Embolic Stroke due to Pulmonary Vein Thrombosis: A Late Complication of Lung Transplantation.

Pulmonary vein thrombosis (PVT) is a rare condition seen almost exclusively in the first two weeks after lung transplantation or lobectomy. Subsequent embolic phenomena are uncommon. Herein, a 47-year-old male with a history of bilateral lung transplantation presented with transient episodes of acute dysphasia and right arm weakness. Brain MRI revealed cortical infarcts in the territory of the left middle cerebral artery. Transesophageal echocardiogram demonstrated a thrombus in the left lower pulmonary vein. This represents the latest manifestation of a PVT reported in the literature-6 years after redo transplantation and 13 years after the original surgery. Investigation for PVT should be considered in any patient with previous lung transplantation that presents with systemic emboli.

Secretory component mediates Candida albicans binding to epithelial cells.

OBJECTIVES: Candida albicans attaches to oral surfaces via a number of mechanisms including adherence mediated by salivary components adsorbed to the C. albicans cell surface. Our goal was to identify the salivary molecules involved. MATERIALS AND METHODS: Biotinylated salivary polypeptides that were bound by C. albicans were detected in extracts from washed, saliva-treated yeast cells by polyacrylamide gel electrophoresis and electroblot or immunoblot transfer analysis and purified by electroelution. Purified material was tested for the ability to promote the adherence of radiolabelled C. albicans yeast cells to cultured epithelial monolayers. RESULTS: Three of the polypeptides bound by C. albicans cells were identified as components of secretory IgA, including secretory component. Using non-denaturing polyacrylamide gel electrophoresis, we demonstrated that secretory component could be detected in its free form in saliva, and was bound by yeast cells. Secretory component which was purified by electroelution from non-denaturing PAGE-separated saliva, without detectable complete IgA, promoted adherence of yeast cells to cultured epithelial monolayers in a dose-dependent fashion. CONCLUSION: These results indicate that despite the inhibitory effect on adherence of IgA specific to C. albicans, IgA components, in particular secretory component, also promote binding to cultured epithelial monolayers.

In vitro expression of Candida albicans alcohol dehydrogenase genes involved in acetaldehyde metabolism.

Alcohol consumption is a risk factor for oral cancer, possibly via its conversion to acetaldehyde, a known carcinogen. The oral commensal yeast Candida albicans may be one of the agents responsible for this conversion intra-orally. The alcohol dehydrogenase (Adh) family of enzymes are involved in acetaldehyde metabolism in yeast but, for C. albicans it is not known which family member is responsible for the conversion of ethanol to acetaldehyde. In this study we determined the expression of mRNAs from three C. albicans Adh genes (CaADH1, CaADH2 and CaCDH3) for cells grown in different culture media at different growth phases by Northern blot analysis and quantitative reverse transcription polymerase chain reaction. CaADH1 was constitutively expressed under all growth conditions but there was differential expression of CaADH2. CaADH3 expression was not detected. To investigate whether CaAdh1p or CaAdh2p can contribute to alcohol catabolism in C. albicans, each gene from the reference strain C. albicans SC5314 was expressed in Saccharomyces cerevisiae. Cell extracts from an CaAdh1p-expressing S. cerevisiae recombinant, but not an CaAdh2p-expressing recombinant, or an empty vector control strain, possessed ethanol-utilizing Adh activity above endogenous S. cerevisiae activity. Furthermore, expression of C. albicans Adh1p in a recombinant S. cerevisiae strain in which the endogenous ScADH2 gene (known to convert ethanol to acetaldehyde in this yeast) had been deleted, conferred an NAD-dependent ethanol-utilizing, and so acetaldehyde-producing, Adh activity. We conclude that CaAdh1p is the enzyme responsible for ethanol use under in vitro growth conditions, and may contribute to the intra-oral production of acetaldehyde.

Detection of Candida albicans ADH1 and ADH2 mRNAs in human archival oral biopsy samples.

OBJECTIVES: The aim of this study was to investigate the relationship between expression of Candida albicans alcohol dehydrogenases (ADH) genes in archival formalin-fixed paraffin-embedded (FFPE) samples from biopsies of leukoplakia. MATERIALS AND METHODS: Archival FFPE samples were obtained from four sample groups: normal oral mucosa, non-dysplastic leukoplakia, chronic hyperplastic candidosis (CHC), and non-CHC dysplastic leukoplakia. The presence of C. albicans was determined by periodic acid Schiff staining and by immunocytochemistry. C. albicans ADH1 and ADH2 mRNAs were detected using reverse transcription PCR. RESULTS: Candida albicans was detected in FFPE samples diagnosed as CHC (the histological diagnoses had been made by specialist oral pathologists, using uniform criteria), but not in any other sample group, including the non-dysplastic leukoplakias. RT-PCR confirmed a significant correlation between the expression of CaADH1 mRNA (P = 0.000), but not for CaADH2 mRNA (P = 0.056) in archival FFPE samples (n = 31) from biopsies of leukoplakia. CONCLUSIONS: Candida albicans was the predominant species in the lesions diagnosed as CHC, and the presence of C. albicans in CHC lesions was associated with a high expression of C. albicans ADH1 mRNA. There was no association between the presence of Candida and malignant transformation in the cases examined; however, the number of cases was limited and further studies are needed to further elucidate the role of C. albicans ADH1 in the pathogenesis of oral squamous cell carcinoma.

Adherence of Candida albicans to silicone is promoted by the human salivary protein SPLUNC2/PSP/BPIFA2.

Interactions between Candida albicans, saliva and saliva-coated oral surfaces are initial events in the colonization of the oral cavity by this commensal yeast, which can cause oral diseases such as candidiasis and denture stomatitis. Candida albicans also colonizes silicone voice prostheses, and the microbial biofilm formed can impair valve function, necessitating frequent prosthesis replacement. We have previously shown that saliva promoted binding of C. albicans cells to silicone in vitro, and that the selective binding of specific salivary proteins to voice prosthesis silicone mediated attachment of C. albicans cells. The C. albicans cells adhered to a polypeptide (or polypeptides) of ~36 kDa eluted from saliva-treated silicone. We show here that a protein of similar size was identified in replicate blots of the eluate from saliva-treated silicone when the blots were probed with antibodies to human SPLUNC2, a salivary protein with reported microbial agglutination properties. In addition, SPLUNC2 was depleted from saliva that had been incubated with silicone coupons. To determine whether SPLUNC2 is a yeast-binding protein, SPLUNC2 cDNA was expressed in Escherichia coli. Purified recombinant His-tagged protein (SPLUNC2r) bound to silicone as demonstrated by immunoblot analysis of an eluate from SPLUNC2r-treated silicone coupons and (35) S-radiolabelled C. albicans cells adhered in a dose-dependent manner to SPLUNC2r-coated silicone. We conclude that SPLUNC2 binds to silicone and acts as a receptor for C. albicans adherence to, and subsequent colonization of, voice prosthesis silicone.

Characterization of two Candida albicans surface mannoprotein adhesins that bind immobilized saliva components.

Salivary components, including the basic proline-rich proteins (bPRP), act as receptors for the adherence of Candida albicans, and this interaction may be important for oral colonization and the development of mucosal Candida infections. Treatment of C. albicans cells with agents affecting either proteinacious or carbohydrate surface macromolecules reduced their adherence in in vitro assays of C. albicans adherence to saliva-coated hydroxyapatite beads and to membrane-immobilized salivary bPRP. In order to identify C. albicans adhesins that bind saliva receptors, yeast cell surface material was extracted by mild glucanase treatment, and was shown to competitively inhibit ( > 50%) the adherence of C. albicans yeast cells in both assays. Concanavalin A sepharose affinity chromatography was used to partially purify glycosylated components of the extract, and two polypeptides (97.4 and 35 kDa) were further purified by preparative SDS PAGE separation and electro-elution. The 97.4 and 35 kDa polypetides each possessed greater adherence-inhibitory specific activity (> 100-fold and > 30-fold respectively) than the original glucanase extract from C. albicans yeast cells. The 35 kDa putative surface protein was identified by N-terminal sequencing and immunoblotting, as the 1,3-beta glucosyltransferase, Bgl2p.

Saliva promotes Candida albicans adherence to human epithelial cells.

Adhesion of Candida cells to oral surfaces is an initial event in pathogenesis. Since specific immobilized salivary components mediate the binding of Candida albicans to hydroxyapatite, we hypothesized that saliva may also promote adherence to oral epithelia via a similar mechanism. In an in vitro model, C. albicans ATCC 10261 yeast cells adhered in a saturable manner to monolayers of three cultured human epithelial cell lines (A549, HEp-2, and HET-1A). The addition of whole saliva to the assay promoted the binding of C. albicans to all cell lines in a dose-dependent manner, but pre-incubation of the epithelial cells with pooled whole saliva had no effect on subsequent adherence. Pre-incubation of the yeast cells with pooled whole saliva, however, significantly enhanced (by up to 120%, P < 0.05) binding to epithelial cell monolayers, and pooled saliva that had been pre-incubated with C. albicans yeast cells was defective in promoting yeast adherence. There was a negative correlation (r = 0.68, P < 0.005) between specific IgA titers against whole cells of C. albicans and adherence-promoting activities for individual saliva samples. The adhesion-inhibitory effect of specific anti-C. albicans IgA was reversed by depletion of IgA from saliva by affinity chromatography. Factors in whole saliva, therefore, bound to the yeast cells, counter the C. albicans-specific salivary IgA inhibitory effect on adhesion and promote the adherence of C. albicans yeast cells to cultured epithelial cells.

The pavA gene of Streptococcus pneumoniae encodes a fibronectin-binding protein that is essential for virulence.

Streptococcus pneumoniae colonizes the nasopharynx in up to 40% of healthy subjects, and is a leading cause of middle ear infections (otitis media), meningitis and pneumonia. Pneumococci adhere to glycosidic receptors on epithelial cells and to immobilized fibronectin, but the bacterial adhesins mediating these reactions are largely uncharacterized. In this report we describe a novel pneumococcal protein PavA, which binds fibronectin and is associated with pneumococcal adhesion and virulence. The pavA gene, present in 64 independent isolates of S. pneumoniae tested, encodes a 551 amino acid residue polypeptide with 67% identical amino acid sequence to Fbp54 protein in Streptococcus pyogenes. PavA localized to the pneumococcal cell outer surface, as demonstrated by immunoelectron microscopy, despite lack of conventional secretory or cell-surface anchorage signals within the primary sequence. Full-length recombinant PavA polypeptide bound to immobilized human fibronectin in preference to fluid-phase fibronectin, in a heparin-sensitive interaction, and blocked binding of wild-type pneumococcal cells to fibronectin. However, a C-terminally truncated PavA' polypeptide (362 aa residues) failed to bind fibronectin or block pneumococcal cell adhesion. Expression of pavA in Enterococcus faecalis JH2-2 conferred > sixfold increased cell adhesion levels to fibronectin over control JH2-2 cells. Isogenic mutants of S. pneumoniae, either abrogated in PavA expression or producing a 42 kDa C-terminally truncated protein, showed up to 50% reduced binding to immobilized fibronectin. Inactivation of pavA had no effects on growth rate, cell morphology, cell-surface physico-chemical properties, production of pneumolysin, autolysin, or surface proteins PspA and PsaA. Isogenic pavA mutants of encapsulated S. pneumoniae D39 were approximately 104-fold attenuated in virulence in the mouse sepsis model. These results provide evidence that PavA fibronectin-binding protein plays a direct role in the pathogenesis of pneumococcal infections.

Intracellular compartmentalization of DNA fragments in cultured airway epithelial cells mediated by cationic lipids.

PURPOSE: The amount and intracellular distribution of DNA fragments (491-bp) was characterized after transfection in vitro with a commercially available cationic lipid. Localization of fragment to the nucleus, its subcellular distribution, and integrity within the cells was determined for various times after transfection. METHODS: Cystic fibrosis (CF) airway epithelial cells were transfected with 32P and FITC labeled single-stranded (ss) or double-stranded (ds) DNA fragments complexed with Lipofectamine at various charge ratios. RESULTS: A 511 (+/-) charge ratio was found to be the optimal ratio for transfection of both ss-and dsDNA. After a 5 h exposure, 7.51 +/- 0.89% of the radioactivity was associated with the nuclear fraction whereas only 1.07 +/- 0.23%, was found in the nuclear fraction when dsDNA was used. The nuclear radioactivity detected after a 24 h exposure was only 1/3 of that after 5 h. Analysis of fragment stability in the cytosolic and nuclear fractions showed the presence of intact fragment in each subcellular compartment. No intranuclear/intracellular fragment could be detected in control experiments with naked DNA. Conclusions. The results from these experiments indicate that small fragments of DNA can be efficiently and rapidly transferred intact to the cell nucleus using cationic lipids and that ssDNA fragments are more effective than dsDNA fragments for nuclear delivery.

Binding properties of Streptococcus gordonii SspA and SspB (antigen I/II family) polypeptides expressed on the cell surface of Lactococcus lactis MG1363.

The oral bacterium Streptococcus gordonii expresses two cell wall-associated polypeptides, designated SspA (1,542 amino acid residues) and SspB (1,462 amino acid residues), that have 70% sequence identity. These polypeptides are members of the antigen I/II family of oral streptococcal adhesins and mediate the binding of streptococci to salivary glycoproteins, collagen, and other oral microorganisms such as Actinomyces naeslundii. To determine if SspA and SspB have differential binding properties, the coding sequences of the sspA and sspB genes were cloned into expression plasmid vector pTREX1-usp45LS to generate pTREX1-sspA and pTREX1-sspB, respectively, and the Ssp polypeptides were displayed on the cell surface of Lactococcus lactis MG1363. Lactococcal cells expressing similar levels of surface SspA or SspB polypeptide were then compared for their abilities to adhere to a range of antigen I/II polypeptide substrates. More than twice as many L. lactis cells expressing SspA bound to immobilized salivary agglutinin glycoprotein (SAG) as did L. lactis cells expressing SspB. In contrast, lactococci expressing SspB adhered twice as well as lactococci producing SspA to collagen type I and to Candida albicans. The binding of A. naeslundii to lactococci was only weakly enhanced by surface expression of Ssp polypeptides. L. lactis(pTREX1-sspB) cells bound in greater numbers to SAG than did Enterococcus faecalis JH2-2 cells expressing SspB from pAM401EB-5. The results suggest that SspA and SspB have markedly different binding affinities for their oral substrates and thus may function to promote site diversity in colonization by S. gordonii.

Candida albicans binding to the oral bacterium Streptococcus gordonii involves multiple adhesin-receptor interactions.

Candida albicans binds to several species of oral streptococci, in particular Streptococcus gordonii, through recognition of a streptococcal cell wall polysaccharide receptor (A. R. Holmes, P. K. Gopal, and H. F. Jenkinson, Infect. Immun. 63:1827-1834, 1995). We now show that isogenic cell surface protein mutants of S. gordonii DL1, unaltered in expression of cell wall polysaccharide, are reduced in ability to support adherence of C. albicans cells in a solid-phase assay. Inactivation of the S. gordonii cshA and cshB genes, encoding high-molecular-mass cell surface polypeptides, and inactivation of the sspA and sspB genes, encoding antigen I/II salivary adhesins, resulted in 40 and 79% reductions, respectively, in adherence of C. albicans cells. Inactivation of the S. gordonii scaA gene encoding a cell surface lipoprotein had no effect on C. albicans adherence. Polyclonal antiserum to streptococcal antigen I/II protein SpaP and antibodies specific to the amino-terminal nonrepetitive (NR) domain of CshA both inhibited adherence of C. albicans to S. gordonii cells. Conversely antibodies to the amino acid repeat block repetitive (R) domain of CshA, or to ScaA, did not inhibit C. albicans adherence. Immobilized recombinant polypeptide fragments of CshA comprising NR domain or R domain sequences both supported adherence of C. albicans cells. Expression of S. gordonii SspB protein on the surface of Enterococcus faecalis conferred on the enterococcal cells the ability to bind C. albicans, and this was ablated by antigen I/II antiserum. Collectively the results suggest that interaction of C. albicans with S. gordonii is mediated by a complement of adhesin-receptor interactions that involves two families of streptococcal multifunctional polypeptide adhesins, bacterial cell wall polysaccharide, and as yet unidentified yeast cell surface components.

Cell surface polypeptide CshA mediates binding of Streptococcus gordonii to other oral bacteria and to immobilized fibronectin.

Isogenic mutants of Streptococcus gordonii DL1 (Challis) in which the genes encoding high-molecular-mass cell surface polypeptides CshA and/or CshB were inactivated were deficient in binding to four strains of Actinomyces naeslundii and two strains of Streptococcus oralis. Lactose-sensitive interactions of S. gordonii with A. naeslundii ATCC 12104 and PK606 were associated with expression of cshA but not of cshB. Lactose-insensitive interactions of S. gordonii with A. naeslundii T14V and WVU627, and with S. oralis C104 and 34, were dependent on expression of cshA and cshB. S. gordonii DL1 cells bound to immobilized human fibronectin (Fn), but not to soluble Fn, in a dose-dependent manner, and binding was noninhibitable by heparin. S. gordonii cshA and cshB mutants were also deficient in binding to immobilized human Fn. Antibodies to an NH2-terminal nonrepetitive region (amino acid residues 42 to 886) of recombinant CshA inhibited binding of S. gordonii DL1 cells to A. naeslundii T14V and PK606 and to immobilized Fn. Conversely, antibodies to an amino acid repeat block segment of the COOH-terminal domain (amino acid residues 2026 to 2508) were not inhibitory to adherence. Assays using CshA-specific antibodies revealed that surface expression of CshA was reduced in cshB mutants. The results suggest that CshA acts as a multifunctional adhesin in S. gordonii and that major adhesion-mediating sequences are specified within the nonrepetitive NH2-terminal region of the polypeptide.

Tandem genes encode cell-surface polypeptides SspA and SspB which mediate adhesion of the oral bacterium Streptococcus gordonii to human and bacterial receptors.

The highly conserved antigen I/II family of polypeptides produced by oral streptococci are believed to be colonization determinants and may mediate adhesion of bacterial cells to salivary glycoproteins absorbed to cells and tissues in the human oral cavity. Streptococcus gordonii is shown to express, on the cell surface, two antigen I/II polypeptides designated SspA and SspB (formerly Ssp-5) that are the products of tandemly arranged chromosomal genes. The structure and arrangement of these genes is similar in two independently isolated strains, DL1 and M5, of S. gordonii. The mature polypeptide sequences of M5 SspA (1539 amino acid (aa) residues) and SspB (1462 aa residues) are almost wholly conserved (98% identical) in the C-terminal regions (from residues 796 in SspA and 719 in SspB, to the respective C-termini), well-conserved (84%) at the N-terminal regions (residues 1-429), and divergent (only 27% identical residues) within the intervening central regions. Insertional inactivation of the sspA gene in S. gordonii DL1 resulted in reduced binding of cells to salivary agglutinin glycoprotein (SAG), human erythrocytes, and to the oral bacterium Actinomyces naeslundii. Further reductions in streptococcal cell adhesion to SAG and to two strains of A. naeslundii were observed when both sspA and sspB genes were inactivated. The results suggest that both SspA and SspB polypeptides are involved in adhesion of S. gordonii cells to human and bacterial receptors.

Peanut sensitivity as a cause of burning mouth.

A patient with a history of a burning tongue together with discomfort of the labial and buccal mucosae was given an elimination diet and skin patch tests to determine the allergen in her diet. The patient was identified as being intolerant of an aqueous peanut extract. Three allergens in peanut butter were identified, the one with greatest reactivity being a heat-stable, water-soluble, nonglycosylated protein with a molecular weight in excess of 10 kD. Modification of her diet has resulted in resolution of the oral problem.