The genetic background determines material-induced bone formation through the macrophage-osteoclast axis.

Osteoinductive materials are characterized by their ability to induce bone formation in ectopic sites. Thus, osteoinductive materials hold promising potential for repairing bone defects. However, the mechanism of material-induced bone formation remains unknown, which limits the design of highly potent osteoinductive materials. Here, we demonstrated a genetic background link among macrophage polarization, osteoclastogenesis and material-induced bone formation. The intramuscular implantation of an osteoinductive material in FVB/NCrl (FVB) mice resulted in more M2 macrophages at week 1, more osteoclasts at week 2 and increased bone formation after week 4 compared with the results obtained in C57BL/6JOlaHsd (C57) mice. Similarly, in vitro, with a greater potential to form M2 macrophages, monocytes derived from FVB mice formed more osteoclasts than those derived from C57 mice. A transcriptomic analysis identified Csf1, Cxcr4 and Tgfbr2 as the main genes controlling macrophage-osteoclast coupling, which were further confirmed by related inhibitors. With such coupling, macrophage polarization and osteoclast formation of monocytes in vitro successfully predicted in vivo bone formation in four other mouse strains. Considering material-induced bone formation as an example of acquired heterotopic bone formation, the current findings shed a light on precision medicine for both bone regeneration and the treatment of pathological heterotopic bone formation.

Focused ultrasound-mediated small-molecule delivery to potentiate immune checkpoint blockade in solid tumors.

In the last decade, immune checkpoint blockade (ICB) has revolutionized the standard of treatment for solid tumors. Despite success in several immunogenic tumor types evidenced by improved survival, ICB remains largely unresponsive, especially in "cold tumors" with poor lymphocyte infiltration. In addition, side effects such as immune-related adverse events (irAEs) are also obstacles for the clinical translation of ICB. Recent studies have shown that focused ultrasound (FUS), a non-invasive technology proven to be effective and safe for tumor treatment in clinical settings, could boost the therapeutic effect of ICB while alleviating the potential side effects. Most importantly, the application of FUS to ultrasound-sensitive small particles, such as microbubbles (MBs) or nanoparticles (NPs), allows for precise delivery and release of genetic materials, catalysts and chemotherapeutic agents to tumor sites, thus enhancing the anti-tumor effects of ICB while minimizing toxicity. In this review, we provide an updated overview of the progress made in recent years concerning ICB therapy assisted by FUS-controlled small-molecule delivery systems. We highlight the value of different FUS-augmented small-molecules delivery systems to ICB and describe the synergetic effects and underlying mechanisms of these combination strategies. Furthermore, we discuss the limitations of the current strategies and the possible ways that FUS-mediated small-molecule delivery systems could boost novel personalized ICB treatments for solid tumors.

The nano-windmill exerts superior anti-inflammatory effects via reducing choline uptake to inhibit macrophage activation.

Macrophages' activation plays a central role during the development and progression of inflammation, while the regulation of metabolic reprogramming of macrophages has been recently identified as a novel strategy for anti-inflammatory therapies. Our previous studies have found that tetrahedral framework nucleic acid (tFNA) plays a mild anti-inflammatory effect by inhibiting macrophage activation, but the specific mechanism remains unclear. Here, by metabolomics and RNA sequencing, choline uptake is identified to be significantly repressed by decreased slc44a1 expression in tFNA-treated activated macrophages. Inspired by this result, combined with the excellent delivery capacities of tFNA, siR-slc44a1 is loaded into the tFNA to develop a new tFNA-based small interfering RNA (siRNA) delivery system named 'nano-windmill,' which exhibits a synergetic role by targeting slc44a1, finally blowing up the anti-inflammatory effects of tFNA to inhibit macrophages activation via reducing choline uptake. By confirming its anti-inflammatory effects in chronic (periodontitis) and acute (sepsis) inflammatory disease, the tFNA-based nanomedicine developed for inflammatory diseases may provide broad prospects for tFNA upgrading and various biological applications such as anti-inflammatory.

A Mechanically Reinforced Super Bone Glue Makes a Leap in Hard Tissue Strong Adhesion and Augmented Bone Regeneration.

Existing bone tissue engineering strategies aim to achieve minimize surgical trauma, stabilize the injured area, and establish a dynamic osteogenic microenvironment. The cutting-edge bone glue developed in this study satisfies these criteria. Inspired by the excellent adhesive properties of mussels, herein, a super osteogenic glue (L-DPZ) that integrates poly(vinyl alcohol), L-dopa amino acid, and zeolitic imidazolate framework-8 characterized by catechol-metal coordination is used to successfully adhere to hard tissue with a maximum adhesive strength of 10 MPa, which is much higher than those of commercial and previously reported bone glues. The stable hard tissue adhesion also enables it to adhere strongly to luxated or broken teeth, Bio-Oss (a typical bone graft material), and splice fragments from comminuted fractures of the rabbit femur. Then, it is testified that the L-DPZ hydrogels exhibit satisfactory biocompatibility, stable degradability, and osteogenic ability in vitro. Moreover, the ability to anchor Bio-Oss and sustained osteogenesis of L-DPZ result in satisfactory healing in calvarial bone defect models in rabbits, as observed by increased bone thickness and the ingrowth of new bone tissue. These results are expected to demonstrate solutions to clinical dilemmas such as comminuted bone fracture fixation, bone defect reconstruction, and teeth dislocation replantation.

Microbial Analysis of Obturators During Maxillofacial Prosthodontic Treatment Over an 8-Year Period.

The aim of the study was to investigate the microbial colonization (by Candida species, anaerobic and facultative anaerobic bacteria) of maxillary obturators used for the restoration of maxillary defects, including during radiotherapy.Retrospective cohort study.Fifteen patients requiring a maxillary obturator prosthesis had swabs of their obturators and adjacent tissues taken at different stages of their treatment over a period of 8 years.Identification of microbial species from the swabs was carried out using randomly amplified polymorphic DNA polymerase chain reaction (RAPD PCR) analysis, checkerboard DNA-DNA hybridization, CHROMagar Candida chromogenic agar, and DNA sequencing.Candida species were detected in all patients and all patients developed mucositis and candidiasis during radiotherapy which was associated with an increase in colonization of surfaces with Candida spp., particularly C albicans. Microbial colonization increased during radiotherapy and as an obturator aged, and decreased following a reline, delivery of a new prosthesis, or antifungal treatment during radiotherapy.Microbial colonization of maxillary obturators was related to the stage of treatment, age of the obturator material, radiotherapy and antifungal medications, and antifungal treatment may be recommended if C albicans colonization of palatal tissues is greater than 105 colony-forming units per cm2 following the first week of radiotherapy.

Prevalence of halitosis among young adults in Dunedin, New Zealand.

OBJECTIVES: To investigate the prevalence of halitosis in young adults. METHODS: Young adults (n = 372; mean age = 21.0 ± 2.6 years old, range = 18-30 years) in Dunedin, New Zealand, were recruited into the cross sectional study after providing informed consent. The prevalence of halitosis was determined using both objective measurements (parts per billion [ppb] volatile sulphur compounds [VSCs] in the exhaled air) and subjective measurements (self-reported halitosis questionnaire, tongue coating index, and organoleptic assessment). RESULTS: Volatile sulphur compounds measurements indicated that the prevalence of halitosis (values ≥140 ppb) was 31.2%; 25.6% of participants self-reported halitosis. The organoleptic assessment revealed that 14.3% of the participants had a score of ≥2. A positive correlation was found between the VSC measurements and organoleptic assessment (p < 0.05). No significant relationship was found between self-reported halitosis and either organoleptic assessment or VSC measurements. Self-reported dry mouth, smoking, oral hygiene index, DMFT index, and tongue coating score were significantly associated with the organoleptic assessment (p < 0.05). The self-reported dry mouth, mouth breathing and tongue coating score were significantly associated with the VSC scores (p < 0.05). CONCLUSION: Halitosis, as represented by VSC, was found in 31.2% of the participants. VSC scores and organoleptic assessment were positively correlated. There was no significant relationship between self-reported halitosis and either organoleptic assessment or VSC measurements.

Complex Interactions Between Circulating Fatty Acid Levels, Desaturase Activities, and the Risk of Gestational Diabetes Mellitus: A Prospective Cohort Study.

Objective: Maternal abnormal fatty acid desaturation has previously been linked to gestational diabetes mellitus (GDM). However, few studies have investigated this relationship longitudinally throughout pregnancy. In this study, we investigated the relationship between GDM and desaturase activities across the pregnancy trimesters. Methods: A total of 661 women (GDM = 189, non-GDM = 472) were selected from the Complex Lipids in Mothers and Babies (CLIMB) cohort study. Clinical information and maternal serum were collected at 11-14, 22-28, and 32-34 weeks of gestation. Totally, 20 serum fatty acids were quantified using gas chromatography-mass spectrometry (GC-MS) analysis at each timepoint. Polyunsaturated fatty acid (PUFA) product-to-precursor ratios were used to estimate desaturase and elongase activities including delta-5 desaturase, delta-6 desaturase, stearoyl-CoA desaturase, and elongase. Results: After adjusting for major potential confounders including maternal age, BMI, primiparity, smoking, and alcohol consumption, we observed a significant increase in the levels of γ-linolenic acid (GLA) and eicosatrienoic acid (DGLA) in the first trimester of women with GDM, whereas GLA and DGLA were reduced in the third trimester, when compared to the non-GDM group. Arachidonic acid (AA) showed an upward trend in the GDM group throughout pregnancy. Estimated delta-6 desaturase and delta-5 desaturase activity were elevated in the first trimester (OR = 1.40, 95% CI 1.03-1.91; OR = 0.56, 95% CI 0.32-0.96) but attenuated in the third trimester (OR = 0.78, 95% CI 0.58-1.07; OR = 2.64, 95% CI 1.46-4.78) in GDM pregnancies, respective to controls. Estimated delta-9-18 desaturase activity (OR = 3.70, 95% CI 1.49-9.19) was increased in women with GDM in later pregnancy. Conclusions: Our study highlights the potential importance of fatty acid desaturase activities, particularly estimated delta-5 desaturase and delta-9-18 desaturase in the pathophysiology of GDM. These findings may have applications for the early diagnosis and management of GDM.

CD39/CD73 Dysregulation of Adenosine Metabolism Increases Decidual Natural Killer Cell Cytotoxicity: Implications in Unexplained Recurrent Spontaneous Abortion.

Unexplained recurrent spontaneous abortion (URSA) is believed to be associated with impaired immunosuppression at the maternal-fetal interface, but the detailed molecular mechanism remains unclear. The ATP-adenosine metabolic pathway regulated by CD39/CD73 has recently been recognized to be important in immunosuppression. This study aimed to investigate the regulation of decidual natural killer (dNK) cells and fetal extravillous trophoblast (EVT) cells by CD39 and CD73 in URSA, as well as the possible regulatory mechanism of CD39/CD73 via the TGF-ß-mTOR-HIF-1α pathway using clinical samples and cell models. Fewer CD39+ and CD73+ cells were found in the URSA decidual and villous tissue, respectively. Inhibition of CD39 on dNK cells transformed the cells to an activated state with increased toxicity and decreased apoptosis, and changed their cytokine secretion, leading to impaired invasion and proliferation of the co-cultured HTR8/SVneo cells. Similarly, inhibition of CD73 on HTR8/SVneo cells decreased the adenosine concentration in the cell culture media, increased the proportion of CD107a+ dNK cells, and decreased the invasion and proliferation capabilities of the HTR8/SVneo cells. In addition, transforming growth factor-ß (TGF-ß) triggered phosphorylation of mammalian target of rapamycin (mTOR) and Smad2/Smad3, which subsequently activated hypoxia-inducible factor-1α (HIF-1α) to induce the CD73 expression on the HTR8/SVneo cells. In summary, reduced numbers of CD39+ and CD73+ cells at the maternal-fetal interface, which may be due to downregulated TGF-ß-mTOR-HIF-1α pathway, results in reduced ATP-adenosine metabolism and increased dNK cytotoxicity, and potentially contributes to URSA occurrences.

Engineering a Cysteine-Deficient Functional Candida albicans Cdr1 Molecule Reveals a Conserved Region at the Cytosolic Apex of ABCG Transporters Important for Correct Folding and Trafficking of Cdr1.

Pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters of the ABCG family are eukaryotic membrane proteins that pump an array of compounds across organelle and cell membranes. Overexpression of the archetype fungal PDR transporter Cdr1 is a major cause of azole antifungal drug resistance in Candida albicans, a significant fungal pathogen that can cause life-threatening invasive infections in immunocompromised individuals. To date, no structure for any PDR transporter has been solved. The objective of this project was to investigate the role of the 23 Cdr1 cysteine residues in the stability, trafficking, and function of the protein when expressed in the eukaryotic model organism, Saccharomyces cerevisiae The biochemical characterization of 18 partially cysteine-deficient Cdr1 variants revealed that the six conserved extracellular cysteines were critical for proper expression, localization, and function of Cdr1. They are predicted to form three covalent disulfide bonds that stabilize the large extracellular domains of fungal PDR transporters. Our investigations also revealed a novel nucleotide-binding domain motif, GX2[3]CPX3NPAD/E, at the peripheral cytosolic apex of ABCG transporters that possibly contributes to the unique ABCG transport cycle. With this knowledge, we engineered an "almost cysteine-less," yet fully functional, Cdr1 variant, Cdr1P-CID, that had all but the six extracellular cysteines replaced with serine, alanine, or isoleucine (C1106I of the new motif). It is now possible to perform cysteine-cross-linking studies that will enable more detailed biochemical investigations of fungal PDR transporters and confirm any future structure(s) solved for this important protein family.IMPORTANCE Overexpression of the fungal pleiotropic drug resistance (PDR) transporter Cdr1 is a major cause of antifungal drug resistance in Candida albicans, a significant fungal pathogen that can cause life-threatening invasive infections in immunocompromised individuals. To date, no structure for any PDR ABC transporter has been solved. Cdr1 contains 23 cysteines; 10 are cytosolic and 13 are predicted to be in the transmembrane or the extracellular domains. The objective of this project was to create, and biochemically characterize, CDR1 mutants to reveal which cysteines are most important for Cdr1 stability, trafficking, and function. During this process we discovered a novel motif at the cytosolic apex of PDR transporters that ensures the structural and functional integrity of the ABCG transporter family. The creation of a functional Cys-deficient Cdr1 molecule opens new avenues for cysteine-cross-linking studies that will facilitate the detailed characterization of an important ABCG transporter family member.

The Role of Biofilms and Material Surface Characteristics in Microbial Adhesion to Maxillary Obturator Materials: A Literature Review.

BACKGROUND: Maxillofacial prosthetics includes restoration of maxillary defects resulting from resection of palate and nasosinus neoplasms with obturator prostheses which may be colonized by microorganisms and function as a reservoir of infection. Patients with neoplasms commonly also require radiotherapy that can result in changes in saliva quality and quantity and changes in the oral microbial flora. The altered flora, in individuals immunocompromised from cancer therapy, increases their risk of prosthesis-related infections. OBJECTIVES: In this review article, we explore microbial biofilms, their main components, mechanisms of microbial adhesion, and stages of biofilm development. We also discuss the different materials that are used for manufacturing maxillary obturators, their characteristic features, and how these can affect microbial adhesion. Furthermore, we shed some light on the factors that affect microbial adhesion to the surface of maxillary obturators including tissue proteins, protein adsorption, and the acquired enamel pellicle. CONCLUSIONS: The conclusions drawn from this literature review are that it is imperative to minimize the risk of local and systemic infections in immunocompromised patients with cancer having maxillary defects. It is also important to determine the role of saliva in microbial adhesion to obturator materials as well as develop materials that have a longer life span with surface characteristics that promote less microbial adhesion than current materials.

Newly identified motifs in Candida albicans Cdr1 protein nucleotide binding domains are pleiotropic drug resistance subfamily-specific and functionally asymmetric.

An analysis of Candida albicans ABC transporters identified conserved related α-helical sequence motifs immediately C-terminal of each Walker A sequence. Despite the occurrence of these motifs in ABC subfamilies of other yeasts and higher eukaryotes, their roles in protein function remained unexplored. In this study we have examined the functional significance of these motifs in the C. albicans PDR transporter Cdr1p. The motifs present in NBD1 and NBD2 were subjected to alanine scanning mutagenesis, deletion, or replacement of an entire motif. Systematic replacement of individual motif residues with alanine did not affect the function of Cdr1p but deletion of the M1-motif in NBD1 (M1-Del) resulted in Cdr1p being trapped within the endoplasmic reticulum. In contrast, deletion of the M2-motif in NBD2 (M2-Del) yielded a non-functional protein with normal plasma membrane localization. Replacement of the motif in M1-Del with six alanines (M1-Ala) significantly improved localization of the protein and partially restored function. Conversely, replacement of the motif in M2-Del with six alanines (M2-Ala) did not reverse the phenotype and susceptibility to antifungal substrates of Cdr1p was unchanged. Together, the M1 and M2 motifs contribute to the functional asymmetry of NBDs and are important for maturation of Cdr1p and ATP catalysis, respectively.

Drug resistance is conferred on the model yeast Saccharomyces cerevisiae by expression of full-length melanoma-associated human ATP-binding cassette transporter ABCB5.

ABCB5, an ATP-binding cassette (ABC) transporter, is highly expressed in melanoma cells, and may contribute to the extreme resistance of melanomas to chemotherapy by efflux of anti-cancer drugs. Our goal was to determine whether we could functionally express human ABCB5 in the model yeast Saccharomyces cerevisiae, in order to demonstrate an efflux function for ABCB5 in the absence of background pump activity from other human transporters. Heterologous expression would also facilitate drug discovery for this important target. DNAs encoding ABCB5 sequences were cloned into the chromosomal PDR5 locus of a S. cerevisiae strain in which seven endogenous ABC transporters have been deleted. Protein expression in the yeast cells was monitored by immunodetection using both a specific anti-ABCB5 antibody and a cross-reactive anti-ABCB1 antibody. ABCB5 function in recombinant yeast cells was measured by determining whether the cells possessed increased resistance to known pump substrates, compared to the host yeast strain, in assays of yeast growth. Three ABCB5 constructs were made in yeast. One was derived from the ABCB5-ß mRNA, which is highly expressed in human tissues but is a truncation of a canonical full-size ABC transporter. Two constructs contained full-length ABCB5 sequences: either a native sequence from cDNA or a synthetic sequence codon-harmonized for S. cerevisiae. Expression of all three constructs in yeast was confirmed by immunodetection. Expression of the codon-harmonized full-length ABCB5 DNA conferred increased resistance, relative to the host yeast strain, to the putative substrates rhodamine 123, daunorubicin, tetramethylrhodamine, FK506, or clorgyline. We conclude that full-length ABCB5 can be functionally expressed in S. cerevisiae and confers drug resistance.

Response of human periodontal ligament cells to baicalin.

BACKGROUND: Periodontitis is the most common cause of tooth loss in adults. Periodontal ligament cell (PLC)-based therapy is considered one of the most promising methods in periodontal tissue regeneration. The traditional Chinese medicine baicalin has been shown to possess antimicrobial and anti-inflammatory activities and enhance cell proliferation and alkaline phosphatase activity. The aim of this study is to investigate the response of human PLCs (HPLCs) to baicalin. METHODS: The effect of baicalin on cultured HPLC proliferation was measured with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The effect of baicalin on the expression of osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL), core binding factor α1 (Cbfα1), and osteocalcin (OC) was determined by quantitative real-time polymerase chain reaction and immunodetection. RESULTS: Baicalin at a concentration of 0.01 µg/mL promoted HPLC proliferation, upregulated OPG messenger RNA (mRNA) and protein expression, and downregulated RANKL mRNA and protein expression. In addition to reducing the RANKL/OPG expression ratio significantly, it also increased Cbfα1 and OC mRNA and protein expression. CONCLUSION: Baicalin showed multifaceted regulation of genes with important roles in tissue growth and differentiation, and thus it has the potential to be a promising candidate for HPLC-based periodontal regeneration therapy.

Heterologous expression of Candida albicans Pma1p in Saccharomyces cerevisiae.

Candida albicans is a major cause of opportunistic and life-threatening systemic fungal infections, especially in the immunocompromised. The plasma membrane proton-pumping ATPase (Pma1p) is an essential enzyme that generates the electrochemical gradient required for cell growth. We expressed C. albicans Pma1p (CaPma1p) in Saccharomyces cerevisiae to facilitate screening for inhibitors. Replacement of S. cerevisiae PMA1 with C. albicans PMA1 gave clones expressing CaPma1p that grew slowly at low pH. CaPma1p was expressed at significantly lower levels and had lower specific activity than the native Pma1p. It also conferred pH sensitivity, hygromycin B resistance, and low levels of glucose-dependent proton pumping. Recombination between CaPMA1 and the homologous nonessential ScPMA2 resulted in chimeric suppressor mutants that expressed functional CaPma1p with improved H(+) -ATPase activity and growth rates at low pH. Molecular models of suppressor mutants identified specific amino acids (between 531 and 595 in CaPma1p) that may affect regulation of the activity of Pma1p oligomers in S. cerevisiae. A modified CaPma1p chimeric construct containing only 5 amino acids from ScPma2p enabled the expression of a fully functional enzyme for drug screens and structural resolution.

Adhesion of yeast and bacteria to oral surfaces.

Colonization of surfaces in the human body by microorganisms is an early, essential, step in the initiation of infectious disease. We have developed in vitro assays to investigate interactions between yeast or bacterial cells and human tissues, fluids, or prostheses. Such assays can be used to identify the adhesins, ligands, and receptors involved in these interactions, for example by determining which components of the microbe or human tissue/fluid interfere with adherence in the assay. The assays can also be applied to finding ways of preventing adhesion, and subsequent disease, by investigating the effects of different conditions and added compounds on adherence in the in vitro assays. We describe six assays for measuring adhesion of the oral yeast Candida albicans, a common commensal and opportunistic pathogen, or the bacterium Staphylococcus epidermidis, which is not normally pathogenic but is known to form biofilms on medical prostheses. The assays described represent two approaches to investigating adhesion; retention at a fixed time point following liquid washes; and retention against a continuous flow of medium.

ABC transporter Cdr1p contributes more than Cdr2p does to fluconazole efflux in fluconazole-resistant Candida albicans clinical isolates.

Fluconazole (FLC) remains the antifungal drug of choice for non-life-threatening Candida infections, but drug-resistant strains have been isolated during long-term therapy with azoles. Drug efflux, mediated by plasma membrane transporters, is a major resistance mechanism, and clinically significant resistance in Candida albicans is accompanied by increased transcription of the genes CDR1 and CDR2, encoding plasma membrane ABC-type transporters Cdr1p and Cdr2p. The relative importance of each transporter protein for efflux-mediated resistance in C. albicans, however, is unknown; neither the relative amounts of each polypeptide in resistant isolates nor their contributions to efflux function have been determined. We have exploited the pump-specific properties of two antibody preparations, and specific pump inhibitors, to determine the relative expression and functions of Cdr1p and Cdr2p in 18 clinical C. albicans isolates. The antibodies and inhibitors were standardized using recombinant Saccharomyces cerevisiae strains that hyper-express either protein in a host strain with a reduced endogenous pump background. In all 18 C. albicans strains, including 13 strains with reduced FLC susceptibilities, Cdr1p was present in greater amounts (2- to 20-fold) than Cdr2p. Compounds that inhibited Cdr1p-mediated function, but had no effect on Cdr2p efflux activity, significantly decreased the resistance to FLC of seven representative C. albicans isolates, whereas three other compounds that inhibited both pumps did not cause increased chemosensitization of these strains to FLC. We conclude that Cdr1p expression makes a greater functional contribution than does Cdr2p to FLC resistance in C. albicans.

Antifungal saponins from Paris polyphylla Smith.

Three steroidal saponins, including one new and two known compounds, were isolated from the rhizomes of Paris polyphylla Smith. One- and two-dimensional NMR, LC-MS, and interpretation of hydrolytic cleavage experiments led to the identification of the structure of the new saponin as ( 25R)-spirost-5-ene-3 beta,17 alpha-diol (pennogenin) 3- O-{ O- alpha- L-rhamnopyranosyl-(1-->2)- O-[ O- beta-xylopyranosyl-(1-->5)- alpha- L-arabinofuranosyl-(1-->4)]- beta- D-glucopyranoside}. The isolated saponins were evaluated for their antifungal activity against Cladosporium cladosporioides and Candida species and showed comparable activity to chemicals used in some commercial products.

Production from dairy cows of semi-industrial quantities of milk-protein concentrate (MPC) containing efficacious anti-Candida albicans IgA antibodies.

Bovine milk antibodies directed against human pathogenic organisms have potential as prophylactic or therapeutic treatments of disorders affecting mucosal surfaces. The cow, however, does not naturally secrete high levels of IgA in milk, the predominant immunoglobulin of the mucosal immune system. We have patented an immunisation protocol that results in increased production of IgA in ruminant milk and in this study established that our protocol can be used on a scale sufficient to produce semi-industrial quantities of milk for processing. Cows were immunised with a common pathogenic yeast, Candida albicans and responded with high levels of antigen-specific IgA antibodies in their milk. The spray-dried milk-protein concentrate (85% protein) powder was shown to reduce adherence of Cand. albicans cells in in vitro adherence assays, demonstrating an ability to retain efficacy through the processing. These results suggest that this milk product may be of therapeutic value if the reduction in Cand. albicans adhesion observed in vitro translates to reduced colonisation in vivo.

Amino acid residues affecting drug pump function in Candida albicans--C. albicans drug pump function.

Membrane-located drug transporters are important components in the multidrug resistance of microbial cells and human tissues. In fungi, clinically important resistance to antifungal drugs most often results from the over-expression of efflux pump proteins in the plasma membrane of the resistant cell. This review describes studies of the ATP binding cassette (ABC) family of membrane efflux pumps in the opportunistic human pathogen Candida albicans and, in particular, examines how changes in the polypeptide sequence can affect pump function. The identification of amino acid residues affecting pump function can provide new insights into efflux pump mechanisms and the relationship between structure and function. Such information will be important for the design of pump inhibitors which could supplement existing antifungal drugs.

Candida albicans binds to saliva proteins selectively adsorbed to silicone.

Explanted voice prostheses obtained from 5 patients at the time of prosthesis replacement were consistently colonized by yeast, in particular Candida albicans. A simple, reproducible, in vitro model of C. albicans adherence to saliva-coated voice prosthesis silicone was developed. Whole saliva promoted adherence of C. albicans to silicone in a dose-dependent manner. Saliva rinses from voice prosthesis patients also promoted binding of C. albicans to silicone in vitro (mean adherence 14.9% +/- 2.8% of input C. albicans cells). This was significantly higher than C. albicans adherence to silicone in the absence of saliva (P < .001) or adherence promoted by saliva rinses from healthy volunteers (P < .005). Polyacrylamide gel electrophoresis analysis and a blot overlay adherence assay revealed that certain salivary proteins were selectively adsorbed to silicone and that C. albicans yeast cells adhered specifically to the adsorbed salivary proteins.