NADPH oxidase of human dendritic cells: role in Candida albicans killing and regulation by interferons, dectin-1 and CD206

Human monocyte-derived DC express the enzyme NADPH oxidase, responsible for ROS production. We show that Candida albicans did not activate NADPH oxidase in DC, and was poorly killed by these cells. However, Candida-killing activity increased upon DC stimulation with the NADPH oxidase activator PMA and was further enhanced by DC treatment with IFN-alpha or IFN-gamma. This fungicidal activity took place at high DC-to-Candida ratio, but decreased at low DC-to-yeast ratio, when Candida inhibited the NADPH oxidase by contrasting the assembly of the enzyme on DC plasma membrane. The NADPH oxidase inhibitor diphenyliodonium chloride abrogated the PMA-dependent DC candidacidal capacity. Engagement of beta-glucan receptor dectin-1 induced NADPH oxidase activation in DC that was depressed by mannose-binding receptor CD206 co-stimulation. Candida was internalized by DC through mannose-binding receptors, but not through dectin-1, thus explaining why Candida did not elicit NADPH oxidase activity. Our results indicate that NADPH oxidase is involved in DC Candida-killing activity, which is increased by IFN. However, Candida escapes the oxidative damage by inhibiting NADPH oxidase and by entering DC through receptors not involved in NADPH oxidase activation.     

C‐type lectin receptors of the Dectin‐1 cluster: Physiological roles and involvement in disease

C‐type lectin receptors (CLRs) are essential for multicellular existence, having diverse functions ranging from embryonic development to immune function. One subgroup of CLRs is the Dectin‐1 cluster, comprising of seven receptors including MICL, CLEC‐2, CLEC‐12B, CLEC‐9A, MelLec, Dectin‐1, and LOX‐1. Reflecting the larger CLR family, the Dectin‐1 cluster of receptors has a broad range of ligands and functions, but importantly, is involved in numerous pathophysiological processes that regulate health and disease. Indeed, these receptors have been implicated in development, infection, regulation of inflammation, allergy, transplantation tolerance, cancer, cardiovascular disease, arthritis, and other autoimmune diseases. In this mini‐review, we discuss the latest advancements in elucidating the function(s) of each of the Dectin‐1 cluster CLRs, focussing on their physiological roles and involvement in disease.     

Barley beta-Glucan and Zymosan induce Dectin-1 and Toll-like receptor 2 co-localization and anti-leishmanial immune response in Leishmania donovani-infected BALB/c mice

Toll-like receptors (TLRs), TLR2 in particular, are shown to recognize various glycans and glycolipid ligands resulting in various immune effector functions. As barley β-glucan and zymosan are the glycans implicated in immunomodulation, we examined whether these ligands interact with Dectin-1, a lectin-type receptor for glycans, and TLR2 and induce immune responses that can be used against Leishmania infection in a susceptible host. The binding affinity of barley β-glucan and zymosan with Dectin-1 and TLR2 was studied in silico. Barley β-glucan- and zymosan-induced dectin-1 and TLR2 co-localization was studied by confocal microscopy and co-immunoprecipitation. These ligands-induced signalling and effector functions were assessed by Western blot analyses and various immunological assays. Finally, the anti-leishmanial potential of barley β-glucan and zymosan was tested in Leishmania donovani -infected macrophages and in L. donovani-infected BALB/c mice. Both barley β-glucan and zymosan interacted with TLR2 and dectin-1, but with a much stronger binding affinity for the latter, and therefore induced co-localization of these two receptors on BALB/c-derived macrophages. Both ligandsactivated MyD88- and Syk-mediated downstream pathways for heightened inflammatory responses in L. donovani-infected macrophages. These two ligands induced T cell–dependent host protection in L. donovani-infected BALB/c mice. These results establish a novel modus operandi of β-glucans through dectin-1 and TLR2 and suggest an immuno-modulatory potential against infectious diseases.     

Oral treatment with yolk antibodies for the prevention of C. albicans infections in chemotherapy treated children. A feasibility study

Cancer patients treated with chemotherapy often become neutropenic, which predisposes for oral and systemic C. albicans infections. The purpose of this feasibility study was to find out if gargling with anti-C. albicans immunoglobulin Y from egg yolk can prevent oral candidiasis in children, treated for acute lymphatic leukemia. Four patients gargled with the antibodies once a day during the induction phase of chemotherapy. None of four patients treated with IgY got any C. albicans infection. In the non-treated control and in a historic group, seven of thirteen patients had suspected C. albicans infection. This study indicates that anti-C. albicans IgY may prevent oral candidiasis in immunocompromized children.     

Secreted aspartic proteases of Candida albicans activate the NLRP3 inflammasome

In a recent report, we demonstrated that distinct members of the secreted aspartic protease (Sap) family of Candida albicans are able to induce secretion of proinflammatory cytokines by human monocytes, independently of their proteolytic activity and specific pH optima. In particular, C. albicans Sap2 and Sap6 potently induced IL‐1β, TNF‐α, and IL‐6 production. Here, we demonstrate that Sap2 and Sap6 proteins trigger IL‐1β and IL‐18 production through inflammasome activation. This occurs via NLRP3 and caspase‐1 activation, which cleaves pro‐IL‐1β into secreted bioactive IL‐1β, a cytokine that was induced by Saps in monocytes, in monocyte‐derived macrophages and in dendritic cells. Downregulation of NLRP3 by RNA interference strongly reduced the secretion of bioactive IL‐1β. Inflammasome activation required Sap internalization via a clathrin‐dependent mechanism, intracellular induction of K⁺ efflux, and ROS production. Inflammasome activation of monocytes induced by Sap2 and Sap6 differed from that induced by LPS‐ATP in several aspects. Our data reveal novel immunoregulatory mechanisms of C. albicans and suggest that Saps contribute to the pathogenesis of candidiasis by fostering rather than evading host immunity.     

Plasma-derived mannose-binding lectin shows a direct interaction with C1-inhibitor

MBL-deficiency has been associated with an increased frequency and severity of infection, in particular in children and under immunocompromized conditions. In an open uncontrolled safety and pharmacokinetic MBL-substitution study using plasma-derived MBL (pdMBL) in MBL-deficient pediatric oncology patients, we found that despite MBL trough levels above 1.0 μg/ml MBL functionality was not efficiently restored upon ex vivo testing.     

Compound deletion of the rhoGAP C1 and V2 vasopressin receptor genes in a patient with nephrogenic diabetes insipidus

The function of small GTPases is fine‐tuned by a complex network of regulatory proteins such as GTPase‐activating proteins. The C1 gene at Xq28 encodes a protein assumed to function as a Rho GTPase‐activating protein (rhoGAP). Characterization of the molecular defect causing X‐linked nephrogenic diabetes insipidus (NDI) in a patient revealed a submicroscopic deletion of a 21.5‐kb genomic fragment encompassing the entire arginine‐vasopressin V2 receptor gene (AVPR2) and most of the C1 gene locus. In the absence of detailed information about the physiological relevance and specific functions of rhoGAP C1, a thorough clinical and laboratory investigation of the patient was performed. Besides clearly defined NDI symptoms caused by deletion of the AVPR2 gene, no major morphological abnormalities as determined by physical examination, radiography, ultrasound, and computed tomographic scan were detected. Extensive analysis of blood chemical, enzyme, and hormone values over a period of 16 years showed no deviations from normal ranges. On the basis of our observations, the rhoGAP C1 protein is not essential for normal development in the human. Because of a predominant expression pattern of the C1 gene in hematopoietic cells, we focused on immunologic and hematologic laboratory parameters of the affected boy and the mother who was found to be heterozygous. Differential white cell counts, including lymphocyte typing, determination of lymphokines, cytokines, and immunoglobulins, as well as numerous leukocyte function tests, showed no pathological findings. Therefore, we postulate that the loss of rhoGAP C1 function is most likely compensated by other members of the GAP family. Hum Mutat 14:163–174, 1999. © 1999 Wiley‐Liss, Inc.     

IgG1 anti-P2 as a marker of response to interferon in patients with chronic hepatitis C.

To study the relations of antibody production to long-term outcomes after interferon (IFN) treatment in patients with chronic hepatitis C (CH-C), we used ELISA to measure the levels of antibodies against HCV core protein and peptides. Samples from 21 complete responders and 36 non-responders were collected before IFN therapy, soon after the end of IFN therapy and 6 months later. Using a set of 19 synthesized HCV core peptide antigens, we found that anti-P2 (11-25a.a.) was the most prevalent of all IgG antibodies (93%: 39/42). Among complete responders, IgG1 anti-P2 levels had fallen by the end of IFN therapy (from 79.8 +/- 60.4-46.1 +/- 44.2: P < 0.01), and were lower still 6 months after the end of IFN therapy (31.0 +/- 35.2: P < 0.001); this change was the greatest of all antibodies studied. Among the non-responders, there was no change within the follow-up period. Soon after the end of IFN therapy, IgG1 anti-P2 levels were more than 30% lower than the initial value in more than two-thirds of the complete responders, but in only one-third of the non-responders (14/20 vs. 8/25: P < 0.05). Six months after the end of IFN therapy, IgG1 anti-P2 levels were more than 30% lower than the initial value in more than 85% of the complete responders, but in only 12% of the non-responders (17/20 vs. 3/25: P < 0.001). In conclusion, the changes in levels of IgG1 anti-P2 paralleled the activity of chronic hepatitis C after IFN therapy, and IgG1 anti-P2 levels may be markers of the efficacy of IFN therapy.     

Histopathological analysis of initial cellular response in TLR‐2 deficient mice experimentally infected by Leishmania (L.) amazonensis

Tegumentary leishmaniasis is an important public health problem in several countries. The capacity of the Leishmania species, at the initial moments of the infection, to invade and survive inside the host cells involves the interaction of surface molecules that are crucial in determining the evolution of the disease. Using C57BL/6 wild-type and TLR-2^−/− mice infected with L. (L.) amazonensis, we demonstrated that TLR-2^−/− mice presented eosinophilic granuloma in the ear dermis, different from C57BL/6 wild-type mice that presented a cellular profile characterized mainly by mononuclear cell infiltrates, besides neutrophils and eosinophils, during the two first week of infection. When the parasite load was evaluated, we found that the absence of TLR-2 lead to a significant reduction of the infection in deficient mice, when compared with C57BL/6 mice which were more susceptible to the infection. Using TLR-2 deficient mice, it was possible to show that the absence of this receptor determined the reduction of the parasite load and the recruitment of inflammatory cells during the two first weeks after L. (L.) amazonensis infection.     

Identification of 58 novel mutations in Niemann-Pick disease type C: correlation with biochemical phenotype and importance of PTC1-like domains in NPC1

The two known complementation groups of Niemann-Pick Type C disease, NPC1 and NPC2, result from non-allelic protein defects. Both the NPC1 and NPC2 (HE1) gene products are intimately involved in cholesterol and glycolipid trafficking and/or transport. We describe mutation analysis on samples from 143 unrelated affected NPC patients using conformation sensitive gel electrophoresis and DNA sequencing as the primary mutation screening methods for NPC1 and NPC2, respectively. These methods are robust, sensitive, and do not require any specialized laboratory equipment. Analyses identified two NPC1 mutations for 115 (80.4%) patients, one NPC1 mutation for 10 (7.0%) patients, two NPC2 mutations for five (3.5%) patients, one NPC2 mutation for one (0.7%) patient, and no mutations for 12 (8.4%) patients. Thus, mutations were identified on 251 of 286 (88%) disease alleles, including 121 different mutations (114 in NPC1 and seven in NPC2), 58 of which are previously unreported. The most common NPC1 mutation, I1061T, was detected on 18% of NPC alleles. Other NPC1 mutations were mostly private, missense mutations located throughout the gene with clustering in the cysteine-rich luminal domain. Correlation with biochemical data suggests classification of several mutations as severe and others as moderate or variable. The region between amino acids 1038 and 1253, which shares 35% identity with Patched 1, appears to be a hot spot for mutations. Additionally, a high percentage of mutations were located at amino acids identical to the NPC1 homolog, NPC1L1. Biochemical complementation analysis of cases negative for mutations revealed a high percentage of equivocal results where the complementation group appeared to be non-NPC1 and non-NPC2. This raises the possibilities of an additional NPC complementation group(s) or non-specificity of the biochemical testing for NPC. These caveats must be considered when offering mutation testing as a clinical service.     

家族性慢性良性天疱疮一家系ATP2C1基因突变筛查

目的 对家族性慢性良性天疱疮一家系的ATP2C1基因突变进行检测.方法 采用聚合酶链反应扩增该家系4例患者和80名健康对照个体ATP2C1基因的全部外显子,直接测序法进行DNA测序,80名健康对照者为无亲缘关系的正常人.结果 在该家系所有患者中均检测出ATP2C1基因第163位碱基由C突变为T,导致终止密码子的提前出现.该突变位点在该家系正常人及健康对照人群中皆未发现.结论 该突变可影响ATP2C1基因的转录和翻译,在中国汉族人家族性慢性良性天疱疮家系中首次报道.     

Homeodomain interacting protein kinase (HPK‐1) is required in the soma for robust germline proliferation in C. elegans

Background: Tightly regulated pathways maintain the balance between proliferation and differentiation within stem cell populations. In Caenorhabditis elegans, the germline is the only tissue that is maintained by stem‐like cells into adulthood. In the current study, we investigated the role played by a member of the Homeodomain interacting protein kinase (HIPK) family of serine/threonine kinases, HPK‐1, in the development and maintenance of the C. elegans germline. Results: We report that HPK‐1 is required for promotion of germline proliferation during development and into adulthood. Additionally, we show that HPK‐1 is required in the soma for regulation of germline proliferation. We also show that HPK‐1 is a predominantly nuclear protein expressed in several somatic tissues including germline‐interacting somatic cells. Conclusions: Our observations are consistent with a conserved role for HIPKs in the control of cellular proliferation and identify a new context for such control in germ cell proliferation. Developmental Dynamics 242:1250–1261, 2013. © 2013 Wiley Periodicals, Inc.     

Apolipoprotein E/C1/C4/C2 gene cluster diversity in two native Andean populations: Aymaras and Quechuas

The APOE/C1/C4/C2 gene cluster presents high relevance in lipid metabolism and, therefore, has important epidemiological implications. Here, we study for the first time the variation patterns of 25 polymorphisms (10 short tandem repeats, STRs, and 15 single nucleotide polymorphismas, SNPs) in two native Andean samples from Bolivia (45 Aymaras and 45 Quechuas) as well as one European sample (n = 41) as external reference. We estimated diversity parameters, linkage disequilibrium patterns, population structure, and possible selective effects. In general, diversity was low and could be partly attributed to selection (probably due to its physiological importance), since the APOE/C1/C4/C2 region was highly conserved compared to the flanking genes in both Bolivians and Europeans. Moreover, the lower gene diversity in Bolivians compared to Europeans for some markers might indicate different demographic histories. Regarding the APOE isoforms, in addition to ?3 (94%) and ?4 (5%), isoform ?2 (1%) was also detected in Bolivians. In relation to previous hypotheses, our results support that genetic drift or founder effects rather than selection for increased cholesterol absorption are the main factors that have shaped the distribution of APOE isoforms observed in South America.     

TEG-1 CD2BP2 regulates stem cell proliferation and sex determination in the C. elegans germ line and physically interacts with the UAF-1 U2AF65 splicing factor

Background : For a stem cell population to exist over an extended period, a balance must be maintained between self‐renewing (proliferating) and differentiating daughter cells. Within the Caenorhabditis elegans germ line, this balance is controlled by a genetic regulatory pathway, which includes the canonical Notch signaling pathway. Results : Genetic screens identified the gene teg‐1 as being involved in regulating the proliferation versus differentiation decision in the C. elegans germ line. Cloning of TEG‐1 revealed that it is a homolog of mammalian CD2BP2, which has been implicated in a number of cellular processes, including in U4/U6.U5 tri‐snRNP formation in the pre‐mRNA splicing reaction. The position of teg‐1 in the genetic pathway regulating the proliferation versus differentiation decision, its single mutant phenotype, and its enrichment in nuclei, all suggest TEG‐1 also functions as a splicing factor. TEG‐1, as well as its human homolog, CD2BP2, directly bind to UAF‐1 U2AF65, a component of the U2 auxiliary factor. Conclusions : TEG‐1 functions as a splicing factor and acts to regulate the proliferation versus meiosis decision. The interaction of TEG‐1 CD2BP2 with UAF‐1 U2AF65, combined with its previously described function in U4/U6.U5 tri‐snRNP, suggests that TEG‐1 CD2BP2 functions in two distinct locations in the splicing cascade. Developmental Dynamics 241:505–521, 2012.© 2012 Wiley Periodicals, Inc.