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PROBLEM: Early-onset preeclampsia (EOPE) is a severe gestational hypertensive disorder with significant feto-maternal morbidity and mortality due to uteroplacental insufficiency. Circulating extracellular vesicles of placental origin (EV-P) are known to be involved in the pathophysiology of EOPE and might serve as an ideal reservoir for its specific biomarkers. Therefore, we aimed to characterize and perform comparative proteomics of circulating EV-P from healthy pregnant and EOPE women before delivery. METHOD OF STUDY: The EV-P from both groups were isolated using immunoaffinity and were characterized using transmission electron microscopy, dynamic light scattering, nanoparticle tracking analysis, and immunoblotting. Following IgG albumin depletion, the pooled proteins that were isolated from EV-P of both groups were subjected to quantitative TMT proteomics. RESULTS: Circulating term EV-P isolated from both groups revealed â¼150 nm spherical vesicles containing CD9 and CD63 along with placental PLAP and HLA-G proteins. Additionally, the concentration of EOPE-derived EV-P was significantly increased. A total of 208 proteins were identified, with 26 among them being differentially abundant in EV-P of EOPE women. This study linked the pathophysiology of EOPE to 19 known and seven novel proteins associated with innate immune responses such as complement and TLR signaling along with hemostasis and oxygen homeostasis. CONCLUSION: The theory suggesting circulating EVs of placental origin could mimic molecular information from the parent organ-"the placenta"-is strengthened by this study. The findings pave the way for possible discovery of novel prognostic and predictive biomarkers as well as provide insight into the mechanisms driving the pathogenesis of EOPE.
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Vesículas Extracelulares , Hemostasia , Imunidade Inata , Placenta , Pré-Eclâmpsia , Proteômica , Humanos , Feminino , Gravidez , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/imunologia , Pré-Eclâmpsia/imunologia , Pré-Eclâmpsia/metabolismo , Adulto , Placenta/metabolismo , Placenta/imunologia , Biomarcadores/metabolismoRESUMO
INTRODUCTION: Spontaneous abortion (SAB) affects approximately 10% of clinically recognized pregnancies. Fetal trophobalst invasion and remodeling of maternal spiral arteries is reported to be dependent on crosstalk between HLA-C/HLA-G expressed on extra villous trophoblast (EVTs)and Killer cell Immunoglobin like receptors (KIRs) of decidual NK (dNK). Immune dysfunction in decidua contributes to early miscarriage. METHODOLOGY: The study used mother neonate paired cord blood and term placenta samples (n = 46), elective abortus (n = 17,gestational age = 10-12 weeks of pregnancy) and SAB abortus (n = 24, gestational age = 12-15 weeks of pregnancy) for HLA-G, KIR2D and HLA-C. In addition, term placenta was collected from women with history of spontaneous pregnancy loss (n = 24) and women with history of live birth (n = 32). SSP-PCR was used for genotyping, RT-PCR for gene expression, copy number variation (CNVs) and HLA-C allotyping and ELISA for protein expression studies. RESULTS: Membrane bound HLA-G4 isoform proportion was higher 39.28%, p = 0.02) in term placenta. SAB abortus had higher proportion of HLA-G3 (50%),while elective abortus exhibited higher proportion of soluble isoforms (HLA-G5, = 5.9, HLA-G6 = 5.9%, HLA-G7 = 11.8%). Higher inhibitory KIR2DL1 content and copy numbers with lower HLA-C2 in SAB contrasted with higher copy numbers of KIR2DS1(p = 0.001), KIR2DS1+/2DL1+- HLA-C2 combined genotype in healthy placenta. Elevated KIR2D protein levels (p = 0.001), and concurrently, HLA-C levels were upregulated in healthy placenta. CONCLUSION: Our data supports lower cognate receptor ligand KIR2DS1+/2DL1+ HLA-C2 together with predominance of HLA-G3 isoform in SAB as confounding factors in spontaneous pregnancy loss. HLA-G isoforms and expression differed between first trimester abortus and term placenta suggesting temporal modulation and marks novelty of the study.
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Aborto Espontâneo , Antígenos HLA-C , Antígenos HLA-G , Feminino , Humanos , Lactente , Recém-Nascido , Gravidez , Aborto Espontâneo/genética , Aborto Espontâneo/metabolismo , Decídua/metabolismo , Variações do Número de Cópias de DNA , Antígenos HLA-C/genética , Antígenos HLA-G/genética , Antígenos HLA-G/metabolismo , Células Matadoras Naturais , Placenta/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Trofoblastos/metabolismoRESUMO
Pregnancy is an immunologically regulated, complex process. A tightly controlled complement system plays a crucial role in the successful establishment of pregnancy and parturition. Complement inhibitors at the feto-maternal interface are likely to prevent inappropriate complement activation to protect the fetus. In the present study, we aimed to understand the role of Factor H (FH), a negative regulator of complement activation, in normal pregnancy and in a model of pathological pregnancy, i.e. preeclampsia (PE). The distribution and expression of FH was investigated in placental tissues, various placental cells, and in the sera of healthy (CTRL) or PE pregnant women via immunohistochemistry, RT-qPCR, ELISA, and Western blot. Our results showed a differential expression of FH among the placental cell types, decidual stromal cells (DSCs), decidual endothelial cells (DECs), and extravillous trophoblasts (EVTs). Interestingly, FH was found to be considerably less expressed in the placental tissues of PE patients compared to normal placental tissue both at mRNA and protein levels. Similar results were obtained by measuring circulating FH levels in the sera of third trimester CTRL and PE mothers. Syncytiotrophoblast microvesicles, isolated from the placental tissues of PE and CTRL women, downregulated FH expression by DECs. The present study appears to suggest that FH is ubiquitously present in the normal placenta and plays a homeostatic role during pregnancy.
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Placenta , Pré-Eclâmpsia , Feminino , Humanos , Gravidez , Fator H do Complemento/metabolismo , Células Endoteliais/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Trofoblastos/metabolismoRESUMO
In brief: Circulating extracellular vesicles of placental/amniochorionic origin carry placental/amniochorionic proteins and nucleic acids with the potential to facilitate non-invasive diagnosis of pregnancy-related disorders. The study reports an improvised method for the enriched isolation of extracellular vesicles of placental/amniochorionic origin using the two markers, PLAP and HLA-G. Abstract: Extracellular vesicles (EVs) are membrane-bound nanovesicles secreted from the cells into extracellular space and body fluids. They are considered 'fingerprints of parent cells', which can reflect their physiological and functional states. During pregnancy, EVs are produced by the syncytiotrophoblasts and extravillous trophoblasts and are released into the maternal bloodstream. In the present study, placental alkaline phosphatase (PLAP)-specific extracellular vesicles were isolated from maternal serum-derived EVs (SDE) across pregnancy. Transmission electron microscopy and dynamic light scattering analysis showed that the isolated EVs exhibited a spherical morphology with ~30-150 nm size range. Nanoparticle tracking analysis indicated that the concentration of PLAP+ serum-derived EVs (PLAP+-SDE) increased across the gestation. PLAP+-SDE contained DNA with LINE1 promoter methylation pattern. C19 miRNA cluster miRNAs (miR 515-5p, 519e and 520f) were present in PLAP+-SDE along with other miRNAs (miR-133-3p, miR210-3p and miR-223-3p). PLAP+-SDE confirmed the presence of EV markers (CD63 and CD9), along with placental proteins (PLAP and cullin 7). A modified novel strategy to extract an enriched population of circulating placental/amniochorionic EVs was devised employing an additional marker of extravillous trophoblasts, human leukocyte antigen G (HLA-G), along with PLAP. The isolated pooled placental/amniochorionic (PLAP+&HLA-G+) serum-derived EVs (PP-SDE) showed ~two-fold increased protein levels of HLA-G in the third-trimester pregnant women compared to the non-pregnant controls. Future studies will be focused on validation of this novel strategy to isolate an enriched population of placental/amniochorionic EVs to facilitate a better understanding of placental physiology and pathophysiology.
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Vesículas Extracelulares , MicroRNAs , Proteínas da Gravidez , Gravidez , Feminino , Humanos , Placenta/metabolismo , Antígenos HLA-G/metabolismo , Vesículas Extracelulares/metabolismo , Trofoblastos/metabolismo , MicroRNAs/metabolismo , Proteínas da Gravidez/metabolismoRESUMO
Pre-eclampsia (PE), a multifactorial de novo hypertensive pregnancy disorder, is one of the leading causes of foeto-maternal morbidity and mortality. Currently, antihypertensive drugs are the first-line therapy for PE and evidence suggests that low-dose aspirin initiated early in high risk pregnancies may reduce the risk of development or severity of PE. However, an early prediction of this disorder remains an unmet clinical challenge. Several potential serum biomarkers associated with maternal immunoregulation and placental angiogenesis have been evaluated but are ineffective and inconsistent for early prediction. Although placental biomarkers would be more specific and sensitive in predicting the risk of PE, accessing the placenta during pregnancy is not feasible. Circulating placental exosomes (pEXO), originating from foeto-maternal interface, are being evaluated as the placenta's surrogate and the best source of non-invasive placental biomarkers. pEXO appear in the maternal circulation starting from six weeks of gestation and its dynamic biological cargo across pregnancy is associated with successful pregnancy outcomes. Therefore, monitoring changes in pEXO expression profiles could provide new insights into the prediction, diagnosis and treatment of PE. This narrative review comprehensively summarizes the available literature on the candidate predictive circulating biomarkers evaluated for PE to date. In particular, the review elucidates the current knowledge of distinct molecular signatures emanating from pEXO in pre-eclamptic women to support the discovery of novel early predictive biomarkers for effective intervention and management of the disease.
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Exossomos , Hipertensão , Pré-Eclâmpsia , Gravidez , Feminino , Humanos , Placenta/metabolismo , Pré-Eclâmpsia/diagnóstico , Exossomos/metabolismo , Resultado da Gravidez , BiomarcadoresRESUMO
PROBLEM: Surfactant protein D (SP-D), a multimeric collectin expressed by testicular mucosal epithelia and is positively regulated by testosterone. It exerts antimicrobial effects, modulates inflammation and rescued spermatogenesis in a murine model. Various cytokines and chemokines, including MCP-1, play a key role in regulating the inflammation in rat and human testis. The study aimed to investigate the role of SP-D and involvement of chemokines and cytokines in the male infertility associated with urogenital infections or inflammation. METHOD OF STUDY: The cross-sectional study evaluated levels of SP-D, testosterone, estradiol and the cytokines/chemokines including MCP-1 in the serum and semen samples of fertile and infertile Indian men with and without urogenital infections/inflammation (n = 76). RESULTS: Both fertile and infertile males with urogenital infection/inflammation had significantly lower levels of SP-D and higher levels of the chemokine, Monocyte chemoattractant protein 1 (MCP-1) in the serum and seminal plasma. Seminal plasma of these males exhibited significantly higher proportion of proteolytically degraded forms of SP-D. The serum SP-D levels positively correlated with testosterone/estradiol (TE) ratio. There was no significant correlation between the SP-D levels in seminal plasma and sperm count/motility. With a significant area under the Receiver Operating Characteristic curves, the serum and seminal plasma SP-D levels exhibited significant potential to predict infertility with high sensitivity and specificity in men with genital infections/inflammation. CONCLUSIONS: The circulating and seminal plasma SP-D levels were decreased in men with urogenital infection and inflammation. This could be due to their engagement at the site of infection, dysregulated expression owing to the altered hormonal profile and increased proteolytic degradation.
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Infertilidade Masculina , Infecções do Sistema Genital , Humanos , Masculino , Animais , Camundongos , Ratos , Sêmen/metabolismo , Proteína D Associada a Surfactante Pulmonar , Quimiocina CCL2/metabolismo , Infecções do Sistema Genital/metabolismo , Estudos Transversais , Testículo/metabolismo , Testosterona/metabolismo , Inflamação/metabolismo , Quimiocinas/metabolismo , Estradiol/metabolismoRESUMO
Cell surface proteins carrying N-glycans play important roles in inter- and intracellular processes including cell adhesion, development, and cellular recognition. Dysregulation of the glycosylation machinery has been implicated in various diseases, and investigation of global differential cell surface proteome effects due to the loss of N-glycosylation will provide comprehensive insights into their pathogenesis. Cell surface proteins isolated from Parent Pro-5 CHO cells (W5 cells), two CHO mutants with loss of N-glycosylation function derived from Pro-5 CHO (Lec1 and Lec4 cells), were subjected to proteome analysis via high-resolution LCMS. We identified 44 and 43 differentially expressed membrane proteins in Lec1 and Lec4 cells, respectively, as compared to W5 cells. The defective N-glycosylation mutants showed increased abundance of integrin subunits in Lec1 and Lec4 cells at the cell surface. We also found significantly reduced levels of IGF-1R (Insulin like growth factor-1 receptor); a receptor tyrosine kinase; and the GTPase activating protein IQGAP1 (IQ motif-containing GTPase activating protein), a highly conserved cytoplasmic scaffold protein) in Lec1 and Lec4 cells. In silico docking studies showed that the IQ domain of IQGAP1 interacts with the kinase domain of IGF-1R. The integrin signaling and insulin growth factor receptor signaling were also enriched according to GSEA analysis and pathway analysis of differentially expressed proteins. Significant reductions of phosphorylation of ERK1 and ERK2 in Lec1 and Lec4 cells were observed upon IGF-1R ligand (IGF-1 LR3) stimulation. IGF-1 LR3, known as Long arginine3-IGF-1, is a synthetic protein and lengthened analog of insulin-like growth factor 1. The work suggests a novel mechanism for the activation of IGF-1 dependent ERK signaling in CHO cells, wherein IQGAP1 plausibly functions as an IGF-1R-associated scaffold protein. Appropriate glycosylation by the enzymes MGAT1 and MGAT5 is thus essential for processing of cell surface receptor IGF-1R, a potential binding partner in IQGAP1 and ERK signaling, the integral components of the IGF pathway.
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Fator de Crescimento Insulin-Like I , Animais , Cricetinae , Células CHO , Cricetulus , Proteínas Ativadoras de GTPase/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Integrinas/metabolismo , Fosforilação , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , GlicosilaçãoRESUMO
Invasive aspergillosis (IA) is a life-threatening fungal infection for immunocompromised hosts. It is, therefore, necessary to understand the immune pathways that control this infection. Although the primary infection site is the lungs, aspergillosis can disseminate to other organs through unknown mechanisms. Herein we have examined the in vivo role of various complement pathways as well as the complement receptors C3aR and C5aR1 during experimental systemic infection by Aspergillus fumigatus, the main species responsible for IA. We show that C3 knockout (C3-/-) mice are highly susceptible to systemic infection of A. fumigatus. Intriguingly, C4-/- and factor B (FB)-/- mice showed susceptibility similar to the wild-type mice, suggesting that either the complement pathways display functional redundancy during infection (i.e., one pathway compensates for the loss of the other), or complement is activated non-canonically by A. fumigatus protease. Our in vitro study substantiates the presence of C3 and C5 cleaving proteases in A. fumigatus. Examination of the importance of the terminal complement pathway employing C5-/- and C5aR1-/- mice reveals that it plays a vital role in the conidial clearance. This, in part, is due to the increased conidial uptake by phagocytes. Together, our data suggest that the complement deficiency enhances the susceptibility to systemic infection by A. fumigatus.
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Aspergilose , Aspergillus fumigatus , Animais , Complemento C5/genética , Complemento C5/metabolismo , Fator B do Complemento/genética , Pulmão , Camundongos , Esporos FúngicosRESUMO
Lung surfactant protein D (SP-D) and Dendritic cell-specific intercellular adhesion molecules-3 grabbing non-integrin (DC-SIGN) are pathogen recognising C-type lectin receptors. SP-D has a crucial immune function in detecting and clearing pulmonary pathogens; DC-SIGN is involved in facilitating dendritic cell interaction with naïve T cells to mount an anti-viral immune response. SP-D and DC-SIGN have been shown to interact with various viruses, including SARS-CoV-2, an enveloped RNA virus that causes COVID-19. A recombinant fragment of human SP-D (rfhSP-D) comprising of α-helical neck region, carbohydrate recognition domain, and eight N-terminal Gly-X-Y repeats has been shown to bind SARS-CoV-2 Spike protein and inhibit SARS-CoV-2 replication by preventing viral entry in Vero cells and HEK293T cells expressing ACE2. DC-SIGN has also been shown to act as a cell surface receptor for SARS-CoV-2 independent of ACE2. Since rfhSP-D is known to interact with SARS-CoV-2 Spike protein and DC-SIGN, this study was aimed at investigating the potential of rfhSP-D in modulating SARS-CoV-2 infection. Coincubation of rfhSP-D with Spike protein improved the Spike Protein: DC-SIGN interaction. Molecular dynamic studies revealed that rfhSP-D stabilised the interaction between DC-SIGN and Spike protein. Cell binding analysis with DC-SIGN expressing HEK 293T and THP- 1 cells and rfhSP-D treated SARS-CoV-2 Spike pseudotypes confirmed the increased binding. Furthermore, infection assays using the pseudotypes revealed their increased uptake by DC-SIGN expressing cells. The immunomodulatory effect of rfhSP-D on the DC-SIGN: Spike protein interaction on DC-SIGN expressing epithelial and macrophage-like cell lines was also assessed by measuring the mRNA expression of cytokines and chemokines. RT-qPCR analysis showed that rfhSP-D treatment downregulated the mRNA expression levels of pro-inflammatory cytokines and chemokines such as TNF-α, IFN-α, IL-1ß, IL- 6, IL-8, and RANTES (as well as NF-κB) in DC-SIGN expressing cells challenged by Spike protein. Furthermore, rfhSP-D treatment was found to downregulate the mRNA levels of MHC class II in DC expressing THP-1 when compared to the untreated controls. We conclude that rfhSP-D helps stabilise the interaction between SARS- CoV-2 Spike protein and DC-SIGN and increases viral uptake by macrophages via DC-SIGN, suggesting an additional role for rfhSP-D in SARS-CoV-2 infection.
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COVID-19 , Proteína D Associada a Surfactante Pulmonar , Enzima de Conversão de Angiotensina 2 , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Quimiocinas , Chlorocebus aethiops , Citocinas , Células HEK293 , Humanos , Inflamação , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Proteína D Associada a Surfactante Pulmonar/genética , RNA Mensageiro , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , Células VeroRESUMO
Surfactant protein D (SP-D), a pattern recognition molecule, is emerging as a potent anti-tumoural innate immune defense molecule in a range of cancers. Previously, SP-D expression was found to be significantly downregulated at the malignant sites of human prostate adenocarcinoma and associated with an increasing Gleason score and severity. We recently reported selective induction of intrinsic apoptosis by a recombinant fragment of human SP-D (rfhSP-D) in the human Prostate cancer (PCa) biopsy explants and cells with glucose regulated protein of 78 (GRP78) as one of the key interacting partners. The present study evaluated the expression of SP-D in early and advanced stages of PCa using transgenic adenocarcinoma of mouse prostate (TRAMP) model. Both early and late stages of PCa showed significantly decreased SP-D mRNA expression and increased proteolytic degradation of SP-D protein. Systemic and tumoural immunophenotyping of TRAMP model revealed increased serine proteases producing granulocytes and polymorphonuclear myeloid-derived suppressor cells (PMN MDSCs) in the late stage; the serine proteases secreted by these cells could be involved in the degradation of SP-D. Susceptibility of rfhSP-D to elastase-mediated proteolysis provided the rationale to use an elastase-inhibitor to sustain intact rfhSP-D in the tumour microenvironment. The study revealed an immunomodulatory potential of rfhSP-D and elastase inhibitor, sivelestat, to induce macrophage polarization towards M1 with downregulation of PMN MDSCs in ex-vivo cultured TRAMP tumours. Furthermore, rfhSP-D induced immunogenic cell death in murine PCa cells and in TRAMP explants. The findings highlight that SP-D plays an anti-tumourigenic role in PCa by inducing immunogenic cell death and immunomodulation while the prostate tumour milieu adversely impacts SP-D by inhibiting its transcription, and enhancing its proteolytic degradation. Transformation of an immunologically "cold tumour" into a "hot tumour" implicates therapeutic potential of rfhSP-D in PCa.
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Adenocarcinoma , Neoplasias da Próstata , Adenocarcinoma/patologia , Animais , Humanos , Imunomodulação , Masculino , Camundongos , Elastase Pancreática , Próstata/patologia , Neoplasias da Próstata/patologia , Proteína D Associada a Surfactante Pulmonar , Serina Proteases , Tensoativos , Microambiente TumoralRESUMO
BACKGROUND: Dysregulated serum levels of Mannan binding lectin (MBL) has a probable role in Systemic Lupus Erythematosus (SLE) pathogenesis. OBJECTIVE: To evaluate the association between serum MBL levels in SLE patients from western India with the severity of disease Methods: SLE patients (n=70) from Western India were included. Based on MBL levels, patients were classified into four categories, viz. low (<100 ng/ml), mild (100-500 ng/ml), moderate (500-1000 ng/ml) and high (>1000 ng/ml). Correlation of serum MBL levels with disease severity was assessed using the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). MBL levels and circulating immune complex levels were detected by ELISA. C3, C4 and CRP levels were detected by nephelometer. RESULTS: Serum MBL levels of SLE patients (1954 ± 202.4 ng/ml) was lower than that of healthy controls (2388 ± 205.0 ng/ ml). There was no significant correlation between MBL levels with severity of SLE on the basis of ACR criteria and SLEDAI scores (p> 0.05). No significant difference was observed among MBL levels and SLE patients with (1847 ± 246.7) or without (1900 ± 246.8) Lupus Nephritis. SLE patients without infections (n= 33) had low MBL levels (1700 ± 301.0 ng/ ml) as compared with SLE patients with infection (n= 37) (2189 ± 284.6 ng/ ml) (p=0.30) Conclusion: Present study indicated that low MBL levels were not associated with disease severity, haematological manifestations and infections among SLE patients from Western India.
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Lúpus Eritematoso Sistêmico , Nefrite Lúpica , Lectina de Ligação a Manose , Ensaio de Imunoadsorção Enzimática , Humanos , Lúpus Eritematoso Sistêmico/complicações , Lectina de Ligação a Manose/sangue , Índice de Gravidade de DoençaRESUMO
Humoral immunity plays a defensive role against invading microbes. However, it has been largely overlooked with respect to Aspergillus fumigatus, an airborne fungal pathogen. Previously, we have demonstrated that surfactant protein D (SP-D), a major humoral component in human lung-alveoli, recognizes A. fumigatus conidial surface exposed melanin pigment. Through binding to melanin, SP-D opsonizes conidia, facilitates conidial phagocytosis, and induces the expression of protective pro-inflammatory cytokines in the phagocytic cells. In addition to melanin, SP-D also interacts with galactomannan (GM) and galactosaminogalactan (GAG), the cell wall polysaccharides exposed on germinating conidial surfaces. Therefore, we aimed at unravelling the biological significance of SP-D during the germination process. Here, we demonstrate that SP-D exerts direct fungistatic activity by restricting A. fumigatus hyphal growth. Conidial germination in the presence of SP-D significantly increased the exposure of cell wall polysaccharides chitin, α-1,3-glucan and GAG, and decreased ß-1,3-glucan exposure on hyphae, but that of GM was unaltered. Hyphae grown in presence of SP-D showed positive immunolabelling for SP-D. Additionally, SP-D treated hyphae induced lower levels of pro-inflammatory cytokine, but increased IL-10 (anti-inflammatory cytokine) and IL-8 (a chemokine) secretion by human peripheral blood mononuclear cells (PBMCs), compared to control hyphae. Moreover, germ tube surface modifications due to SP-D treatment resulted in an increased hyphal susceptibility to voriconazole, an antifungal drug. It appears that SP-D exerts its anti-A. fumigatus functions via a range of mechanisms including hyphal growth-restriction, hyphal surface modification, masking of hyphal surface polysaccharides and thus altering hyphal immunostimulatory properties.
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Aspergillosis, candidiasis, and cryptococcosis are the most common cause of mycoses-related disease and death among immune-compromised patients. Adhesins are cell-surface exposed proteins or glycoproteins of pathogens that bind to the extracellular matrix (ECM) constituents or mucosal epithelial surfaces of the host cells. The forces of interaction between fungal adhesins and host tissues are accompanied by ligand binding, hydrophobic interactions and protein-protein aggregation. Adherence is the primary and critical step involved in the pathogenesis; however, there is limited information on fungal adhesins compared to that on the bacterial adhesins. Except a few studies based on screening of proteome for adhesin identification, majority are based on characterization of individual adhesins. Recently, based on their characteristic signatures, many putative novel fungal adhesins have been predicted using bioinformatics algorithms. Some of these novel adhesin candidates have been validated by in-vitro studies; though, most of them are yet to be characterised experimentally. Morphotype specific adhesin expression as well as tissue tropism are the crucial determinants for a successful adhesion process. This review presents a comprehensive overview of various studies on fungal adhesins and discusses the targetability of the adhesins and adherence phenomenon, for combating the fungal infection in a preventive or therapeutic mode.
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The complement system is designed to recognise and eliminate invading pathogens via activation of classical, alternative and lectin pathways. Human properdin stabilises the alternative pathway C3 convertase, resulting in an amplification loop that leads to the formation of C5 convertase, thereby acting as a positive regulator of the alternative pathway. It has been noted that human properdin on its own can operate as a pattern recognition receptor and exert immune functions outside its involvement in complement activation. Properdin can bind directly to microbial targets via DNA, sulfatides and glycosaminoglycans, apoptotic cells, nanoparticles, and well-known viral virulence factors. This study was aimed at investigating the complement-independent role of properdin against Influenza A virus infection. As one of the first immune cells to arrive at the site of IAV infection, we show here that IAV challenged neutrophils released properdin in a time-dependent manner. Properdin was found to directly interact with haemagglutinin, neuraminidase and matrix 1 protein Influenza A virus proteins in ELISA and western blot. Furthermore, modelling studies revealed that properdin could bind HA and NA of the H1N1 subtype with higher affinity compared to that of H3N2 due to the presence of an HA cleavage site in H1N1. In an infection assay using A549 cells, properdin suppressed viral replication in pH1N1 subtype while promoting replication of H3N2 subtype, as revealed by qPCR analysis of M1 transcripts. Properdin treatment triggered an anti-inflammatory response in H1N1-challenged A549 cells and a pro-inflammatory response in H3N2-infected cells, as evident from differential mRNA expression of TNF-α, NF-κB, IFN-α, IFN-ß, IL-6, IL-12 and RANTES. Properdin treatment also reduced luciferase reporter activity in MDCK cells transduced with H1N1 pseudotyped lentiviral particles; however, it was increased in the case of pseudotyped H3N2 particles. Collectively, we conclude that infiltrating neutrophils at the site of IAV infection can release properdin, which then acts as an entry inhibitor for pandemic H1N1 subtype while suppressing viral replication and inducing an anti-inflammatory response. H3N2 subtype can escape this immune restriction due to altered haemagglutinin and neuraminindase, leading to enhanced viral entry, replication and pro-inflammatory response. Thus, depending on the subtype, properdin can either limit or aggravate IAV infection in the host.
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Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/imunologia , Neutrófilos/imunologia , Properdina/imunologia , Animais , Cães , Humanos , Células Madin Darby de Rim Canino/imunologia , Células Madin Darby de Rim Canino/virologiaRESUMO
The innate immune system is comprised of both cellular and humoral players that recognise and eradicate invading pathogens. Therefore, the interplay between retroviruses and innate immunity has emerged as an important component of viral pathogenesis. HIV-1 infection in humans that results in hematologic abnormalities and immune suppression is well represented by changes in the CD4/CD8 T cell ratio and consequent cell death causing CD4 lymphopenia. The innate immune responses by mucosal barriers such as complement, DCs, macrophages, and NK cells as well as cytokine/chemokine profiles attain great importance in acute HIV-1 infection, and thus, prevent mucosal capture and transmission of HIV-1. Conversely, HIV-1 has evolved to overcome innate immune responses through RNA-mediated rapid mutations, pathogen-associated molecular patterns (PAMPs) modification, down-regulation of NK cell activity and complement receptors, resulting in increased secretion of inflammatory factors. Consequently, epithelial tissues lining up female reproductive tract express innate immune sensors including anti-microbial peptides responsible for forming primary barriers and have displayed an effective potent anti-HIV activity during phase I/II clinical trials.
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Infecções por HIV , Soropositividade para HIV , HIV-1 , Feminino , Genitália Feminina , Humanos , Imunidade InataRESUMO
Human SP-D is a potent innate immune molecule whose presence at pulmonary mucosal surfaces allows its role in immune surveillance against pathogens. Higher levels of serum SP-D have been reported in the patients with severe acute respiratory syndrome coronavirus (SARS-CoV). Studies have suggested the ability of human SP-D to recognise spike glycoprotein of SARS-CoV; its interaction with HCoV-229E strain leads to viral inhibition in human bronchial epithelial (16HBE) cells. Previous studies have reported that a recombinant fragment of human SP-D (rfhSP-D) composed of 8 Gly-X-Y repeats, neck and CRD region, can act against a range of viral pathogens including influenza A Virus and Respiratory Syncytial Virus in vitro, in vivo and ex vivo. In this context, this study was aimed at examining the likely protective role of rfhSP-D against SARS-CoV-2 infection. rfhSP-D showed a dose-responsive binding to S1 spike protein of SARS-CoV-2 and its receptor binding domain. Importantly, rfhSP-D inhibited interaction of S1 protein with the HEK293T cells overexpressing human angiotensin converting enzyme 2 (hACE2). The protective role of rfhSP-D against SARS-CoV-2 infection as an entry inhibitor was further validated by the use of pseudotyped lentiviral particles expressing SARS-CoV-2 S1 protein; ~0.5 RLU fold reduction in viral entry was seen following treatment with rfhSP-D (10 µg/ml). These results highlight the therapeutic potential of rfhSP-D in SARS-CoV-2 infection and merit pre-clinical studies in animal models.
Assuntos
COVID-19/prevenção & controle , Vírus da Influenza A/fisiologia , Proteína D Associada a Surfactante Pulmonar/metabolismo , Mucosa Respiratória/fisiologia , Vírus Sinciciais Respiratórios/fisiologia , Vírion/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Células HEK293 , Humanos , Imunidade Inata , Ligação Proteica , Proteína D Associada a Surfactante Pulmonar/genética , Proteínas Recombinantes/genética , Mucosa Respiratória/virologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , Internalização do VírusRESUMO
Coronavirus disease (COVID-19) is an acute infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human SP-D (surfactant protein D) is known to interact with the spike protein of SARS-CoV, but its immune surveillance against SARS-CoV-2 is not known. The current study aimed to examine the potential of a recombinant fragment of human SP-D (rfhSP-D) as an inhibitor of replication and infection of SARS-CoV-2. The interaction of rfhSP-D with the spike protein of SARS-CoV-2 and human ACE-2 (angiotensin-converting enzyme 2) receptor was predicted via docking analysis. The inhibition of interaction between the spike protein and ACE-2 by rfhSP-D was confirmed using direct and indirect ELISA. The effect of rfhSP-D on replication and infectivity of SARS-CoV-2 from clinical samples was assessed by measuring the expression of RdRp gene of the virus using quantitative PCR. In silico interaction studies indicated that three amino acid residues in the receptor-binding domain of spike protein of SARS-CoV-2 were commonly involved in interacting with rfhSP-D and ACE-2. Studies using clinical samples of SARS-CoV-2-positive cases (asymptomatic, n = 7; symptomatic, n = 8) and negative control samples (n = 15) demonstrated that treatment with 1.67 µM rfhSP-D inhibited viral replication by â¼5.5-fold and was more efficient than remdesivir (100 µM) in Vero cells. An approximately two-fold reduction in viral infectivity was also observed after treatment with 1.67 µM rfhSP-D. These results conclusively demonstrate that the rfhSP-D mediated calcium independent interaction between the receptor-binding domain of the S1 subunit of the SARS-CoV-2 spike protein and human ACE-2, its host cell receptor, and significantly reduced SARS-CoV-2 infection and replication in vitro.
Assuntos
COVID-19/metabolismo , Proteína D Associada a Surfactante Pulmonar , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus , Replicação Viral , Adulto , Animais , Chlorocebus aethiops , Feminino , Humanos , Masculino , Ligação Proteica , Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Células VeroRESUMO
C-type lectin receptors (CLRs) belong to the family of pattern recognition receptors (PRRs). They have a critical role to play in the regulation of a range of physiological functions including development, respiration, angiogenesis, inflammation, and immunity. CLRs can recognize distinct and conserved exogenous pathogen-associated as well as endogenous damage-associated molecular patterns. These interactions set off downstream signaling cascades, leading to the production of inflammatory mediators, activation of effector immune cells as well as regulation of the developmental and physiological homeostasis. CLR signaling must be tightly controlled to circumvent the excessive inflammatory burden and to maintain the cellular homeostasis. Recently, MicroRNAs (miRNAs) have been shown to be important regulators of expression of CLRs and their downstream signaling. The delicate balance between miRNAs and CLRs seems crucial in almost all aspects of multicellular life. Any dysregulations in the miRNA-CLR axes may lead to tumorigenesis or inflammatory diseases. Here, we present an overview of the current understanding of the central role of miRNAs in the regulation of CLR expression, profoundly impacting upon homeostasis and immunity, and thus, development of therapeutics against immune disorders.