Oral biology of human immunodeficiency virus-infected individuals in Hong Kong

published_or_final_versionDentistryDoctoralDoctor of Philosoph

Purpurin alters the architecture of Candida albicans biofilms

Objectives: To investigate the in vitro effects of purpurin on Candida albicans hyphal development and biofilm formation. Methods: C. albicans SC 5314 biofilms were prepared in polystyrene, flat bottom 96-well microtiter plates. The cell suspension (100 µl, 107cells/ml in YNB) was transferred to the wells and incubated at 37oC, 75 rpm for 1.5 h. After the removal of loosely adherent cells by washing, fresh YNB (200 µl) was added to the wells and incubated for 24 h for biofilm formation. Thereafter, the wells were washed with PBS and purpurin (200 µl, 5µg/ml in YNB) was added and incubated at similar conditions for 24 h. The susceptibility of C. albicans biofilms to purpurin was assessed semi-quantitatively by XTT reduction assay, and the effects of purpurin on hyphal development and biofilm architecture were qualitatively assessed by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Results: XTT assay indicated that the susceptibility of C. albicans biofilms to purpurin was dose-dependent. At 5µg/ml, biofilm viability was reduced by ~22% and by ~46% at high concentration (20µg/ml) (P \u3c 0.05). The SEM and CLSM results confirmed the inhibitory effect of purpurin on C. albicans architecture. Purpurin-treated C. albicans biofilms were scanty, and exclusively consisted of blastospores compared to its control which was relatively confluent, and rich in hyphae. Conclusions: Purpurin inhibits hyphal development in C. albicans and disintegrates the biofilm. Its ability to inhibit these candidal virulence factors may shed light on further development of antifungal strategies against this human fungal pathogen

Reduced level of induced endocytosis in Candida albicans sap7 mutants

Objective: To investigate the effect of C. albicans SAP7 gene in induced endocytosis in Candida-epithelial cell interactions. Method: In this study, C. albicans sap7 mutant strain was constructed in our laboratory by a PCR – based gene disruption method. In the endocytosis assay, the number of C. albicans cells that were cell-associated with and endocytosed by OKF6/TERT2 was determined using differential fluorescence assay with minor modifications. Briefly, conflucent OKF6/TERT2 cells were infected with 1×105 Candida cells for 3 h incubation. After removing nonadherent organisms, the cells were fixed with 3% paraformaldehyde. The adherent but not endocytosed Candida cells were stained by a polyclonal rabbit anti-Candida antibody conjugated with red fluorescence Alexa Fluor 568. Afterwards, cells were permeated with 0.5% Triton X-100. All cell-associated organisms (including adherent and endocytosed organisms) were labeled with anti-Candida antibody conjugated with green fluorescence Alexa Fluor 488. The number of endocytosis organisms was determined by subtracting the number of adherent organisms (which is red fluorescence) from the number of cell-associated organisms (which is green fluorescence) observed under CLSM. In each well, at least 100 organisms were examined, and this assay was conducted in triplicate on three separate occasions. Result: Deletion of the SAP7 gene in C. albicans attenuated the induced endocytosis in C. albicans - epithelial cell interactions. Compared with the wild type SC5314, C. albicans sap7 mutant strain significantly reduced the induced endocytosis by 47.8% (p \u3c 0.01). Conclusion: SAP7 gene contributes to C. albicans induced endocytosis of oral epithelial cells

Candida albicans SAP9: a gene with novel function in hyphal development in Candida albicans

Objectives: To investigate the role of C. albicans Sap9 in yeast-to-hyphal morphological transition. Methods: In this study, SAP9 null mutant strain (Dsap9::hisG/Dsap9::hisG-URA3-hisG) was created by URA blaster method. Ura- phenotype strain was selected in synthetic complete glucose medium containing 0.1% 5-fluoroorotic acid, and complemented by URA3. Hyphal development of fungal cells (1×106 cfu/ml) was evaluated in both liquid and solid hyphal-inducing media, including Lee’s medium (pH 7.0), RPMI-1640, YPD+10% fetal bovine serum (FBS) and Spider’s medium at 37℃. To analyze the mRNA expression of hyphal-specific genes, fungal cells were prepared in the YPD containing 10% FBS at 37 ℃. Total RNA was extracted by SV Total RNA Isolation System and was reverse transcribed into cDNA using Superscirpt II. Gene expression was determined by Fast SYBR Green Master Mix, with the program: 95 ℃ incubation for 20s, and then 40 cycles of 95 ℃ for 1s and 60 ℃ for 20s. Student’s t test (P value \u3c 0.05) was used for statistical analysis. Results: Compared with wild type SC5314 strain, SAP9 null mutant strain was defective in the hyphal development in both liquid and solid hyphal-inducting media. Gene expression analysis indicated that the expression level of transcription factor gene EFG1 was down-regulated by about 40% (P Conclusions: C. albicans SAP9 might play a pivotal role in hyphal development in C. albicans via the cellular events mediated by EFG1

Potential role of Candida albicans secreted aspartic protease 9 in serum induced-hyphal formation and interaction with oral epithelial cells

© 2019 Elsevier Ltd Introduction: Candida albicans possesses the ability to switch rapidly between yeast to hyphal forms. Hyphal formation is a remarkable pathogenic characteristic, which allows C. albicans to invade into host cells. Objectives: This study was to investigate the role of the C. albicans SAP9 gene in hyphal formation and invasion ability. Methods: The morphology of fungal cells in the hyphal-inducing liquid media (YPD+10% fetal bovine serum) was observed by the microscopy. And the morphology of the colony on solid agar plates of YPD+10% fetal bovine serum was photographed by the digital camera. The mRNA expressions of hypha-associated genes in serum medium were also analyzed by real time PCR. Then for the interaction between C. albicans and oral epithelial cells, endocytosis essay, invasion essay and damage assay were performed to compare the differences between the sap9Δ/Δ mutant strain and wild type strain. Results: Compared with the wild type strain, the sap9Δ/Δ mutant strain exhibited a deficient yeast-to-hyphal morphological transition under serum hyphal-inducing conditions. Furthermore, the SAP9 knockout strain revealed a significant down-regulation of the expression of EFG1 (~40%), which is a transcription factor gene that mediates hyphae formation in C. albicans. Compared with the wild type strain, a 70% reduction in the endocytosis of the sap9Δ/Δ mutant strain by host cells was observed, as well as a 25% attenuation of active penetration and a 40% attenuation of host cell damage (P \u3c 0.05). Conclusions: Our data strongly suggests that C. albicans Sap9 is a potential hyphal-associated factor that responds to serum hyphal-inducing stimuli via a cAMP-protein kinase A pathway mediated by EFG1, and contributes to the process of invasion of Candida into the epithelial cells, leading to host cell damage

Efficacy and safety of sparsentan versus irbesartan in patients with IgA nephropathy (PROTECT): 2-year results from a randomised, active-controlled, phase 3 trial

BackgroundSparsentan, a novel, non-immunosuppressive, single-molecule, dual endothelin angiotensin receptor antagonist, significantly reduced proteinuria versus irbesartan, an angiotensin II receptor blocker, at 36 weeks (primary endpoint) in patients with immunoglobulin A nephropathy in the phase 3 PROTECT trial's previously reported interim analysis. Here, we report kidney function and outcomes over 110 weeks from the double-blind final analysis.MethodsPROTECT, a double-blind, randomised, active-controlled, phase 3 study, was done across 134 clinical practice sites in 18 countries throughout the Americas, Asia, and Europe. Patients aged 18 years or older with biopsy-proven primary IgA nephropathy and proteinuria of at least 1·0 g per day despite maximised renin–angiotensin system inhibition for at least 12 weeks were randomly assigned (1:1) to receive sparsentan (target dose 400 mg oral sparsentan once daily) or irbesartan (target dose 300 mg oral irbesartan once daily) based on a permuted-block randomisation method. The primary endpoint was proteinuria change between treatment groups at 36 weeks. Secondary endpoints included rate of change (slope) of the estimated glomerular filtration rate (eGFR), changes in proteinuria, a composite of kidney failure (confirmed 40% eGFR reduction, end-stage kidney disease, or all-cause mortality), and safety and tolerability up to 110 weeks from randomisation. Secondary efficacy outcomes were assessed in the full analysis set and safety was assessed in the safety set, both of which were defined as all patients who were randomly assigned and received at least one dose of randomly assigned study drug. This trial is registered with ClinicalTrials.gov, NCT03762850.FindingsBetween Dec 20, 2018, and May 26, 2021, 203 patients were randomly assigned to the sparsentan group and 203 to the irbesartan group. One patient from each group did not receive the study drug and was excluded from the efficacy and safety analyses (282 [70%] of 404 included patients were male and 272 [67%] were White) . Patients in the sparsentan group had a slower rate of eGFR decline than those in the irbesartan group. eGFR chronic 2-year slope (weeks 6–110) was −2·7 mL/min per 1·73 m2 per year versus −3·8 mL/min per 1·73 m2 per year (difference 1·1 mL/min per 1·73 m2 per year, 95% CI 0·1 to 2·1; p=0·037); total 2-year slope (day 1–week 110) was −2·9 mL/min per 1·73 m2 per year versus −3·9 mL/min per 1·73 m2 per year (difference 1·0 mL/min per 1·73 m2 per year, 95% CI −0·03 to 1·94; p=0·058). The significant reduction in proteinuria at 36 weeks with sparsentan was maintained throughout the study period; at 110 weeks, proteinuria, as determined by the change from baseline in urine protein-to-creatinine ratio, was 40% lower in the sparsentan group than in the irbesartan group (−42·8%, 95% CI −49·8 to −35·0, with sparsentan versus −4·4%, −15·8 to 8·7, with irbesartan; geometric least-squares mean ratio 0·60, 95% CI 0·50 to 0·72). The composite kidney failure endpoint was reached by 18 (9%) of 202 patients in the sparsentan group versus 26 (13%) of 202 patients in the irbesartan group (relative risk 0·7, 95% CI 0·4 to 1·2). Treatment-emergent adverse events were well balanced between sparsentan and irbesartan, with no new safety signals.InterpretationOver 110 weeks, treatment with sparsentan versus maximally titrated irbesartan in patients with IgA nephropathy resulted in significant reductions in proteinuria and preservation of kidney function

Sparsentan in patients with IgA nephropathy: a prespecified interim analysis from a randomised, double-blind, active-controlled clinical trial

Background: Sparsentan is a novel, non-immunosuppressive, single-molecule, dual endothelin and angiotensin receptor antagonist being examined in an ongoing phase 3 trial in adults with IgA nephropathy. We report the prespecified interim analysis of the primary proteinuria efficacy endpoint, and safety. Methods: PROTECT is an international, randomised, double-blind, active-controlled study, being conducted in 134 clinical practice sites in 18 countries. The study examines sparsentan versus irbesartan in adults (aged ≥18 years) with biopsy-proven IgA nephropathy and proteinuria of 1·0 g/day or higher despite maximised renin-angiotensin system inhibitor treatment for at least 12 weeks. Participants were randomly assigned in a 1:1 ratio to receive sparsentan 400 mg once daily or irbesartan 300 mg once daily, stratified by estimated glomerular filtration rate at screening (30 to 1·75 g/day). The primary efficacy endpoint was change from baseline to week 36 in urine protein-creatinine ratio based on a 24-h urine sample, assessed using mixed model repeated measures. Treatment-emergent adverse events (TEAEs) were safety endpoints. All endpoints were examined in all participants who received at least one dose of randomised treatment. The study is ongoing and is registered with ClinicalTrials.gov, NCT03762850. Findings: Between Dec 20, 2018, and May 26, 2021, 404 participants were randomly assigned to sparsentan (n=202) or irbesartan (n=202) and received treatment. At week 36, the geometric least squares mean percent change from baseline in urine protein-creatinine ratio was statistically significantly greater in the sparsentan group (-49·8%) than the irbesartan group (-15·1%), resulting in a between-group relative reduction of 41% (least squares mean ratio=0·59; 95% CI 0·51-0·69; p<0·0001). TEAEs with sparsentan were similar to irbesartan. There were no cases of severe oedema, heart failure, hepatotoxicity, or oedema-related discontinuations. Bodyweight changes from baseline were not different between the sparsentan and irbesartan groups. Interpretation: Once-daily treatment with sparsentan produced meaningful reduction in proteinuria compared with irbesartan in adults with IgA nephropathy. Safety of sparsentan was similar to irbesartan. Future analyses after completion of the 2-year double-blind period will show whether these beneficial effects translate into a long-term nephroprotective potential of sparsentan. Funding: Travere Therapeutics

Efficacy and safety of sparsentan versus irbesartan in patients with IgA nephropathy (PROTECT): 2-year results from a randomised, active-controlled, phase 3 trial

Background Sparsentan, a novel, non-immunosuppressive, single-molecule, dual endothelin angiotensin receptor antagonist, significantly reduced proteinuria versus irbesartan, an angiotensin II receptor blocker, at 36 weeks (primary endpoint) in patients with immunoglobulin A nephropathy in the phase 3 PROTECT trial's previously reported interim analysis. Here, we report kidney function and outcomes over 110 weeks from the double-blind final analysis. Methods PROTECT, a double-blind, randomised, active-controlled, phase 3 study, was done across 134 clinical practice sites in 18 countries throughout the Americas, Asia, and Europe. Patients aged 18 years or older with biopsy-proven primary IgA nephropathy and proteinuria of at least 1·0 g per day despite maximised renin–angiotensin system inhibition for at least 12 weeks were randomly assigned (1:1) to receive sparsentan (target dose 400 mg oral sparsentan once daily) or irbesartan (target dose 300 mg oral irbesartan once daily) based on a permuted-block randomisation method. The primary endpoint was proteinuria change between treatment groups at 36 weeks. Secondary endpoints included rate of change (slope) of the estimated glomerular filtration rate (eGFR), changes in proteinuria, a composite of kidney failure (confirmed 40% eGFR reduction, end-stage kidney disease, or all-cause mortality), and safety and tolerability up to 110 weeks from randomisation. Secondary efficacy outcomes were assessed in the full analysis set and safety was assessed in the safety set, both of which were defined as all patients who were randomly assigned and received at least one dose of randomly assigned study drug. This trial is registered with ClinicalTrials.gov, NCT03762850. Findings Between Dec 20, 2018, and May 26, 2021, 203 patients were randomly assigned to the sparsentan group and 203 to the irbesartan group. One patient from each group did not receive the study drug and was excluded from the efficacy and safety analyses (282 [70%] of 404 included patients were male and 272 [67%] were White) . Patients in the sparsentan group had a slower rate of eGFR decline than those in the irbesartan group. eGFR chronic 2-year slope (weeks 6–110) was −2·7 mL/min per 1·73 m2 per year versus −3·8 mL/min per 1·73 m2 per year (difference 1·1 mL/min per 1·73 m2 per year, 95% CI 0·1 to 2·1; p=0·037); total 2-year slope (day 1–week 110) was −2·9 mL/min per 1·73 m2 per year versus −3·9 mL/min per 1·73 m2 per year (difference 1·0 mL/min per 1·73 m2 per year, 95% CI −0·03 to 1·94; p=0·058). The significant reduction in proteinuria at 36 weeks with sparsentan was maintained throughout the study period; at 110 weeks, proteinuria, as determined by the change from baseline in urine protein-to-creatinine ratio, was 40% lower in the sparsentan group than in the irbesartan group (−42·8%, 95% CI −49·8 to −35·0, with sparsentan versus −4·4%, −15·8 to 8·7, with irbesartan; geometric least-squares mean ratio 0·60, 95% CI 0·50 to 0·72). The composite kidney failure endpoint was reached by 18 (9%) of 202 patients in the sparsentan group versus 26 (13%) of 202 patients in the irbesartan group (relative risk 0·7, 95% CI 0·4 to 1·2). Treatment-emergent adverse events were well balanced between sparsentan and irbesartan, with no new safety signals. Interpretation Over 110 weeks, treatment with sparsentan versus maximally titrated irbesartan in patients with IgA nephropathy resulted in significant reductions in proteinuria and preservation of kidney function.</p