Stephen Challacombe

Candida albicans Secreted Aspartyl Proteinases in Virulence and Pathogenesis

SUMMARY Candida albicans is the most common fungal pathogen of humans and has developed an extensive repertoire of putative virulence mechanisms that allows successful colonization and infection of the host under suitable predisposing conditions. Extracellular proteolytic activity plays a central role in Candida pathogenicity and is produced by a family of 10 secreted aspartyl proteinases (Sap proteins). Although the consequences of proteinase secretion during human infections is not precisely known, in vitro, animal, and human studies have implicated the proteinases in C. albicans virulence in one of the following seven ways: (i) correlation between Sap production in vitro and Candida virulence, (ii) degradation of human proteins and structural analysis in determining Sap substrate specificity, (iii) association of Sap production with other virulence processes of C. albicans, (iv) Sap protein production and Sap immune responses in animal and human infections, (v) SAP gene expression during Candida infections, (vi) modulation of C. albicans virulence by aspartyl proteinase inhibitors, and (vii) the use of SAP-disrupted mutants to analyze C. albicans virulence. Sap proteins fulfill a number of specialized functions during the infective process, which include the simple role of digesting molecules for nutrient acquisition, digesting or distorting host cell membranes to facilitate adhesion and tissue invasion, and digesting cells and molecules of the host immune system to avoid or resist antimicrobial attack by the host. We have critically discussed the data relevant to each of these seven criteria, with specific emphasis on how this proteinase family could contribute to Candida virulence and pathogenesis.

American College of Rheumatology classification criteria for Sjögren's syndrome: a data-driven, expert consensus approach in the Sjögren's International Collaborative Clinical Alliance cohort.

We propose new classification criteria for Sjögrens syndrome (SS), which are needed considering the emergence of biologic agents as potential treatments and their associated comorbidity. These criteria target individuals with signs/symptoms suggestive of SS.

Denture Plaque and Adherence of Candida Albicans To Denture-Base Materials in Vivo and in Vitro

The aim of this paper is to review our understanding of the mechanisms and clinical significance of adhesion of C. albicans to denture-base materials in relation to denture plaque and denture-related stomatitis. Earlier reports in the literature of a 65% prevalence level of denture-related stomatitis seem to be exaggerated. More recent studies indicate that denture-related stomatitis is considerably less common, particularly in normal healthy subjects. The etiology of the condition is discussed in this review, and although much of the literature supports the view that the condition is strongly associated with C. albicans, this is not always so. In some subjects, the cause appears to be related to a non-specific plaque. This review also considers the role of denture plaque in the pathogenesis of denture-related stomatitis, the sequential development of denture plaque, and its colonization by Candida organisms. Designing controlled in vivo studies is difficult, and as a consequence, many investigators have had to resort to in vitro studies. The majority of these studies have attempted to investigate the hydrophobicity of C. albicans, relating the surface free-energy of denture-base materials, particularly acrylic resin, to that of the organism. Surprisingly little work has been directed at surface roughness and how it affects retention of organisms. Further, no attention has been paid to the properties and character of the surface, other than average surface roughness, as it affects adhesion. A comparison of results from in vitro studies on the effect on adhesion of pre-coating the surfaces of denture-base materials with saliva has produced equivocal conclusions. This is largely due to little standardization of experimental protocols between studies, particularly in the collection and handling of the saliva used. In conclusion, the review strongly supports the suggestion that adherence of C. albicans to denture-base materials in vitro is related to the hydrophobicity of the organism. The clinical significance of the observation and the mechanisms for the development and maturation of denture plaque are yet to be understood. There is a clear need for further investigation of other factors that may moderate the adhesion of organisms and subsequent colonization of denture-base materials.

Human papillomaviruses in oral carcinoma and oral potentially malignant disorders: a systematic review

OBJECTIVES Human papillomavirus (HPV) in oral carcinoma (OSCC) and potentially malignant disorders (OPMD) is controversial. The primary aim was to calculate pooled risk estimates for the association of HPV with OSCC and OPMD when compared with healthy oral mucosa as controls. We also examined the effects of sampling techniques on HPV detection rates. METHODS Systematic review was performed using PubMed (January 1966-September 2010) and EMBASE (January 1990-September 2010). Eligible studies included randomized controlled, cohort and cross-sectional studies. Pooled data were analysed by calculating odds ratios, using a random effects model. Risk of bias was based on characteristics of study group, appropriateness of the control group and prospective design. RESULTS Of the 1121 publications identified, 39 cross-sectional studies met the inclusion criteria. Collectively, 1885 cases and 2248 controls of OSCC and 956 cases and 675 controls of OPMD were available for analysis. Significant association was found between pooled HPV-DNA detection and OSCC (OR = 3.98; 95% CI: 2.62-6.02) and even for HPV16 only (OR = 3.86; 95% CI: 2.16-6.86). HPV was also associated with OPMD (OR = 3.87; 95% CI: 2.87-5.21). In a subgroup analysis of OPMD, HPV was also associated with oral leukoplakia (OR = 4.03; 95% CI: 2.34-6.92), oral lichen planus (OR = 5.12; 95% CI: 2.40-10.93), and epithelial dysplasia (OR = 5.10; 95% CI: 2.03-12.80). CONCLUSIONS The results suggest a potentially important causal association between HPV and OSCC and OPMD.

Flow Rates of Resting Whole and Stimulated Parotid Saliva in Relation to Age and Gender

Dry mouth is a common feature in the elderly, but it is not clear what proportion of incidences are related to functional disturbances and whether age per se and gender play a role. The aim of this study was to determine the effects of age and gender on salivary flow rates. The effect of age on unstimulated (resting) whole and stimulated parotid saliva flow rates was determined in 116 unmedicated, healthy individuals. The subjects were divided into four age groups: 20-39 years (group A), 40-59 years (group B), 60-79 years (group C), and 80 years and over (group D). A significant decrease in the secretion rates of unstimulated whole saliva in relation to age was observed in the study population (p < 0.001). However, the flow rates of stimulated parotid saliva were not significantly different in the four age groups. Females had significantly lower mean flow rates than males for both unstimulated (resting) whole saliva (p < 0.005) and stimulated parotid saliva (p < 0.05). In the study as a whole, significant negative correlations were found between either the DMF index (decayed, missing, and filled teeth) or the DMFS index (decayed, missing, and filled tooth surfaces) and the flow rates of unstimulated whole saliva (p < 0.02), but no relationship to stimulated parotid saliva flow rates was apparent. The results suggest that elderly subjects have no impairment in their ability to respond to sialogogues but that resting saliva rates are significantly lower than in younger individuals and may contribute to the increase in oral mucosal diseases seen in the elderly.

ADHERENCE OF CANDIDA ALBICANS TO DENTURE-BASE MATERIALS WITH DIFFERENT SURFACE FINISHES

OBJECTIVES To assess the in vitro adherence of Candida albicans to heat-cured hard and soft denture-base materials with varying surface roughness, and to observe the effect of a mixed salivary pellicle on candidal adhesion to these surfaces. METHODS In vitro adhesion assays on heat-cured acrylic resin (Trevalon), Molloplast B and Novus using the type strain of C. albicans (NCPF 3153A). Surfaces for the assays were prepared using clinically appropriate rotary instruments. Unstimulated, pooled and clarified whole saliva was used to assess its effect on adhesion. RESULTS Significantly greater adhesion of C. albicans to rough rather than smooth surfaces was found (P < 0.001), as well as increased adhesion to the machined soft lining materials compared with acrylic. Pre-coating denture-base materials with saliva reduced candidal adhesion on all materials. CONCLUSIONS Rough surfaces on denture-base materials promote the adhesion of C. albicans in vitro. However, saliva reduces adhesion of C. albicans and thus diminishes the effect of surface roughness and free surface energy differences between materials.

A Biphasic Innate Immune MAPK Response Discriminates between the Yeast and Hyphal Forms of Candida albicans in Epithelial Cells

Summary Discriminating between commensal and pathogenic states of opportunistic pathogens is critical for host mucosal defense and homeostasis. The opportunistic human fungal pathogen Candida albicans is also a constituent of the normal oral flora and grows either as yeasts or hyphae. We demonstrate that oral epithelial cells orchestrate an innate response to C. albicans via NF-κB and a biphasic MAPK response. Activation of NF-κB and the first MAPK phase, constituting c-Jun activation, is independent of morphology and due to fungal cell wall recognition. Activation of the second MAPK phase, constituting MKP1 and c-Fos activation, is dependent upon hypha formation and fungal burdens and correlates with proinflammatory responses. Such biphasic response may allow epithelial tissues to remain quiescent under low fungal burdens while responding specifically and strongly to damage-inducing hyphae when burdens increase. MAPK/MKP1/c-Fos activation may represent a “danger response” pathway that is critical for identifying and responding to the pathogenic switch of commensal microbes.

Quantitative expression of the Candida albicans secreted aspartyl proteinase gene family in human oral and vaginal candidiasis

A quantitative real-time RT-PCR system was established to identify which secreted aspartyl proteinase (SAP) genes are most highly expressed and potentially contribute to Candida albicans infection of human epithelium in vitro and in vivo. C. albicans SC5314 SAP1-10 gene expression was monitored in organotypic reconstituted human epithelium (RHE) models, monolayers of oral epithelial cells, and patients with oral (n=17) or vaginal (n=17) candidiasis. SAP gene expression was also analysed in Deltasap1-3, Deltasap4-6, Deltaefg1 and Deltaefg1/cph1 mutants to determine whether compensatory SAP gene regulation occurs in the absence of distinct proteinase gene subfamilies. In monolayers, RHE models and patient samples SAP9 was consistently the most highly expressed gene in wild-type cells. SAP5 was the only gene significantly upregulated as infection progressed in both RHE models and was also highly expressed in patient samples. Interestingly, the SAP4-6 subfamily was generally more highly expressed in oral monolayers than in RHE models. SAP1 and SAP2 expression was largely unchanged in all model systems, and SAP3, SAP7 and SAP8 were expressed at low levels throughout. In Deltasap1-3, expression was compensated for by increased expression of SAP5, and in Deltasap4-6, expression was compensated for by SAP2: both were observed only in the oral RHE. Both Deltasap1-3 and Deltasap4-6 mutants caused RHE tissue damage comparable to the wild-type. However, addition of pepstatin A reduced tissue damage, indicating a role for the Sap family as a whole in inducing epithelial damage. With the hypha-deficient mutants, RHE tissue damage was significantly reduced in both Deltaefg1/cph1 and Deltaefg1, but SAP5 expression was only dramatically reduced in Deltaefg1/cph1 despite the absence of hyphal growth in both mutants. This indicates that hypha formation is the predominant cause of tissue damage, and that SAP5 expression can be hypha-independent and is not solely controlled by the Efg1 pathway but also by the Cph1 pathway. This is believed to be the first study to fully quantify SAP gene expression levels during human mucosal infections; the results suggest that SAP5 and SAP9 are the most highly expressed proteinase genes in vivo. However, the overall contribution of the Sap1-3 and Sap4-6 subfamilies individually in inducing epithelial damage in the RHE models appears to be low.

The severity of cutaneous and oral pemphigus is related to desmoglein 1 and 3 antibody levels.

Background Pemphigus vulgaris (PV) and foliaceus (PF) are characterized by antibodies to the desmosomal proteins desmoglein 3 (Dsg3) and desmoglein 1 (Dsg1), respectively. Past studies using indirect immunofluorescence (IIF) as a measure of pemphigus antibody levels have failed to demonstrate consistently a relationship between disease severity and IIF titres. However, IIF is not able to measure separately Dsg1 and 3 antibodies, unlike enzyme‐linked immunosorbent assays (ELISA), which utilize recombinant proteins.

Activation-induced cytidine deaminase expression in follicular dendritic cell networks and interfollicular large B cells supports functionality of ectopic lymphoid neogenesis in autoimmune sialoadenitis and MALT lymphoma in Sjögren's syndrome

Demonstration of ectopic germinal center-like structures (GC-LSs) in chronically inflamed tissues in patients with autoimmune disorders is a relatively common finding. However, to what extent ectopic lymphoid structures behave as true GC and are able to support class switch recombination (CSR) and somatic hypermutation (SHM) of the Ig genes is still debated. In addition, no information is available on whether CSR and SHM can take place in the absence of GCs at extrafollicular sites in an ectopic lymphoid tissue. In this study, we show that in salivary glands (SGs) of Sjögren’s syndrome (SS) activation-induced cytidine deaminase (AID), the enzyme responsible for CSR and SHM is invariably expressed within follicular dendritic cell (FDC) networks but is not detectable in SGs in the absence of ectopic GC-LSs, suggesting that FDC networks play an essential role in sustaining the Ag-driven B cell proliferation within SS-SGs. We also show that the recently described population of interfollicular large B cells selectively expresses AID outside ectopic GC in the T cell-rich areas of periductal aggregates. Finally, we report that AID retains its exclusive association with numerous, residual GCs in parotid SS-MALT lymphomas, whereas neoplastic marginal zone-like B cells are consistently AID negative. These results strongly support the notion that ectopic lymphoid structures in SS-SGs express the molecular machinery to support local autoantibody production and B cell expansion and may play a crucial role toward lymphomagenesis.