Résumé
Phaeohypomycosis is a rare cutaneous and subcutaneous fungal infection caused by dematiaceous fungi. They have a widespread global distribution occasionally affecting humans. A 26-year-old woman presented with multiple skin lesions over her face and extremities for last 7 years, unresponsive to systemic amphotericin B and itraconazole. Further investigations revealed CARD9 mutation and phaeohyphomycosis caused by the pigmented fungus Exserohilum rosatratum. Lesions subsequently improved with oral flucytosine and itraconazole
Résumé
Objective:To explore genetic etiology and evaluate antifungal immunity in a patient with recurrent cervical lymphadenitis caused by Candida albicans. Methods:Next-generation sequencing was performed to screen susceptibility genes for mycosis in a patient with recurrent cervical lymphadenitis caused by Candida albicans and his parents. Peripheral blood mononuclear cells (PBMCs) and neutrophils were extracted from the patient and 6 healthy controls, and subjected to in vitro co-culture with Candida albicans. Western blot analysis was performed to determine the expression of caspase recruitment domain-containing protein 9 (CARD9) in PBMCs of the patient, enzyme-linked immunosorbent assay to detect levels of tumor necrosis factor-α (TNF-α), interleukin (IL) -6, IL-17A, IL-1β and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the co-culture medium, and a colony-counting method was used to detect the survival rate of Candida albicans after treatment with neutrophils. Statistical analysis was carried out by using t test for comparisons between two groups. Results:Two compound heterozygous mutations were identified in the CARD9 gene of the patient, including c.68C>A (p.S23X) in exon 2 inherited from his father and c.820dupG (p.D274Gfs*61) in exon 6 inherited from his mother. Western blot analysis showed that the relative expression level of CARD9 protein in the PBMCs was 0.41 ± 0.07 in the healthy control group, but CARD9 expression was absent in the patient. After stimulation with heat-inactivated Candida albicans spores, the levels of TNF-α, IL-6, IL-17A, IL-1β and GM-CSF secreted by PBMCs of the patient were significantly lower than those by PBMCs of the healthy controls (all P < 0.001). After 30- and 120-minute in vitro co-culture with neutrophils, the survival rates of Candida albicans were significantly higher in the patient (78.00%, 74.00%, respectively) than in the healthy controls (70.91% ± 1.75%, 34.55% ± 5.35%, t = 3.74, 6.99, respectively, both P < 0.05) . Conclusion:Compound heterozygous mutations were identified in the CARD9 gene of the patient with recurrent cervical lymphadenitis caused by Candida albicans, which led to the absence of CARD9 protein expression, and the patient had a defect in the immunity against Candida albicans.
Résumé
Resumen La candidemia es la micosis invasora más frecuente en los pacientes internados en hospitales de alta complejidad en el mundo. La infección fúngica en el sistema nervioso central constituye una complicación potencialmente mortal que agrava el pronóstico de los pacientes. El presente artículo aborda aspectos relevantes sobre las características clínicas de esta enfermedad, los mecanismos de invasión del hongo, la respuesta inmunitaria local frente a Candida albicans y el impacto de los defectos genéticos en receptores de la inmunidad innata, que aumentan la susceptibilidad a la neurocandidiasis.
Sujets)
Humains , Infections du système nerveux central , Candidose invasive , Candida albicans , Candidose invasive/diagnosticRésumé
Background: C-type lectin domain family 4 member D (CLEC4D) can induce Th17 cell differentiation and regulate IL-17A expression in systemic fungal infection, and whether CLEC4D plays a role in intestinal immunity has not been reported at home and abroad. Aims: To investigate the expressions and clinical significance of CLEC4D and CARD9 in colon tissue in patients with inflammatory bowel disease (IBD). Methods: A total of 48 IBD patients from October 2016 to June 2018 at the Second Affiliated Hospital of Zhengzhou University were enrolled, including 36 patients with ulcerative colitis (UC) and 12 patients with Crohn's disease (CD). And 30 adjacent normal colon tissues in patients with colon cancer were served as controls. Immunohistochemistry was used to detect the expressions of CLEC4D and CARD9 in colon tissue. And their correlations with clinical characteristics were analyzed. Results: The expressions of CLEC4D and CARD9 in UC group and CD group were significantly higher than those in control group (P0.05). The expressions of CLEC4D and CARD9 in UC group were positively correlated with Mayo score and Baron grade (P0.05). The expressions of CLEC4D and CARD9 in IBD patients were positively correlated with CRP (P0.05). Conclusions: The expressions of CLEC4D and CARD9 are elevated in patients with IBD and are closely related to patients' condition. CLEC4D may participate in the intestinal immunity of IBD through the CARD9-related pathway.
Résumé
Objective: To investigate the expression of caspase recruitment domain containing protein 9 (CARD9) protein in breast cancer tissues and its clinical significance. Methods: The expressions of CARD9 mRNA and protein in breast cancer cells, normal breast cells, breast cancer tissues and adjacent normal tissues were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The relationship between CARD9 expression and clinicopathological features was analyzed. The expressions of nuclear factor-κB (NF-κB) signaling pathway-related factors p50, p65, interlukin-1β (IL-1β) and IL-12 mRNA in breast cancer tissues and adjacent normal tissues were detected by real-time fluorescent quantitative PCR. The expressions of p50, p65, inhibitor of NF-κB kinase α (IKKα) and phosphorylated-IKKα (p-IKKα) proteins in breast cancer tissues and adjacent normal tissues were detected by Western blotting. The proliferation of breast cancer cells with CARD9 over-expression was detected by WST-1 assay. Results: The expressions of CARD9 mRNA (P < 0.01) and protein (P = 0.012) in breast cancer tissues were higher than those in the adjacent normal tissues. The expressions of CARD9 mRNA in breast cancer T-47D cells (P = 0.021) and MCF-7 cells (P = 0.014) were higher than that in normal breast Hs578Bst cells. The expressions of CARD9 mRNA and protein in breast cancer ZR-75-1 cells were higher than those in normal breast Hs578Bst cells (P = 0.003 and P = 0.015). The expression of CARD9 protein was positively correlated with the tumor size and estrogen receptor (ER) (both P < 0.015). The expressions of p50 (P < 0.05) and p-IKKα (P < 0.01) in breast cancer tissues were higher than those in the adjacent normal tissues. The expressions of p50 (r = 0.622, P < 0.001), p65 (r = 0.392, P = 0.005), IL-1β (r = 0.685, P < 0.001) and IL-12 (r = 0.556, P < 0.001) mRNAs were positively correlated with the expression of CARD9 mRNA. Enhancing the expression of CARD9 could promote the proliferation of breast cancer cells (P < 0.05). Conclusion: CARD9 may promote the proliferation of breast cancer cells and is involved in the formation and development of breast cancer by activating the NF-κB signaling pathway.
Résumé
Objective To study the mechanism of adaptive immunity against Rhizopus arrihizus (R. arrihizus) infections. Methods Bone marrow derived dendritic cells (BMDCs) were separated from C57BL/6 mice and Card9-/- mice and then were cultured in vitro. Resting spores and swollen spores of R. arrihizus were in vitro co-cultured with BMDCs with or without Syk inhibition. Secretion of cytokines ( IL-23, IL-1βand IL-12) was analyzed by ELISA after 24 hours of culture. Na?ve T cells derived from C57BL/6 mice were in vitro co-cultured with spore-stimulated BMDCs for four days. Levels of IL-17A and IFN-γ in supernatants of cell culture were analyzed by ELISA. Flow cytometry was performed to analyze T cell differ-entiation. Confocal microscopy was used to observe the images of stained β-glucan on the surface of resting and swollen spores. Swollen spores were co-cultured with Dectin-1, Dectin-2, TLR2 and mannose receptor ( MMR) , and the binding results were analyzed by flow cytometry. Results Swollen spores but resting spores could induce the maturation of BMDCs and promote the secretion of cytokines (IL-23, IL-1βand IL-12). Co-culturing T cells with swollen spore-stimulated BMDCs enhanced their differentiation to Th17 and Th1. In addition, swollen spores promoted the secretion of Th1-related cytokine ( IFN-γ) and Th17-related cytokine (IL-17A). Adding Syk inhibitor to Card9-/-BMDCs or wild type BMDCs significantly inhibited the secretion of cytokines and T cell differentiation, especially in the Card9-/- group. β-glucan was overserved on the surface of swollen spores, but not on resting spores. On the surface of swollen spores existed pathogen associated molecular patterns ( PAMPs) that could bind with Dectin-1 and TLR2. Conclusion Swollen spores of R. arrihizus could active BMDCs to secrete cytokines of IL-23, IL-1β and IL-12 and trigger T cell responses in vitro. The possible mechanism might be associated with β-glucan exposed on the surface of swollen spores that binds with Dectin-1. The responses between BMDCs and R. arrihizus are Syk-Card9-dependent.
Résumé
Objective To investigate the mechanism of caspase recruitment domain‐containing protein 9 (CARD9) in the early stage of acute pancreatitis(AP) .Methods Peripheral blood mononuclear cells (PBMC ) of 49 AP patients (33 mild acute pancreatitis (MAP ) patients and 16 severe acute pancreatitis (SAP) patients) were collected on the Day 1st ,3rd and 5th of hospitalization .Twenty healthy volunteers were enrolled in control group .The expression level of CARD9 ,B‐cell lymphoma(Bcl)‐10 ,p38 mitogen‐activated protein kinase (MAPK ) and p65 nuclear factor Kappa B (NF‐κB ) in PBMC of AP patients were detected by Western blotting .The co‐localization ,expression and binding between CARD9 and Bcl‐10 in PBMC of control group ,SAP group and MAP group on the Day 1st hospitalization were determined by cell immune‐fluorescence staining and co‐immuno precipitation method .Single factor analysis of variance and Mann‐Whitney test were performed for data comparison between groups .Pearson method was used for correlation analysis .Results The results of Western blotting indicated that the expression of CARD9 and Bcl‐10 in PBMC of SAP group on the Day 1st ,3rd and 5th of hospitalization (1 .12 ± 0 .05 ,1 .03 ± 0 .03 and 1 .01 ± 0 .01 ;1 .74 ± 0 .08 ,1 .72 ± 0 .10 and 1 .69 ± 0 .11) were all significantly higher than those of control group (0 .33 ± 0 .10 and 1 .02 ± 0 .11) and MAP group (0 .71 ± 0 .02 ,0 .55 ± 0 .06 and 0 .25 ± 0 .07 ;1 .15 ± 0 .03 ,1 .09 ± 0 .07 and 1 .01 ± 0 .04) ,and the differences were statistically significant (F= 35 .76 and 18 .20 ,all P< 0 .05) .The expression of p38 MAPK in PBMC of SAP group on the Day lst ,3rd of hospitalization (1 .88 ± 0 .08 、1 .68 ± 0 .11) were significantly higher than those of MAP group on the Day 1st ,3rd ,5th (0 .86 ± 0 .08 ,0 .77 ± 0 .10 ,0 .73 ± 0 .20) and healthy control group (0 .58 ± 0 .24 , F= 7 .24 ,all P < 0 .01) .The expression of p65 NF‐κB in PBMC of SAP group on the Day 1st ,3rd of hospitalization (1 .64 ± 0 .02 ,1 .55 ± 0 .03) were significantly higher than those of MAP group on the Day 3rd ,5th (1 .06 ± 0 .14 ,0 .87 ± 0 .20) and healthy control group (1 .17 ± 0 .13 ,F= 4 .51 ,all P< 0 .05) .The results of immune‐fluorescence staining indicated that CARD9 and Bcl‐10 co‐localized in nucleus .The results of co‐immuno precipitation showed that the binding degree between CARD9 and Bcl‐10 of SAP group was significantly higher than that of control group and MAP group . Pearson correlative analysis suggested that the level of p65 NF‐κB and p38 MAPK in PBMC of AP patients were positive correlated with the expression of CARD9 (r= 0 .692 and 0 .834 ,both P< 0 .01) .Conclusion CARD9 is positive correlated with NF‐κB and MAPK , which indicates CARD9 induced inflammatory cytokines by activating NF‐κB and MAPK signaling pathways in AP .
Résumé
Objective To compare the Dectin-1 signal transduction pathway and its function on dendritic cells between a female patient with recurrent vulvovaginal candidiasis (RVVC) and a healthy woman,and to explore the possible mechanism for VVC recurrence in this patient.Methods Venous blood samples were collected from a female patient with RVVC and a healthy woman.Then,monocytes were isolated from the blood samples,and were induced to differentiate into dendritic cells (DCs) in vitro.The obtained DCs were divided into three groups to be cultured alone,cocultured with Candida albicans or the combination of Candida albicans and anti-Dectin-1 antibodies for different durations.Flow cytometry was performed to determine the expression levels of CD83,CD86 and CD80 on DCs to evaluate the maturity of DCs,Western blot analysis to measure the protein expressions of Dectin-1,Syk and CARD9 in DCs,and enzyme-linked immunosorbent assay (ELISA) to determine the levels of interleukin (IL)-23,tumor necrosis factor α (TNF-α) and IL-12 in the culture supernatant of DCs.Results After co-culture with Candida albicans for 24 hours,the expressions of CD83,CD86 and CD80 were significantly inhibited on the patient-derived DCs compared with the controlderived DCs.Western blot analysis showed no significant differences in the expression of Dectin-1 between the controland patient-derived DCs,but a decrease in the expressions of phosphorylated-Syk and CARD9 in the patient-derived DCs compared with the control-derived DCs after 2-hour coculture with Candida albicans.After co-culture with Candida albicans for 6 hours,the levels of IL-23,TNF-α and IL-12 were lower in the culture supernatant of patient-derived DCs than in that of control-derived DCs.Furthermore,the anti-Dectin antibody showed no inhibitory effects on the activation of the Syk-dependent signal transduction pathway in or the secretion of the above cytokines by the patient-derived DCs.Conclusion The Dectin-1 signal transduction pathway was abnormal in DCs from the patient with RVVC,which may decelerate the maturation of DCs,inhibit the secretion of IL-23,TNF-o and IL-12 by them,and finally result in a defect in natural mucosal immunity against Candida infection in the host.