15 research outputs found
Cloning and recombinant expression of a 822 bp region of a Pf403 Plasmodium falciparum gene.
No full textThesis (M.Sc.) - University of Natal, Pietermaritzburg, 2003.Malaria is a devastating parasitic disease in humans caused by species in the genus Plasmodium.
With over 100 million cases and at least 1.5 million fatalities each year, the disease accounts for
4-5% of all fatalities in the world. A recent increase in the number of malaria cases in South
Africa has imposed severe costs on the economy and public health.
Immunity to malaria is a multi-component system involving both B and T celllymphocytes.
Pc96 is a 96 kDa antigen identified in the mouse malaria model Plasmodium chabaudi adami. It
is known to be associated with the outer membrane of mouse erythrocytes infected with the
parasite and has shown protective roles in mice challenged with P. chabaudi adami. A specific T
cell clone has been identified that adoptively provides protection to athymic mice infected with
P. chabaudi adami. Antibodies raised against Pc96 identified proteins that induced the
proliferation of the protective T cell clones. At least four other antigens of different species of.
malaria share at least one cross-reactive epitope.
In an attempt to identify a Plasmodiumfalciparum homologue ofPc96, the amino-acid sequence
was used in a BLAST search of the P. falciparum genome database, identifying a 403 kDa
protein with a high degree of homology to Pc96. Sequence alignments indicated a region
spanning 90 amino acids in Pf403 that overlaps the Pc96 amino acid sequence. A 178 kDa
protein in P. yoelii yoelii (Pyy178) was shown to be highly similar to Pc96. Tvcell epitope
prediction programs identified putative T cell epitopes in Pc96 which appear to be conserved in
Pf403 and Pyy178. A casein kinase IT phosphorylation site was also identified in this region and
is conserved in both sequences. PCR primers were designed to amplify regions of the
MAL3P6.11 gene coding for Pf403 from P.falciparum genomic DNA. An 817 bp region in the
MAL3P6.11 gene was amplified. This codes for the region ofPf403 that shows high homology
to Pc96 and contains the conserved T cell epitopes and casein kinase phophorylation site. A
BamHI site was incorporated into the forward primer to facilitate in-frame ligation with cloning
vectors. The PCRproduct obtained was verified by restriction analysis using HindIII and EcoRI
sites within the fragment.
The 817 bp peR product was cloned into the pMOSBlue vector using a blunt-endedPCR cloning
kit, and transformed into MOSBlue competent cells. Recombinants were identified using the uIV
complementation system, and verified by PCR, plasmid DNA isolation, and restriction digestion
analysis. The insertDNA in pMOSBlue was cut out with BamHI and sub-cloned into the BamHI
site in the pMAL-C2x expression vector. Sequencing ofthe construct confirmed the identity of
the cloned insert and showed the sequence to be in frame with the malE gene coding for maltose
binding protein (MBP). The fusion protein, MBP-Pf32 .5, was induced and expressed as a 75 kDa
protein comprising ofthe 32.5 kDa region ofPf403, and MBP (42.5 kDa) and was detected by
anti-MBP antibodies, by western blotting. This recombinant protein has many applications for
further studies involving the characterisation of the Pf403 protein, and the determination of
possible roles that the protein may have in stimulating an immune response during human
malaria infections
IL-10 mediated control of TNF gene regulation in human macrophages
No full textEThOS - Electronic Theses Online ServiceGBUnited Kingdo
Role of STAT3 in glucocorticoid-induced expression of the human IL-10 gene
No full textIn the present report we have determined the molecular mechanisms, which govern the expression of the human IL-10 gene when induced by the glucocorticoid Methyl-Prednisolone (MP). Treatment of cells with MP at 10(-6) M will readily induce IL-10 in CD19+ primary B cells and in a human B cell line. Analysis of the IL-10 promoter showed a robust 18-fold induction and demonstrated that a potential GRE motif was not required, while mutation of the -120 STAT-motif strongly reduced MP-induced trans-activation. A strong induction was also seen with a trimeric STAT-motif and over-expression of dominant-negative STAT3 could block MP induction of IL-10 mRNA. Finally, MP treatment induced binding of STAT3 to the promoter as shown by gelshift, supershift and by chromatin-immunoprecipitation. These data show that glucocorticoid-induced expression of the IL-10 gene is mediated by the transcription factor STAT3
A deletion defining a common Asian lineage of Mycobacterium tuberculosis associates with immune subversion
No full textSix major lineages of Mycobacterium tuberculosis appear preferentially transmitted amongst distinct ethnic groups. We identified a deletion affecting Rv1519 in CH, a strain isolated from a large outbreak in Leicester U.K., that coincidentally defines the East African-Indian lineage matching a major ethnic group in this city. In broth media, CH grew less rapidly and was less acidic and H2O2-tolerant than reference sequenced strains (CDC1551 and H37Rv). Nevertheless, CH was not impaired in its ability to grow in human monocyte-derived macrophages. When compared with CDC1551 and H37Rv, CH induced less protective IL-12p40 and more antiinflammatory IL-10 and IL-6 gene transcription and secretion from monocyte-derived macrophages. It thus appears that CH compensates microbiological attenuation by skewing the innate response toward phagocyte deactivation. Complementation of Rv1519, but none of nine additional genes absent from CH compared with the type strain, H37Rv, reversed the capacity of CH to elicit antiinflammatory IL-10 production by macrophages. The Rv1519 polymorphism in M. tuberculosis confers an immune subverting phenotype that contributes to the persistence and outbreak potential of this lineag
T-Cell Responses
No full textLentiviral vectors deliver antigens to dendritic cells (DCs) in vivo, but they do not trigger DC maturation. We therefore expressed a viral protein that constitutively activates NF-kappaB, vFLIP from Kaposi's sarcoma-associated herpesvirus (KSHV), in a lentivector to mature DCs. vFLIP activated NF-kappaB in mouse bone marrow-derived DCs in vitro and matured these DCs to a similar extent as lipopolysaccharide; costimulatory markers CD80, CD86, CD40, and ICAM-1 were upregulated and tumor necrosis factor alpha and interleukin-12 secreted. The vFLIP-expressing lentivector also matured DCs in vivo. When we coexpressed vFLIP in a lentivector with ovalbumin (Ova), we found an increased immune response to Ova; up to 10 times more Ova-specific CD8(+) T cells secreting gamma interferon were detected in the spleens of vFLIP_Ova-immunized mice than in the spleens of mice immunized with GFP_Ova. Furthermore, this increased CD8(+) T-cell response correlated with improved tumor-free survival in a tumor therapy model. A single immunization with vFLIP_Ova also reduced the parasite load when mice were challenged with OVA-Leishmania donovani. In conclusion, vFLIP from KSHV is a DC activator, maturing DCs in vitro and in vivo. This demonstrates that NF-kappaB activation is sufficient to induce many aspects of DC maturation and that expression of a constitutive NF-kappaB activator can improve the efficacy of a vaccine vector
Bruton's tyrosine kinase regulates TLR7/8-induced TNF transcription via nuclear factor-κB recruitment
No full textTumour necrosis factor (TNF) is produced by primary human macrophages in response to stimulation by exogenous pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) via Toll-like receptor (TLR) signalling. However, uncontrolled TNF production can be deleterious and hence it is tightly controlled at multiple stages. We have previously shown that Bruton's tyrosine kinase (Btk) regulates TLR4-induced TNF production via p38 MAP Kinase by stabilising TNF messenger RNA. Using both gene over-expression and siRNA-mediated knockdown we have examined the role of Btk in TLR7/8 mediated TNF production. Our data shows that Btk acts in the TLR7/8 pathway and mediates Ser-536 phosphorylation of p65 RelA and subsequent nuclear entry in primary human macrophages. These data show an important role for Btk in TLR7/8 mediated TNF production and reveal distinct differences for Btk in TLR4 versus TLR7/8 signalling
IRF5 PROMOTES INFLAMMATORY MACROPHAGE POLARIZATION AND TH1/TH17 RESPONSE
No full textPolymorphisms in the gene encoding the transcription factor IRF5 that lead to higher mRNA expression are associated with many autoimmune diseases. Here we show that IRF5 expression in macrophages was reversibly induced by inflammatory stimuli and contributed to the plasticity of macrophage polarization. High expression of IRF5 was characteristic of M1 macrophages, in which it directly activated transcription of the genes encoding interleukin 12 subunit p40 (IL-12p40), IL-12p35 and IL-23p19 and repressed the gene encoding IL-10. Consequently, those macrophages set up the environment for a potent T helper type 1 (T(H)1)-T(H)17 response. Global gene expression analysis demonstrated that exogenous IRF5 upregulated or downregulated expression of established phenotypic markers of M1 or M2 macrophages, respectively. Our data suggest a critical role for IRF5 in M1 macrophage polarization and define a previously unknown function for IRF5 as a transcriptional repressor
Discoidin Receptor 2 Controls Bone Formation and Marrow Adipogenesis
No full textCell–extracellular matrix (ECM) interactions play major roles in controlling progenitor cell fate and differentiation. The receptor tyrosine kinase, discoidin domain receptor 2 (DDR2), is an important mediator of interactions between cells and fibrillar collagens. DDR2 signals through both ERK1/2 and p38 MAP kinase, which stimulate osteoblast differentiation and bone formation. Here we show that DDR2 is critical for skeletal development and differentiation of marrow progenitor cells to osteoblasts while suppressing marrow adipogenesis. Smallie mice (Ddr2slie/slie), which contain a nonfunctional Ddr2 allele, have multiple skeletal defects. A progressive decrease in tibial trabecular bone volume/total volume (BV/TV) was observed when wild‐type (WT), Ddr2wt/slie, and Ddr2slie/slie mice were compared. These changes were associated with reduced trabecular number (Tb.N) and trabecular thickness (Tb.Th) and increased trabecular spacing (Tb.Sp) in both males and females, but reduced cortical thickness only in Ddr2slie/slie females. Bone changes were attributed to decreased bone formation rather than increased osteoclast activity. Significantly, marrow fat and adipocyte‐specific mRNA expression were significantly elevated in Ddr2slie/slie animals. Additional skeletal defects include widened calvarial sutures and reduced vertebral trabecular bone. To examine the role of DDR2 signaling in cell differentiation, bone marrow stromal cells (BMSCs) were grown under osteogenic and adipogenic conditions. Ddr2slie/slie cells exhibited defective osteoblast differentiation and accelerated adipogenesis. Changes in differentiation were related to activity of runt‐related transcription factor 2 (RUNX2) and PPARγ, transcription factors that are both controlled by MAPK‐dependent phosphorylation. Specifically, the defective osteoblast differentiation in calvarial cells from Ddr2slie/slie mice was associated with reduced ERK/MAP kinase and RUNX2‐S319 phosphorylation and could be rescued with a constitutively active phosphomimetic RUNX2 mutant. Also, DDR2 was shown to increase RUNX2‐S319 phosphorylation and transcriptional activity while also increasing PPARγ‐S112 phosphorylation, but reducing its activity. DDR2 is, therefore, important for maintenance of osteoblast activity and suppression of marrow adipogenesis in vivo and these actions are related to changes in MAPK‐dependent RUNX2 and PPARγ phosphorylation. © 2016 American Society for Bone and Mineral Research.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135235/1/jbmr2893_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135235/2/jbmr2893.pd
Hyaluronan carried by tumor-derived microvesicles induces IL-10 production in classical (CD14<sup>++</sup>CD16<sup>-</sup>) monocytes via PI3K/Akt/mTOR-dependent signalling pathway
No full textTumor-derived microvesicles (TMV) can mimic effects of tumor cells leading to an increased anti-inflammatory cytokine production, such as interleukin 10 (IL-10), by tumor-infiltrating monocytes and macrophages. Yet, the mechanism of IL-10 induction by TMV in monocytes remains unclear. The co-incubation of TMV derived from the human pancreas carcinoma cell line (HPC-4) with human monocytes resulted in a nearly 30-fold increase in IL-10 protein production. This effect operates at the level of transcription since monocytes transduced with an adenovirus containing IL-10-promoter luciferase reporter gene showed a 5-fold induction of luciferase activity after treatment with TMV. Since tumor cells can express hyaluronan (HA), which participates in tumor invasion and metastases, we have tested its effect on IL-10 expression. We showed that HA at the concentration of 100 μg/ml induces IL-10 protein expression and the IL-10 promoter activation in monocytes. Moreover, hyaluronidase treatment of TMV reduced IL-10 protein production by 50% and promoter activity by 40%. Inhibitors of the PI3K/Akt/mTOR pathway reduced both, TMV-induced IL-10 promoter activity and protein production, and the same was observed in monocytes when stimulated by HPC-4 cells or HA. Inhibition of PI3K activity down-regulated phosphorylation of the Akt and (to a lesser extent) mTOR proteins in monocytes following TMV or HA stimulation. When comparing monocyte subsets, TMV induced IL-10 protein and mRNA synthesis only in classical CD14++CD16- but not in CD16-positive monocytes. Our data show that TMV induce IL-10 synthesis in human classical monocytes via HA, which, in turn, activates the PI3K/Akt/mTOR pathway
