Mannan-binding lectin regulates Treg/Th17 cell balance in mice with Candida albicans infection / 中华微生物学和免疫学杂志

Objective To investigate the regulation of Treg/Th17 axis by mannan-binding lectin (MBL) in mice with Candida albicans ( C. albicans ) infection. -ethods MBL gene-knockdown (MBL-/-) C57BL/6 mice and wild-type (WT) C57BL/6 mice were divided into four groups. C. albicans strains (2×107 CFU) were injected intraperitoneally into the mice of infection groups. Paraffin sections of mouse liver and kidney tissues were prepared on 3 d. Histopathological changes were observed with hematox-ylin and eosin ( HE) and Periodic acid-Schiff ( PAS) staining. Flow cytometry was performed to analyze Th17 and Treg cells in mice on 7 d. The levels of IL-10 and IL-17A were measured by ELISA. CD4+T cells were obtained from spleen cells by magnetic sorting. Expression of Foxp3 and RORγt at mRNA and protein levels were detected by qRT-PCR and Western blot, respectively. Results The mouse model of C. albicans infection was established successfully. After infection, the MBL-/- mice had higher percentages of Th17 cells, but lower percentages of Treg cells than the WT mice. ELISA results showed that compared with the WT mice with C. albicans infection, the MBL-/- mice had significantly increased IL-17A and decreased IL-10 after infection. Moreover, the expression of RORγt at both mRNA and protein levels was up-regula-ted, while that of Foxp3 was down-regulated in the MBL-/- mice than in the WT mice following infection. Conclusions MBL regulates the immune balance of Treg/Th17 cells in mice infected with C. albicans through promoting the differentiation of CD4+ T cells into Treg cell subsets and inhibiting the differentiation into Th17 cell subsets.

Effects of mannan-binding lectin on differentiation of Th17 cells / 中华微生物学和免疫学杂志

Objective To investigate the effects of mannan-binding lectin ( MBL) on the differen-tiation of Th17 cells (T helper cell 17, Th17). Methods CD4+T cells were separated in vitro from fresh human cord blood by MACS ( magnetic-activated cell sorting ) separator. Anti-CD3 monoclonal antibody ( McAb) and anti-CD28 McAb were used to stimulate CD4+T cells with IL-6 and TGF-β1 as inducers. Then, these cells were treated with ( MBL group) or without ( control group) different concentrations of MBL. Percentages of Th17 cells in different groups were detected by fluorescence-activated cell sorting( FACS) after 72 hours of culturing. Quantitative real-time PCR ( Q-PCR) was used to analyze the expres-sion of RORγt ( retinoid-related orphan receptor-γ-t) at mRNA level in both control and MBL groups. En-zyme-linked immunosorbent assay (ELISA) was performed to detect the levels of IL-17A in supernatants of cell culture from different groups. FACS was used to detect the percentages of Th17 cells in MBL-/-and wild type ( WT) mice. Results MBL could significantly reduce the percentage of Th17 cells after 72 hours of culturing as compared with the control group. Moreover, MBL could significantly down-regulate the expres-sion of RORγt at mRNA level and decrease the expression of IL-17A. Results of animal experiments showed that the percentages of CD4+RORγt+Th17 cells in MBL-/- mice were significantly higher than those in WT mice. Conclusion MBL can inhibit the differentiation of CD4+T cells to Th17 cells, which is induced by IL-6 and TGF-β1.

Effects of mannan-binding lectin on differentiation of Th17 cells / 中华微生物学和免疫学杂志

Objective To investigate the effects of mannan-binding lectin ( MBL) on the differen-tiation of Th17 cells (T helper cell 17, Th17). Methods CD4+T cells were separated in vitro from fresh human cord blood by MACS ( magnetic-activated cell sorting ) separator. Anti-CD3 monoclonal antibody ( McAb) and anti-CD28 McAb were used to stimulate CD4+T cells with IL-6 and TGF-β1 as inducers. Then, these cells were treated with ( MBL group) or without ( control group) different concentrations of MBL. Percentages of Th17 cells in different groups were detected by fluorescence-activated cell sorting( FACS) after 72 hours of culturing. Quantitative real-time PCR ( Q-PCR) was used to analyze the expres-sion of RORγt ( retinoid-related orphan receptor-γ-t) at mRNA level in both control and MBL groups. En-zyme-linked immunosorbent assay (ELISA) was performed to detect the levels of IL-17A in supernatants of cell culture from different groups. FACS was used to detect the percentages of Th17 cells in MBL-/-and wild type ( WT) mice. Results MBL could significantly reduce the percentage of Th17 cells after 72 hours of culturing as compared with the control group. Moreover, MBL could significantly down-regulate the expres-sion of RORγt at mRNA level and decrease the expression of IL-17A. Results of animal experiments showed that the percentages of CD4+RORγt+Th17 cells in MBL-/- mice were significantly higher than those in WT mice. Conclusion MBL can inhibit the differentiation of CD4+T cells to Th17 cells, which is induced by IL-6 and TGF-β1.

Changes in the Expression and Distribution of Claudins, Increased Epithelial Apoptosis, and a Mannan-Binding Lectin-Associated Immune Response Lead to Barrier Dysfunction in Dextran Sodium Sulfate-Induced Rat Colitis

BACKGROUND/AIMS: This animal study aimed to define the underlying cellular mechanisms of intestinal barrier dysfunction. METHODS: Rats were fed 4% with dextran sodium sulfate (DSS) to induce experimental colitis. We analyzed the sugars in 24-hour urine output by high pressure liquid chromatography. The expression of claudins, mannan-binding lectin (MBL), and MBL-associated serine proteases 2 (MASP-2) were detected in the colonic mucosa by immunohistochemistry; and apoptotic cells in the colonic epithelium were detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method assay. RESULTS: The lactulose and sucralose excretion levels in the urine of rats with DSS-induced colitis were significantly higher than those in the control rats. Mannitol excretion was lower and lactulose/mannitol ratios and sucralose/mannitol ratios were significantly increased compared with those in the control group (p<0.05). Compared with the controls, the expression of sealing claudins (claudin 3, claudin 5, and claudin 8) was significantly decreased, but that of claudin 1 was increased. The expression of pore-forming claudin 2 was upregulated and claudin 7 was downregulated in DSS-induced colitis. The epithelial apoptotic ratio was 2.8%+/-1.2% in controls and was significantly increased to 7.2%+/-1.2% in DSS-induced colitis. The expression of MBL and MASP-2 in the intestinal mucosa showed intense staining in controls, whereas there was weak staining in the rats with colitis. CONCLUSIONS: There was increased intestinal permeability in DSS-induced colitis. Changes in the expression and distribution of claudins, increased epithelial apoptosis, and the MASP-2-induced immune response impaired the intestinal epithelium and contributed to high intestinal permeability.

Reibergrama para evaluar la síntesis intratecal de Lectina de unión a Manosa / Reibergram to evaluate the Mannan binding Lectin intrathecal synthesis

INTRODUCCIÓN: la dinámica particular de las proteínas derivadas del cerebro en el líquido cefalorraquídeo es diferente a la dinámica de las proteínas derivadas de la sangre. OBJETIVO: describir los datos empíricos de la lectina de unión a manosa y brindar una interpretación teórica de la dinámica de esta proteína a través de la confección un nuevo reibergrama. MÉTODOS: la lectina de unión a manosa en suero y líquido cfalorraquídeo, fue medida en 40 adultos normales a través de un ensayo inmunofluorométrico. El criterio diagnóstico estuvo basado en; muestras controles (pacientes normales) y muestras de pacientes con enfermedades que cursaron con disfunción de barrera sangre-líquido cefalorraquídeo. RESULTADOS: el coeficiente de correlación entre la lectina de unión a manosa en el líquido cefalorraquídeo y en el suero, fue muy bajo. El reibergrama de la lectina de unión a manosa se diseñó de acuerdo con procedimientos previos. CONCLUSIONES: bajo cualquier condición de barrera sangre-líquido cefalorraquídeo, el reibergrama puede identificar la ocurrencia de síntesis intratecal de lectina de unión a manosa.

BACKGROUND: The dynamics of brain derived proteins in cerebrospinal fluid is different from the dynamics of blood-derived proteins. Aim: To describe the empirical data for mannan binding lectin and gives a theoretical interpretation of the dynamics of this protein in cerebrospinal fluid through a new reibergram. METHODS: Serum and cerebrospinal fluid mannan binding lectin were measured in 40 normal adults by immunofluorometric assays. The diagnostic criteria were based in; normal control samples defined clinically and diseases with blood-cerebrospinal fluid barrier dysfunction. RESULTS: Correlation coefficient between cerebrospinal fluid MBL and serum MBL was very low. Mannan binding lectin reibergram was designed according with previous procedures. CONCLUSION: Under all conditions of the blood-cerebrospinal fluid barrier, the reibergram can identify the occurrence of intrathecal mannan binding lectin synthesis.

The clinical significance of serum mannan-binding lectin concentration in patients with chronic hepatitis B / 国际检验医学杂志

Objective To explore the concentration of mannan-binding lectin(MBL ) of patients with chronic hepatitis B virus (HBV) .Methods Serum MBL concentrations of 250 patients(case group) with HBV and 150 healthy controls(control group) were measured .Results The serum MBL concentration in case group was higher than that in control group(t=7 .097 ,P<0 .01) . The serum MBL concentration in high HBV-DNA loading group was higher than that in control group(t=7 .179 ,P<0 .01) .The serum MBL concentration in low HBV-DNA loading group was higher than that in control group(t=4 .404 ,P<0 .01) .Conclusion Detection of serum MBL in patients with HBV will be clinically useful for understanding state of an illness and observing the cur-ative effect .

Association between mannose-binding lectin and the sensitivities of common infections in children / 中华微生物学和免疫学杂志

Objective To investigate the association between genetic polymorphisms and protein levels of mannose-binding lectin (MBL) and the sensitivities of common infections in a pediatric Han population lived in Zhejiang Province.Methods MBL genetic polymorphisms of patients and controls were detected by PCR-based sequencing.MBL protein levels were measured using MBL ELISA Kit.Results No mutations at positions +223 and +239 of the exon 1 were detected in either patients or controls.No mutation at position +4 of the promoter was detected in controls.The frequencies of the three genotypes HH,HL,and LL at position-550 of the promoter were different between patients and controls(P＜0.05).The frequencies of genotypes YA and XB relevant to MBL protein levels were also different between patients and controls(P＜0.05).Comparing the frequencies of genotypes YA and XB in separate infectious disease with controls,significant differences were found in the group of RRI and CMV infection.The distributions of serum MBL level frequencies in patients and controls were both characterized by skewed distributions.MBL levels of patients with CMV infection were lower than those of controls(P＜0.05).Inversely,MBL levels of patients with acute respiratory infection and localized abscess were higher than those of controls (P＜O.05).Conclusion Genetic polymorphism of MBL gene is seemed to be relative to the sensitivity of common infections in children.

The role of mannan-binding lectin in inhibition of inflammatory responses induced by peptidoglycan and its mechanism in THP-1/CD14 cells / 中华微生物学和免疫学杂志

Objective To investigate the effects of mannan-binding lectin(MBL) on TNF-α production induced by peptidoglycan (PGN) and its mechanism in human THP-1/CD14 monocytes.Methods The THP-1/CD14 cells were stimulated for 24 h with PGN at the indicated ratios after pretreated with human natural MBL at concentrations ranging from 1 to 20 mg/L for 2 h.The content of TNF-α and IL-6 in culture supernatants were detected by ELISA,and the levels of TNF-α and IL-6 mRNA expressions in these cells were determined by RT-PCR.FACS was used to investigate the interaction of MBL with THP-1/CD14 cells and the impact of MBL on PGN binding to THP-1/CD14 cells.Western blot was used to detect PGN-induced NF-κB translocation in THP-1/CD14 cells.Results ELISA showed that secretion of TNF-α and IL-6 from THP-1/CD14 cells could be induced by PGN ;The productions of TNF-α and IL-6 by THP-1/CD14 cells induced with PGN were profoundly inhibited by MBL at higher concentrations (10-20 mg/L) but not MBL at lower concentrations (1 mg/L).RT-PCR analysis also indicated that the mRNA expressions of TNF-α and IL-6 in THP-1/CD14 cells were decreased by MBL at higher concentration,compared to the corresponding THP-1/CD14 cells stimulated with PGN only.FACS showed that the binding of MBL to THP-1/CD14 cells was evident in a Ca2+-dependent manner.PGN could competitively inhibit the binding of MBL to THP-1/CD14 cells.MBL could competitively inhibit the binding of PGN to THP-1/CD14 cells by binding to THP-1/CD14 cells directly.Similarly,MBL at higher concentration (20 mg/L) decreased the NF-κB translocation in THP-1/CD14 cells.Conclusion MBL may inhibit TNF-α and IL-6 production induced by PGN in THP-1/CD14 cells through NF-κB signaling pathways,suggesting that MBL can play some roles in the regulation of PGN-induced inflammatory response.

Analysis on the relationship between the human EV71 associated HFMD children's clinical manifestation and the genetic polymorphism and serum concentration of mannan binding lectin / 中华微生物学和免疫学杂志

Objective To explore the relationship between the genetic polymorphism and serum concentration of mannan binding lectin (MBL)and the clinical manifestation of the hand-foot-mouth disease (HFMD) children infection by human enterovirus 71 (HEV71).Methods One hundred and thirty-eight children diagnosed as HFMD infected by HEV71 (including 80 mild cases and 58 severe cases) and 40 healthy,symptom-free children were investigated.The concentrations of serum MBL were measured in 40 healthy controls,80 mild HFMD cases and 56 severe HFMD cases at both acute and convalescent phases by a sandwich enzyme immunoassay with a human MBL ELISA kit.And the genomic DNA of all cases were extracted from blood according to standard phenol-chloroform procedure.Six SNPs in the MBL gene(-550G/C,-221G/C and +4C/T of the promoter,CGT52TGT,GGC54GAC,and GGA57GAA of the exon 1) were analyzed by a sequencing-based typing method.Results The MBL serum level of the severe HFMD circulatory respiratory failure group in acute phase was significantly increased compared with severe HFMD encephalitis group,the mild cases and the control,but in the convalescence phase it significantly decreased compared with them.The frequencis of type B/B mutation (+230 of the exon 1),type P/P mutation (+4C/T of the promoter),and type H/H mutation (-550G/C of the promoter) were a significant difference among mild group,severe group and the control(P=0.006,0.043,0.028,respectively).The frequencies of LYPB/LYPB genotype and HYPA/HYPA genotype were a significant difference among mild group,severe groupand the control (P=0.028,0.014,respectively).Conclusion Low MBL protein level as a result genetic polymorphism seems to be correlative with clinical manifestation of HFMD disease.The MBL gene mutation and low MBI.protein level may be used as one of the evaluation method of HFMD severeity.

Mannan-binding lectin inhibits IL-8 and TNF-α production induced by C. albicans in THP1/CD14 cells / 中华微生物学和免疫学杂志

Objective To investigate the effects of mannan-binding lectin (MBL) on IL-8 and TNF-α production induced by Candida albicans ( C. albicans) in human THP1/CD14 monocytes. Methods The THP1/CD14 cells were stimulated for 24 h with heat-inactivated yeast form or hyphal form cells of C. albicans strain at the indicated ratios after pretreated with human natural MBL at concentrations ranging from 1 to 20 mg/L for 2 h. The content of IL-8 and TNF-α in culture supernatants were detected by ELISA,and the levels of IL-8 and TNF-α mRNA expressions in these cells were determined by RT-PCR. Western blot was used to detect C. albicans-induced NF-κB translocation in THP1/CDI4 cells. Results ELISA showed that secretion of IL-8 and TNF-α from THP1/CD14 cells could be induced by both yeast cells and hyphal cells. Hyphal cells proved to be much less efficient than yeast cells in stimulating production of IL-8and TNF-α by THP1/CD14 cells. The productions of IL-8 and TNF-α by THP1/CD14 cells induced with C.albicans were profoundly inhibited by MBL at higher concentrations ( 10-20 mg/L) but not MBL at lower concentrations ( 1 mg/L). RT-PCR analysis also indicated that the mRNA expressions of IL-8 and TNF-αt in THP1/CD14 cells were decreased to various extents by MBL at higher concentration, compared to the corresponding THP1/CD14 cells stimulated with C. albicans only. Similarly, MBL at higher concentration ( 20mg/L) decreased the NF-κB translocation in THP1/CD14 cells. Conclusion MBL may inhibit IL-8 and TNF-α production induced by dimorphism C. albicans in THP1/CD14 cells, suggesting that MBL can play some roles on the regulation of C. albicans immune response.

Association of SP-D, MBL and I-NOS genetic variants with pulmonary tuberculosis.

Background: Pulmonary tuberculosis is caused by Mycobacterium tuberculosis . It is a multifactorial disease with both host as well as pathogen factors contributing to susceptibility and protection from the disease. Various reports have highlighted important roles of lung surfactant protein D (SP-D), mannan-binding lectin (MBL) and I-NOS in innate immune defense against M. tuberculosis Aims : The present study investigated the role of polymorphisms in three candidate genes encoding Lung surfactant protein D, Mannan binding lectin and Inducible Nitric oxide synthase, in susceptibility and protection to pulmonary tuberculosis. Settings and Design : A case-control association study of SNP's in lung surfactant protein D (SP-D), mannan-binding lectin (MBL) and I-NOS with pulmonary tuberculosis in Indian population was carried out. This involved sequencing of all the coding exons of lung surfactant protein D (SP-D) , while, exon 1 (collagen region) and exon 4 (carbohydrate recognition domain) of mannan-binding lectin (MBL) and exons 2, 8 and 16 of I-NOS and their flanking intronic regions for single nucleotide polymorphisms in DNA samples isolated from 30 pulmonary tuberculosis patients and 30 controls of Indian population. Statistical analysis: Various allele frequencies were calculated using online two by two table (home.clara.net/sisa/). Odds ratio and P values were calculated at 95% confidence interval (CI). Results : A total of fourteen single nucleotide polymorphisms (5 in SP-D , 5 in MBL and 4 in I-NOS ) were observed of which four (G459A SP-D , G274T I-NOS , G1011A and T357G MBL ) have not been reported earlier. Four single nucleotide polymorphisms viz. G459A of exon 7 of SP-D ( P =0.00, odds ratio (OR) = 4.96, 2.18 P = 0.00 or= 3.85 1.66 P =0.00 or=4.04, 2.20< OR<7.42) and G274T of intron 16 of I-NOS ( P =0.00 or=4.46, 2.40 Conclusion: The present study has led to identification of 4 SNP's in SP-D , MBL and I-NOS associated with pulmonary tuberculosis in Indian population.

Association of mannan-binding lectin with inflammatory bowel disease / 医学研究生学报

Mannan binding lectin (MBL) is an innate immune mediator belonging to the collectin family known to bind to the surface of many viruses, bacteria and fungi. Many researches show that MBL is associated with many autoimmune diseases. Inflammatory bowel diseases (IBD) including ulcerative colitis and Crohn's disease are related with innate individual immunity. This article reviews the recent researches on the association of MBL with IBD.

Investigation of single nucleotide polymorphisms, haplotypes and genotypes of mannan-binding lectin gene in Bai(Pai) nationality in China / 中国免疫学杂志

Objective:To investigate the single nucleotide polymorphisms(SNP), haplotypes and genotypes of mannan-binding lectin(MBL) gene in the Bai(Pai) nationality from YunNan province, China.Methods:The three SNP sites CGT52TGT, GGC54GAC and GGA57GAA(named alleles D, B and C respectively, wildtype named A) in exon1 of MBL gene of 70 DNA samples of Bai nationality whose three SNP sites, -550G/C, -221C/G and +4C/T(named alleles H/L, X/Y and P/Q respectively), in promoter region of MBL gene had been clear, haplotypes and genotypes of MBL genes were detected and analyzed by sequence specific primer-polymerase chain reaction.Results:It was found that in Bais population, the frequency of alleles B was 0.100, there only were five haplotypes, HYPA, LXPA, LYQA, LYPA and LYPB, whose frequencies were 0.250, 0.107, 0.407, 0.135 and 0.100 respectively, the frequencies of several genotypes were LYPA/LYPA 0.043, LXPA/LYQA 0.143, LYPA/LYPB 0.014, HYPA/LYQA 0.086, LYPA/ LYQA 0.157, HYPA/LYPA 0.014, LYPB/LYQA 0.143, HYPA/LYPB 0.043, LXPA/LXPA 0.014, HYPA/LXPA 0.043, LYQA/LYQA 0.143 and HYPA/HYPA 0.157.Conclusion:In the MBL genes in Bais population, there is the allele B, the polymorphism haplotypes are mostly LYQA and HYPA, and the genotypes, LYPA/LYQA, HYPA/HYPA, LXPA/LYQA, LYPB/LYQA and LYQA/LYQA.