Σκοπός της μελέτης ήταν η αποσαφήνιση των μηχανισμών με τους οποίους η P.aeruginosa διαντιδρά με τους PRRs των μακροφάγων. Από τα δεδομένα μας οι υποδοχείς TLR2 και Mannose receptor συνεργάζονται προς μέγιστη ενεργοποίηση των μακροφάγων σε απάντηση στη λοίμωξη από P.aeruginosa / The aim of the study was to delineate the mechanisms of P.aeruginosa interaction with specific PRRs on macrophages. Our data suggest that TLR2 and Mannose receptor synergize for maximum activation of human macrophages during Pseudomonas infection.

Η λεκτίνη ΜΒL συμμετέχει στην φυσική ανοσία, αφ΄ενός σαν ενεργοποιητής του συστήματος του συμπληρώματος και αφ΄ετέρου σαν οψωνίνη που προσδένεται σε συγκεκριμένες υδατανθρακικές δομές των μικροοργανισμών. Οι λεκτίνες τύπου C, είναι ασβέστιο εξαρτώμενες και φέρουν περιοχή δέσμευσης σε υδατάνθρακες (CRD). Προκειμενου να διασαφηνιστεί περαιτέρω η εξελικτική πορεία της λεκτινικής οδού του συμπληρώματος, απομονώθηκαν, κλωνοποιήθηκαν και χαρακτηρίστηκαν δύο ισομορφές της λεκτίνης ΜBL, οι ΜΒL1 και MBL2, στην ιριδίζουσα πέστροφα (Oncorhynchus mykiss). Οι συναγώμενες αμινοξικές αλληλουχίες των MBL1 και ΜΒL2 είναι 185 και 186 αμινοξέα, αντίστοιχα, παρουσιάζουν μεταξύ τους ταυτοσημία της τάξης του 83% ενώ εμφανίζουν το υψηλότερο σκορ ταυτοσημίας, 43% και 41% αντίστοιχα με τους τελεόστεους ιχθύες Αtlantic salmon και zebrafish. Το σκορ ταυτοσημίας των πρωτεϊνών της πέστροφας με τις αντίστοιχες πρωτεΐνες των πτηνών και των θηλαστικών κυμαίνεται μεταξύ 28 και 32%. Επιπλέον,οι περιοχές CRD των πρωτεϊνών αυτών χαρακτηρίζονται από την ύπαρξη του δομικού μοτίβου EPN που σχετίζεται με την εξειδίκευση της δέσμευσης ως προς την μαννόζη. Τα αποτελέσματα έδειξαν επίσης πως τα μόρια MBL1 και ΜBL2, εκφράζονται, σε επίπεδο mRNA, στο ήπαρ και στον σπλήνα της πέστροφας, αντίστοιχα. / Mannose-binding lectin(MBL) participates in the innate immune system as an activator of the complement system and as an opsonin after binding to certain carbohydrate structures on microorganisms. C-type lectins are all Ca2+ -dependent and they share a tightly folded carbohydrate recognition domain (CRD). In order to stydy the evolution of the complement lectin pathway, we report the isolation and characterization of two mannose-binding lectin isoforms, MBL1 and MBL2, in rainbow trout (Oncorhynchus mykiss). The deduced amino acid sequences of trout MBL1 and MBL2 are 185 and 186aa, respectively, presenting 83% identity to each other. These proteins exhibit the highest identity score 43 and 41% with the atlantic salmon and zebrafish counterparts. The identity with the bird and mammalian MBLs ranges from 28 to 32%. The trout MBL molecules contain in the CRD domain the EPN motif of mannose-binding C-type lectins, which is important for mannose specificity. Trout MBL1 and MBL2 are expressed exclusively in liver and spleen, respectively.

Mannose-binding lectin (MBL) and ficolins are complement-activating proteins, and both play an important role in innate immunity by recognizing specific carbohydrate moieties on the surface of wide range of microorganisms. Previous studies have shown that porcine ficolin-α and MBL-A bind to surface polysaccharides of bacteria cultured in suspension, but their interactions with bacteria in biofilm culture have not been studied. The objectives of this thesis were to determine whether porcine plasma ficolin and MBL bind to Actinobacillus pleuropneumoniae in biofilm cultures. APP serotype 5a (APP5a) was used because it produced pronounced biofilm in plastic culture dishes, in comparison with APP5b that was previously reported to bind ficolin in suspension cultures. N-acetylglucosamine (GlcNAc) in the biofilm produced by APP5a was stained with wheat germ agglutinin conjugated with Alexa Fluor-555 and identified by confocal laser scanning microscopy (CLSM). Dispersin B prevented APP5a biofilm formation indicating the requirement of poly N-acetylglucosamine (PNAG) for bacterial cohesion. Bound purified ficolin or ficolin in plasma both were eluted with GlcNAc from APP5a biofilm cultures. To address preferential binding of ficolin-α to biofilm matrix, ficolin-α was eluted with GlcNAc from extracellular polymeric substances (EPS) in supernatant after pelleting the bacteria. Biotinylated-ficolin that retained GlcNAc-binding activity for APP5b planktonic cultures was shown to bind strongly to APP5a biofilm, as detected by fluorescent NeutrAvidin staining and CLSM, but not in the presence of GlcNAc. Further, MBL-A in ficolin-depleted porcine plasma also bound to APP5a biofilm and was eluted with a sugar solution containing GlcNAc, galactose, mannose and glucose. These studies demonstrate that both porcine ficolin-α and MBL-A bind to biofilm cultures of APP5a in a carbohydrate-dependent manner, and suggest that the production of PNAG in biofilm is a binding target for ficolin. / Natural Sciences and Engineering Research Council of Canada (NSERC)

No description available.

No description available.

The disease listeriosis is caused by Listeria monocytogenes. This common food-borne disease has been responsible for about 0.1 to 10 cases per million inhabitants per year. However, this disease is serious with its high fatality rates of 20% - 30%, and 40% of all cases reported have been in pregnant women suffered from a foetal abortion. Recently the organism has acquired resistance to antibiotic treatment and the development of an alternative treatment is necessary. Class IIa bacteriocins such as leucocin A have been shown to be active against L. monocytogenes. However, the leucocin A receptor molecule responsible for growth inhibition within L. monocytogenes remains unclear. Various studies have implicated the mannose PTS permease (EIIt Man) of L. monocytogenes as the putative receptor for class IIa bacteriocins. The results from studies reviewed indicate that the EIIt Man of L. monocytogenes could be the chiral receptor needed for bacteriocin interaction at the surface of targeted cells. Specifically, the membrane associated IIDMan and IICMan subunits were implicated in direct interaction with class IIa bacteriocins. Our study focused on cloning, expression and purification of the subunits of the mannose PTS permease of L. monocytogenes EGD. Primers were designed to amplify the subunit genes of the mptACD operon. The mptC, mptD and mptAB genes which were then successfully cloned into pET28a expression vector and transformed into E. coli JM109(DE3) host strain. Recombinant plasmids were screened using colony PCR. Subsequently recombinant pET28-C, pET28-D and pET28-AB was once again transformed and expressed in the E. coli BL21(DE3) pLysS expression host strain. After an induction at 30°C for 5 hours, IICMan and IIDMan were found to be expressed in the cell membrane, whilst IIABMan was expressed in the cytosol of the host expression strain. Membrane proteins His-IICMan, His- IIDMan, and cytosol associated His-IIABMan were purified using Ni2+-NTA affinity chromatography. Results for His-IICMan yielded a 28 kDa protein and a 55 kDa co-purified protein. Results for His-IIDMan yielded a 31 kDa protein and a 60 kDa co-purified protein. Results for His-IIABMan yielded a 35 kDa protein and a 68 kDa co-purified protein. A western blot analysis revealed that all proteins purified carried an attached His-tag as detected by an anti-mouse peroxidase conjugate anti-His-tag antibody. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

Mannose binding lectin (MBL) belongs to the collectin family of soluble pattern recognition molecules that elicit diverse biologic activities. Via multiple carbohydrate-recognition domains (CRD), MBL binds to mannose and N-acetyl-glucosamine oligosaccharides present on the surface of bacteria, fungi and yeast. Following pathogen recognition, MBL activates the complement system via MBL associated serine proteases in a manner independent of antibody and C1 complex. Deficiency in function and level of MBL is found in 25% of otherwise apparently healthy individuals, representing the most prevalent innate immune deficiency. MBL deficiency is a risk factor for the development of infections in humans and mice. The role of MBL as a modulator of infection is complex. MBL deficiency may influence proinflammatory cytokine production, expression of leukocyte adhesion molecules, or vascular damage, during the course of infection. Given that dendritic cells (DC) are antigen presenting cells (APC) with potent capacity to respond to microbial stimulation, I hypothesized that MBL deficiency may be reflected in DC functions associated with microbial stimulation. Initially, I investigated the association of MBL with human immune cells and demonstrated that in both MBL-Sufficient (MBL-S) and MBL-Deficient (MBL-D) individuals, MBL was particularly associated with monocytes. RT-PCR analysis demonstrated MBL was not transcribed by monocytes or other immune cells investigated (T, B, and NK cells, CD11c+DC, immature monocyte derived DC [MoDC], LPS matured MoDC, and granulocytes), suggesting MBL association with the cell surface may be via an adapter or co-receptor. Magnetically separated monocytes but not MoDC bound exogenous purified human plasma MBL (hpMBL). Addition of hpMBL (5 -15 µg/mL) did not induce MoDC activation, and MBL added together with lipopolysaccharide (LPS) did not induce MoDC activation above the level induced by LPS only. In the second part of this study, I used the particulate MBL ligand zymosan (Zy) as a pathogenic stimulus in a whole blood model to gain a greater understanding of the consequences of MBL deficiency. I compared surface phenotype, inflammatory cytokine production and antigen presenting capacity of blood myeloid (M)DC of MBL-D and MBL-S individuals following stimulation with Zy and MBL opsonised Zy (MBL-Zy). Blood MDC in MBL-D individuals, unlike their counterpart in MBL-S individuals, displayed unique functional characteristics, including higher production of proinflammatory cytokines IL-6 and TNF-, but poor capacity for allo-T cell effector cell induction. It appeared that stimulation with MBL-Zy reduced elevated production of IL-6 but not TNF- by blood MDC in MBL-D individuals. In the third part, expression microarray analysis was utilised to provide broad information on the genes and potential signalling pathways involved in the MDC responses in MBL-D and MBL-S individuals following stimulation with Zy and MBL-Zy. MBL-S individuals demonstrated greater capacity to induce T cell and NK cell signalling pathways than MBL-D individuals. Further, MBL acted as a regulator of important inflammatory molecules, namely T-cell receptor zeta (CD247), IFN-γ and perforin 1. The data presented in this study provides novel information on blood MDC function in MBL-S and MBL-D individuals in response to pathogen stimulation, and provided insight into mechanisms involved in the increased frequency of infection observed in MBL-D individuals.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2003. / Vita. Bibliography: 160-174.

Universiẗat, Diss., 2003--Düsseldorf.

A leishmaniose visceral (LV) ou calazar é uma doença endêmica, crônica, grave e de alta letalidade se não tratada. Os estudos apontam a proteína Lectina Ligante de Manose (MBL), codificada pelo gene MBL2, como uma peça-chave na imunidade inata, dada a sua função no reconhecimento microbiano, na eliminação, inflamação e morte celular. Neste trabalho realizamos um estudo do tipo caso-controle que teve como objetivo investigar a associação entre variantes no gene MBL2 e a suscetibilidade à LV em indivíduos residentes em áreas endêmicas da Ilha de São Luís-MA. A amostra foi constituída por 322 indivíduos, sendo 161 casos com LV, não aparentados, de ambos os sexos, residentes em áreas endêmicas da doença na Ilha de São Luís e 161 controles saudáveis, não infectados e não aparentados da mesma região. A identificação dos casos de LV se deu por meio do contato constante com os principais hospitais e ambulatórios de referência para a doença na cidade. Também foram feitas buscas de pacientes com LV em ambiente domiciliar, a partir de registros da FUNASA-MA. A análise molecular consistiu na genotipagem de 6 variantes localizadas na região promotora [posições -550 (C>G), -221(G>C), +4(C>T)] e codificadora [códons 52 (C>T), 54 (G>A) e 57 (G>A)] do gene MBL2, através da reação em cadeia da polimerase e sequenciamento automático. A dosagem da proteína MBL no soro foi realizada pelo teste de ELISA. Verificamos que os fenótipos MBL dependem do conjunto de alelos presentes no gene MBL2, sendo nítido o efeito que as variantes defectivas causam nos níveis da proteína. Não encontramos diferença significativa entre casos e controles em relação à distribuição dos genótipos MBL2 e dos níveis séricos de MBL. As frequências alélicas das variantes exônicas na amostra total mostram que o alelo A é o mais comum (74,8%) e que os alelos defectivos (B, C e D) se encontram principalmente em heterozigose (36,6%), o que reforça a ideia de que alelos MBL2 defectivos são mantidos na população por conferirem vantagem seletiva aos heterozigotos. Em relação aos 3 principais polimorfismos existentes na região promotora, verificamos ser a variante -221G (Y) a mais frequente (88%) seguida de +4C (P) (73%) e de -550C (L) (67%). Identificamos oito haplótipos em MBL2 num total de 644 cromossomos avaliados, em 30 combinações diferentes, sendo HYPA e LYQA os mais frequentes e HYPD e HYPB os mais raros. Todos os portadores de combinações de haplótipos homozigotos para alelos defectivos apresentaram níveis séricos de MBL indetectáveis. Os genótipos LYQA/LYQA e HYPA/HYPA apresentaram as maiores concentrações médias de MBL no soro. A combinação entre SNPs no éxon 1 e na região promotora do gene MBL2 resulta em grande variação nas concentrações de MBL em indivíduos saudáveis. Consideramos que o conjunto de dados gerados é uma contribuição valiosa que poderá ser expandida para outros cenários. / Visceral leishmaniasis (VL), also known as kalazar, is an endemic, chronic, severe and highly lethal disease when not treated. Studies have shown that the protein Mannose-biding lectin (MBL), encoded by the gene MBL2, is the major player in innate immune system due its role in microbial recognition, elimination and inflammation as well as in the cell death. In the current work, we conducted a case-control study which aimed to investigate the association between variants in the gene MBL2 and the susceptibility to VL in individuals living in endemic areas of the São Luís - MA. 322 individuals participated in this study. Of these, 161 were VL cases being unrelated individuals of both sexes, and inhabitants from endemic areas of the disease in São Luís. The other 161 individuals were uninfected healthy controls, being unrelated and from the same region. The identification of VL cases occurred by visiting reference hospitals and clinics in the city. VL patients were identified in the household environment through the records of FUNASA-MA. Molecular analysis consisted in genotyping six variants located in the promoter region [positions -550 (C> G), -221 (G> C), +4 (C> T)] and coding region [codons 52 (C> T), 54 (G> A) and 57 (G> A)] of the MBL2 gene by polymerase chain reaction and automated DNA sequencing. The concentrations of MBL protein in the serum was performed by ELISA. We found that MBL phenotypes depend on the number of alleles present in the gene MBL2, being clear the consequence of the defective variants in the protein levels. There was no significant difference between cases and controls regarding the distribution of MBL2 genotypes and MBL serum levels. The allele frequencies of exon variants in the overall sample showed that the A allele is the most common (74.8%) and that the defective alleles (B, C and D) are mainly heterozygous (36.6%). This highlights the idea that defective MBL2 alleles are maintained in the population to confer selective advantage to heterozygotes. Concerning the three main existing polymorphisms in the promoter region, we noticed that the variant-221G (Y) is more frequent (88%) followed by +4 C (P) (73%) and 550C-(L) (67%) variants. We identified eight haplotypes in MBL2 in a total of 644 chromosomes evaluated in 30 different combinations, being the HYPA and LYQA the most frequent haplotypes and HYPD and HYPB the rarest ones. All carriers with combinations of homozygous haplotypes for defective alleles had undetectable serum levels of MBL. Genotypes LYQA / LYQA and HYPA / HYPA had the highest mean concentrations of MBL in the serum. Combination between SNPs in exon 1 and in the promoter region of the gene MBL2 results in a great variation of MBL concentrations in healthy individuals. We consider that the data set that was generated is a valuable contribution that can be expanded to others cenarios.