microscope by incubation bacterial cells with biotinylated bovine lactoferrin and FITC-conjugated avidin. Fluorescence staining showed bovine lactoferrin binding to all tested strains. A lactoferrin-binding protein with a molecular weight of approximately 67 kDa was also detected in the extracted membrane and cytosolic fraction of each B. longum strain by far-Western blot technique using biotinylated lactoferrin and horseradish peroxidase-conjugated streptavidin. Based on these results, we suggest that existence of lactoferrin-binding protein could be a common characteristic in bifidobacteria. It can also be hypothesized that lactoferrin-binding protein in bifidobacteria is not only involved in growth stimulation mechanism but also could play different roles.     

Secreted aspartic proteases as virulence factors of Candida species

Candida infections have emerged as a significant medical problem during the last few decades. Among the different virulence traits of C. albicans, secreted proteolytic activity has been intensively investigated. Pathogenesis of the various forms of candidiasis was shown to be associated with the differential and temporal regulation of the expression of genes coding for secreted aspartic proteases (Sap). These enzymes act as cytolysins in macrophages after phagocytosis of Candida, are present in tissue penetration and are also involved in adherence to epithelial cells. Since the introduction of new antiretroviral therapeutics such as HIV protease inhibitors, oropharyngeal candidiasis is less often observed in AIDS patients. Different HIV aspartic protease inhibitors were able to inhibit the C. albicans Saps involved in adherence. The lower rates of oropharyngeal candidiasis observed in individuals receiving antiretroviral combination therapy could reflect not only an improvement in the immune system but also direct inhibition of Candida Saps by HIV protease inhibitors. Therefore, the development of specific aspartic protease inhibitors might be of interest for the inhibition of candidiasis.     

念珠菌—上皮细胞互作研究进展

念珠菌作为共生菌定植于机体黏膜表面,一般情况下并不引起感染,但当机体出现免疫力下降或微生态失衡等状况时,可引发口咽念珠菌病、外阴阴道念珠菌病等黏膜感染。对于念珠菌黏膜感染的治疗,虽然抗菌药物是不可或缺的因素,但宿主自身的免疫力,尤其是黏膜上皮细胞作为抵御念珠菌感染的第一道防线,发挥着重要作用。本文将念珠菌—上皮细胞相互作用研究进展作一综述。     

Mouse model of oropharyngeal candidiasis

Oropharyngeal candidiasis is a frequent cause of morbidity in patients with defects in cell-mediated immunity or saliva production. Animal models of this infection are important for studying disease pathogenesis and evaluating vaccines and antifungal therapies. Here we describe a simple mouse model of oropharyngeal candidiasis. Mice are rendered susceptible to oral infection by injection with cortisone acetate and then inoculated by placing a swab saturated with Candida albicans sublingually. This process results in a reproducible level of infection, the histopathology of which mimics that of pseudomembranous oropharyngeal candidiasis in humans. By using this model, data are obtained after 5-9 d of work.     

泊沙康唑药动学-药效学及其治疗药物监测研究进展

泊沙康唑为新一代三唑类广谱抗真菌药,临床主要用于侵袭性曲霉菌病、念珠菌病的预防和难治性口咽念珠菌病的治疗,具有抗菌 活性高、耐受性好、不良反应少等特点,但其口服后生物利用度具有较大的个体差异。综述泊沙康唑混悬液的药动学影响因素、不同患者 人群的药动学特征以及群体药动学特征、药动学 / 药效学特性、治疗药物监测对临床疗效和不良反应的重要影响,以指导临床个体化用药, 提高用药的有效性和安全性。     

Molecular Cloning and Expression of Yak (Bos grunniens) Lactoferrin cDNA in Pichia pastoris

cDNA encoding lactoferrin from yak was isolated by RT-PCR and then sequenced. The cloned cDNA (2127 bp) encodes a 709 amino acid precursor molecule of yak lactoferrin with a signal peptide of 19 amino acids. The yak lactoferrin cDNA was expressed in Pichia pastoris. The recombinant protein, purified by Ni-NTA affinity column, had a molecular weight of 76 kDa and reacted with an antibody raised against native bovine lactoferrin. The iron-binding behavior and antimicrobial activity of the purified protein indicated that it was correctly folded and functional.     

Metal complexes of lactoferrin and their effect on the intracellular multiplication of Legionella pneumophila

The action of bovine lactoferrin saturated with iron, zinc and manganese on the intracellular multiplication of Legionella pneumophila in HeLa cells has been tested. The results obtained showed that lactoferrin did not influence the invasive efficiency of Legionella. The intracellular multiplication of the bacterium was inhibited by apo-lactoferrin and by lactoferrin saturated with manganese and zinc, whereas lactoferrin saturated with iron enhanced the intracellular growth. Experiments in parallel were performed with iron, manganese and zinc citrate to test the effect due to the metal ions alone. Even in this condition the addition of an iron chelate enhanced the multiplication of Legionella while the manganese chelate produced a certain inhibition.     

Effect of lactoferrin on Helicobacter felis induced gastritis.

Lactoferrin possesses antibiotic, antiinflammatory, and immune-modulating properties that may be active against the gastritis-, ulcer- and cancer-inducing bacterium Helicobacter pylori. In vitro testing of bovine and human lactoferrin by several laboratories has shown significant bacteriostatic and bactericidal activity. Subsequent in vivo testing of bovine lactoferrin in animal models of H. pylori infection has shown beneficial effects of this agent. Our laboratory has utilized a mouse model that is infected with the feline strain of this bacterium, H. felis. The resulting gastritis that develops in this model and the effects of bovine lactoferrin and recombinant human lactoferrin (from Aspergillus niger var. awamori, Agennix Inc., Houston, Tex.) treatment were assessed by various measures. Infected animals treated with orally administered lactoferrin showed reversals in all parameters. In addition, when recombinant human lactoferrin was used in combination with low doses of amoxicillin or tetracycline, there was an enhancement in gastritis-reducing activity. Possible mechanisms for these effects of lactoferrin are discussed. Lactoferrin has significant, orally active in vivo actions and should be further investigated for clinical situations involving Helicobacter infections where it may have utility when administered alone and also when given in combination with established antibiotic agents.     

Binding of bovine lactoferrin to Streptococcus agalactiae

Bovine lactoferrin is an iron-binding protein present in mammary gland secretions. The exposure of Streptococcus agalactiae to bovine lactoferrin resulted in the binding of this protein to all the 12 strains of bovine origin tested, and also, although to a lesser degree, to the five tested strains of human origin. The interaction of lactoferrin with one high-binding bovine strain (24/60, the prototype NT/X strain) was studied. Binding was time-dependent, dose-dependent, and saturable. The binding of lactoferrin was slightly affected by cultivation conditions, and appeared to be heat-stable. The binding of biotinylated lactoferrin was inhibited by unlabelled lactoferrin but not by bovine serum albumin.     

Inhibitory effect of bovine milk lactoferrin on the interaction between a streptococcal surface protein antigen and human salivary agglutinin

Human whole saliva induces aggregation of Streptococcus mutans cells via an interaction between a surface protein antigen (PAc) of the organism and salivary agglutinin. Bovine milk inhibits the saliva-induced aggregation of S. mutans. In this study, the milk component that possesses inhibitory activity against this aggregation was isolated and found to be lactoferrin. Surface plasmon resonance analysis indicated that bovine lactoferrin binds more strongly to salivary agglutinin, especially to high molecular mass glycoprotein, which is a component of the agglutinin, than to recombinant PAc. The binding of bovine lactoferrin to salivary agglutinin was thermostable, and the optimal pH for binding was 4.0. To identify the saliva-binding region of bovine lactoferrin, 11 truncated bovine lactoferrin fragments were constructed. A fragment corresponding to the C-terminal half of the lactoferrin molecule had a strong inhibitory effect on the saliva-induced aggregation of S. mutans, whereas a fragment corresponding to the N-terminal half had a weak inhibitory effect. Seven shorter fragments corresponding to lactoferrin residues 473-538 also showed a high ability to inhibit the aggregation of S. mutans. These results suggest that residues 473-538 of bovine lactoferrin are important in the inhibition of saliva-induced aggregation of S. mutans.     

Oral candidiasis mimicking an oral squamous cell carcinoma: report of a case

doi: 10.1111/j.1741‐2358.2010.00371.x Oral candidiasis mimicking an oral squamous cell carcinoma: report of a case Oral candidiasis is a significant problem in immune‐compromised patients. The most common forms of mucosal candidiasis are oropharyngeal, oesophageal and vaginal, and more than 90% of HIV positive persons will manifest at least one episode of oropharyngeal candidiasis. Local and systemic factors such as uninterrupted daily use of a prosthesis by patients, smoking habit, as well as high glucose intake may contribute to the development of the lesion. The aim of this article is to report an uncommon case of oral candidiasis presenting an aggressive clinical behaviour in a 64‐year‐old male patient, with a significant smoking habit and a medical history of non‐controlled diabetes. The lesion affected the hard and soft palate of the right side, revealing erythematous and ulcerated areas, elevated borders and central portions resembling necrosis, mimicking the clinical features of oral squamous cell carcinoma. However, the correct diagnosis of oral candidiasis was obtained after histopathological and cytological examinations and the patient was easily treated with traditional antifungal drugs and correction of his glucose levels.     

Bovine lactoferrin region responsible for binding to bifidobacterial cell surface proteins

Bovine lactoferrin promotes bifidobacterial growth. Its binding to bifidobacteria is thought to be responsible for such action. After separating the bovine lactoferrin half molecule and extraction of surface proteins from bifidobacteria, binding profiles were observed by immunoblotting. No binding appeared when lactoferrin C-lobe was reacted with the cell surface proteins on a polyvinylidene difluoride membrane. Conversely, a 50-kDa band appeared when the surface proteins were reacted with either intact or nicked bovine lactoferrin. This result strongly suggests that the binding region could be lactoferrin N-lobe. Interestingly, despite the absence of binding, C-lobe enhanced bifidobacterial growth.     

Rapid purification of porcine colostral whey lactoferrin by affinity chromatography on single-stranded DNA-agarose. Characterization, amino acid composition and N-terminal amino acid sequence

We have determined that the major iron-binding and DNA-binding protein in porcine colostral whey is lactoferrin. This lactoferrin was purified to homogeneity in one chromatographic step using immobilized single-stranded DNA-agarose. Although different in chromatographic behavior from human lactoferrin, the porcine lactoferrin purified in this manner was shown to be homogeneous by high-performance ion-exchange chromatography (Mono-S), immobilized metal ion (Cu2+) affinity chromatography, size-exclusion chromatography (TSK-4000SW), and reverse-phase (phenyl) chromatography. Electrophoresis on SDS-polyacrylamide gradient (10-20%) gels under reducing conditions showed the purified lactoferrin to be a single protein (silver-stained) of 78 kDa. Apolactoferrin purified in this manner bound iron and displayed a UV/VIS absorption spectrum indistinguishable from that of human lactoferrin. The molar absorption coefficient of hololactoferrin was 3.86 x 10(3) M-1 at 465 nm and 1.08 x 10(5) M-1 at 280 nm. Affinity elution analyses of the purified lactoferrin on immobilized DNA revealed that the affinity of this protein for DNA was independent of bound iron. Porcine lactoferrin was recognized by antibodies directed against human lactoferrin and bovine lactoferrin. The amino acid composition and N-terminal amino acid sequence analysis (30 residues) revealed a high degree of sequence homology with human, equine and bovine lactoferrin. These results demonstrate the effectiveness of immobilized DNA as a rapid and simple lactoferrin purification procedure and demonstrate the presence of a lactoferrin in porcine colostral whey with a high degree of sequence homology to human lactoferrin.     

Identification of the bactericidal domain of lactoferrin.

We report the existence of a previously unknown antimicrobial domain near the N-terminus of lactoferrin in a region distinct from its iron-binding sites. A single active peptide representing this domain was isolated following gastric pepsin cleavage of human lactoferrin, and bovine lactoferrin, and sequenced by automated Edman degradation. The antimicrobial sequence was found to consist mainly of a loop of 18 amino acid residues formed by a disulfide bond between cysteine residues 20 and 37 of human lactoferrin, or 19 and 36 of bovine lactoferrin. Synthetic analogs of this region similarly exhibited potent antibacterial properties. The active peptide of bovine lactoferrin was more potent than that of human lactoferrin having effectiveness against various Gram-negative and Gram-positive bacteria at concentrations between 0.3 microM and 3.0 microM, depending on the target strain. The effect of the isolated domain was lethal causing a rapid loss of colony-forming capability. Our studies suggest this domain is the structural region responsible for the bacterial properties of lactoferrin.     

Bovine lactoferrin interacts with cable pili of Burkholderia cenocepacia

In this study we evaluated the ability of lactoferrin, the most abundant antimicrobial protein in airway secretions, to bind the surface structures of a Burkholderia strain cystic fibrosis-isolated. Burkholderia cenocepacia is a gram-negative bacterium involved as respiratory pathogen in cystic fibrosis patient infections. This bacterium possesses filamentous structures, named cable pili that have been proposed as virulence factors because of their ability to bind to respiratory epithelia and mucin. Previously, we demonstrated that bovine lactoferrin was able to influence the efficiency of invasion of different iron-regulated morphological forms of B. cenocepacia. Bovine lactoferrin showed to efficiently inhibit invasion of alveolar epithelial cells by free-living bacteria or iron-induced aggregates or biofilm. Results of the present study demonstrate that bovine lactoferrin is also able to specifically bind to B. cenocepacia cells and show that cable pili are involved in this interaction. The attachment of bovine lactoferrin to pili led to a reduced binding of bacterial cells to mucin. Since cable pili are implicated in mediating the bacterial interactions with mucin and epithelial cells, lactoferrin binding to these structures could play an important role in neutralizing bacterial infection in cystic fibrosis patients.     

Proteolytic activity of bovine lactoferrin

Bovine lactoferrin catalyzes the hydrolysis of synthetic substrates (i.e., Z-aminoacyl-7-amido-4-methylcoumarin). Values of Km and kcat for the bovine lactoferrin catalyzed hydrolysis of Z-Phe-Arg-7-amido-4-methylcoumarin are 50 microM and 0.03 s(-1), respectively, the optimum pH value is 7.5 at 25 degrees C. The bovine lactoferrin substrate specificity is similar to that of trypsin, while the hydrolysis rate is several orders of magnitude lower than that of trypsin. The bovine lactoferrin catalytic activity is irreversibly inhibited by the serine-protease inhibitors PMSF and Pefabloc. Moreover, both iron-saturation of the protein and LPS addition strongly inhibit the bovine lactoferrin activity. Interestingly, bovine lactoferrin undergoes partial auto-proteolytic cleavage at positions Arg415-Lys416 and Lys440-Lys441. pKa shift calculations indicate that several Ser residues of bovine lactoferrin display the high nucleophilicity required to potentially catalyze substrate cleavage. However, a definitive identification of the active site awaits further studies.     

Cryptic domains of a 60 kDa heat shock protein of Helicobacter pylori bound to bovine lactoferrin

Abstract Bovine lactoferrin binds to a 60 kDa heat shock protein of Helicobacter pylori . Binding ability was related to human immunoglobulin G because bovine lactoferrin binding proteins were isolated by extraction of cell surface associated proteins with distilled water, applied on IgG-Sepharose and nickel sulphate chelate affinity chromatography. Binding was demonstrated by Western blot after purified protein was digested with α-chymotrypsin and incubated with peroxidase-labeled bovine lactoferrin. Binding was inhibited by bovine lactoferrin, lactose, rhamnose, galactose, and two iron-containing proteins, ferritin and haptoglobin. Helicobacter pylori binds ferritin and haptoglobin via charge or hydrophobic interactions because this binding was not inhibited by specific and various glycoproteins or carbohydrates. Carbohydrate moieties of bovine lactoferrin molecules seem to be involved in binding because glycoproteins with similar carbohydrate structures strongly inhibited binding. Scatchard plot analysis of the binding of peroxidase-labeled bovine lactoferrin to H. pylori cells yielded a k _d 2.88 × 10⁻⁶ M. In addition, binding of H. pylori cells to bovine lactoferrin was enhanced when bacteria treated with pepsin or α-chymotrypsin after isolation from iron-restricted and iron-containing media.     

Different profiles of induced cotton effects of human and bovine lactoferrin by cibacron blue F3GA binding.

1. The properties of complex formation of lactoferrin with Cibacron Blue F3GA dye have been studied by circular dichroic spectral analyses. The Cotton effects were induced by the interaction of lactoferrin with the dye and occurred in the wavelength range from 300 to 450 nm. 2. The patterns of changes in circular dichroic spectra of lactoferrin induced by the dye were different in bovine and human lactoferrin. 3. Iron ions bound to lactoferrin affected the profiles of induced Cotton effects in human lactoferrin but not in bovine lactoferrin. 4. While the dye bound co-operatively to human apo-lactoferrin, such co-operativeness was not observed in iron-saturated human lactoferrin.