Renal cells activate the platelet receptor CLEC-2 through podoplanin.

We have recently shown that the C-type lectin-like receptor, CLEC-2, is expressed on platelets and that it mediates powerful platelet aggregation by the snake venom toxin rhodocytin. In addition, we have provided indirect evidence for an endogenous ligand for CLEC-2 in renal cells expressing HIV-1. This putative ligand facilitates transmission of HIV through its incorporation into the viral envelope and binding to CLEC-2 on platelets. The aim of the present study was to identify the ligand on these cells which binds to CLEC-2 on platelets. Recombinant CLEC-2 exhibits specific binding to HEK-293T (human embryonic kidney) cells in which the HIV can be grown. Furthermore, HEK-293T cells activate both platelets and CLEC-2-transfected DT-40 B-cells. The transmembrane protein podoplanin was identified on HEK-293T cells and was demonstrated to mediate both binding of HEK-293T cells to CLEC-2 and HEK-293T cell activation of CLEC-2-transfected DT-40 B-cells. Podoplanin is expressed on renal cells (podocytes). Furthermore, a direct interaction between CLEC-2 and podoplanin was confirmed using surface plasmon resonance and was shown to be independent of glycosylation of CLEC-2. The interaction has an affinity of 24.5+/-3.7 microM. The present study identifies podoplanin as a ligand for CLEC-2 on renal cells.

No impact of malaria infection or immunisation on the expression of the immune GTPase GIMAP1 at the protein or mRNA levels (Article no. 53)

GIMAP (GTPase of the immunity-associated protein family) proteins are a family of putative GTPases believed to be regulators of cell death in lymphomyeloid cells. GIMAP1 was the first reported member of this gene family, identified as a gene up-regulated at the RNA level in the spleens of mice infected with the malarial parasite, Plasmodium chabaudi. Methods A monoclonal antibody against mouse GIMAP1 was developed and was used to analyse the expression of the endogenous protein in tissues of normal mice and in defined sub-populations of cells prepared from lymphoid tissues using flow cytometry. It was also used to assess the expression of GIMAP1 protein after infection and/or immunization of mice with P. chabaudi. Real-time PCR analysis was employed to measure the expression of GIMAP1 for comparison with the protein level analysis. Results GIMAP1 protein expression was detected in all lineages of lymphocytes (T, B, NK), in F4/80+ splenic macrophages and in some lymphoid cell lines. Additional evidence is presented suggesting that the strong expression by mature B cells of GIMAP1 and other GIMAP genes and proteins seen in mice may be a species-dependent characteristic. Unexpectedly, no increase was found in the expression of GIMAP1 in P. chabaudi infected mice at either the mRNA or protein level, and this remained so despite applying a number of variations to the protocol. Conclusion The model of up-regulation of GIMAP1 in response to infection/immunization with P. chabaudi is not a robustly reproducible experimental system. The GIMAP1 protein is widely expressed in lymphoid cells, with an interesting increase in expression in the later stages of B cell development. Alternative approaches will be required to define the functional role of this GTPase in immune cells.

The severity of malarial anaemia in Plasmodium chabaudi infections of BALB/c mice is determined independently of the number of circulating parasites

Background: Severe malarial anaemia is a major complication of malaria infection and is multifactorial resulting from loss of circulating red blood cells (RBCs) from parasite replication, as well as immune-mediated mechanisms. An understanding of the causes of severe malarial anaemia is necessary to develop and implement new therapeutic strategies to tackle this syndrome of malaria infection. Methods: Using analysis of variance, this work investigated whether parasite-destruction of RBCs always accounts for the severity of malarial anaemia during infections of the rodent malaria model Plasmodium chabaudi in mice of a BALB/c background. Differences in anaemia between two different clones of P. chabaudi were also examined. Results: Circulating parasite numbers were not correlated with the severity of anaemia in either BALB/c mice or under more severe conditions of anaemia in BALB/c RAG2 deficient mice (lacking T and B cells). Mice infected with P. chabaudi clone CB suffered more severe anaemia than mice infected with clone AS, but this was not correlated with the number of parasites in the circulation. Instead, the peak percentage of parasitized RBCs was higher in CB-infected animals than in AS-infected animals, and was correlated with the severity of anaemia, suggesting that the availability of uninfected RBCs was impaired in CB-infected animals. Conclusion: This work shows that parasite numbers are a more relevant measure of parasite levels in P. chabaudi infection than % parasitaemia, a measure that does not take anaemia into account. The lack of correlation between parasite numbers and the drop in circulating RBCs in this experimental model of malaria support a role for the host response in the impairment or destruction of uninfected RBC in P. chabaudi infections, and thus development of acute anaemia in this malaria model.

Mining gene networks with application to GAW15 problem 1

The genetic analysis workshop 15 (GAW15) problem 1 contained baseline expression levels of 8793 genes in immortalised B cells from 194 individuals in 14 Centre d’Etude du Polymorphisme Humane (CEPH) Utah pedigrees. Previous analysis of the data showed linkage and association and evidence of substantial individual variations. In particular, correlation was examined on expression levels of 31 genes and 25 target genes corresponding to two master regulatory regions. In this analysis, we apply Bayesian network analysis to gain further insight into these findings. We identify strong dependences and therefore provide additional insight into the underlying relationships between the genes involved. More generally, the approach is expected to be applicable for integrated analysis of genes on biological pathways.

Xylo-oligosaccharides alone or in synbiotic combination with Bifidobacterium animalis subsp. lactis induce bifidogenesis and modulate markers of immune function in healthy adults: a double-blind, placebo-controlled, randomised, factorial cross-over study.

Prebiotics, probiotics and synbiotics are dietary ingredients with the potential to influence health and mucosal and systemic immune function by altering the composition of the gut microbiota. In the present study, a candidate prebiotic (xylo-oligosaccharide, XOS, 8 g/d), probiotic (Bifidobacterium animalis subsp. lactis Bi-07, 109 colony-forming units (CFU)/d) or synbiotic (8 g XOS+109 CFU Bi-07/d) was given to healthy adults (25–65 years) for 21 d. The aim was to identify the effect of the supplements on bowel habits, self-reported mood, composition of the gut microbiota, blood lipid concentrations and immune function. XOS supplementation increased mean bowel movements per d (P= 0·009), but did not alter the symptoms of bloating, abdominal pain or flatulence or the incidence of any reported adverse events compared with maltodextrin supplementation. XOS supplementation significantly increased participant-reported vitality (P= 0·003) and happiness (P= 0·034). Lowest reported use of analgesics was observed during the XOS+Bi-07 supplementation period (P= 0·004). XOS supplementation significantly increased faecal bifidobacterial counts (P= 0·008) and fasting plasma HDL concentrations (P= 0·005). Bi-07 supplementation significantly increased faecal B. lactis content (P= 0·007), lowered lipopolysaccharide-stimulated IL-4 secretion in whole-blood cultures (P= 0·035) and salivary IgA content (P= 0·040) and increased IL-6 secretion (P= 0·009). XOS supplementation resulted in lower expression of CD16/56 on natural killer T cells (P= 0·027) and lower IL-10 secretion (P= 0·049), while XOS and Bi-07 supplementation reduced the expression of CD19 on B cells (XOS × Bi-07, P= 0·009). The present study demonstrates that XOS induce bifidogenesis, improve aspects of the plasma lipid profile and modulate the markers of immune function in healthy adults. The provision of XOS+Bi-07 as a synbiotic may confer further benefits due to the discrete effects of Bi-07 on the gut microbiota and markers of immune function.

Targeted activation of toll-like receptors: conjugation of a toll-like receptor 7 agonist to a monoclonal antibody maintains antigen binding and specificity

Therapeutic activation of Toll-like receptors (TLR) has potential for cancer immunotherapy, for augmenting the activity of anti-tumor monoclonal antibodies (mAbs), and for improved vaccine adjuvants. A previous attempt to specifically target TLR agonists to dendritic cells (DC) using mAbs failed because conjugation led to non-specific binding and mAbs lost specificity. We demonstrate here for the first time the successful conjugation of a small molecule TLR7 agonist to an anti-tumour mAb (the anti-hCD 20 rituximab) without compromising antigen specificity. The TLR7 agonist UC-1V150 was conjugated to rituximab using two conjugation methods and yield, molecular substitution ratio, retention of TLR7 activity and specificity of antigen binding were compared. Both conjugation methods produced rituximab-UC-1V150 conjugates with UC-1V150 : rituximab ratio ranging from 1:1 to 3:1 with drug loading quantified by UV spectroscopy and drug substitution ratio verified by MALDI TOF mass spectroscopy. The yield of purified conjugates varied with conjugation method, and dropped as low as 31% using a method previously described for conjugating UC-1V150 to proteins, where a bifunctional crosslinker was firstly reacted with rituximab, and secondly to the TLR7 agonist. We therefore developed a direct conjugation method by producing an amine-reactive UV active version of UC-1V150, termed NHS:UC-1V150. Direct conjugation with NHS:UC-1V150 was quick and simple and gave improved conjugate yields of 65-78%. Rituximab-UC-1V150 conjugates had the expected pro-inflammatory activity in vitro (EC50 28-53 nM) with a significantly increased activity over unconjugated UC-1V150 (EC50 547 nM). Antigen binding and specificity of the rituxuimab-UC-1V150 conjugates was retained, and after incubation with human peripheral blood leukocytes, all conjugates bound strongly only to CD20-expressing B cells whilst no non-specific binding to CD20-negative cells was observed. Selective targeting of Toll-like receptor activation directly within tumors or to DC is now feasible.

Tityus discrepans scorpion venom activates platelets through GPVI and a novel Src-dependent signaling pathway

In humans and other mammals, Tityus discrepans (Td) scorpion envenomation produces a variety of systemic effects including respiratory distress, a generalized inflammatory reaction, modulation of blood pressure, fibrin formation, and platelet activation. For many of these effects, the venom components and underlying mechanisms are not known. In the present study, we demonstrate that Td venom (TdV) stimulates integrin αIIbβ3-dependent aggregation of washed human and mouse platelets downstream of Src kinase activation. The pattern of increase in tyrosine phosphorylation induced by TdV in human platelets is similar to that induced by the collagen receptor GPVI, and includes FcR γ-chain, Syk, and PLC γ 2. Confirmation of GPVI activation by TdV was achieved by expression of human GPVI in chicken DT40 B cells and use of a reporter assay. To our surprise, TdV was able to activate mouse platelets deficient in the GPVI-FcR γ-chain complex through a pathway that was also dependent on Src kinases. TdV therefore activates platelets through GPVI and a second, as yet unidentified Src kinase-dependent pathway.

Epstein-Barr virus: clinical and epidemiological revisits and genetic basis of oncogenesis

Epstein-Barr virus (EBV) is classified as a member in the order herpesvirales, family herpesviridae, subfamily gammaherpesvirinae and the genus lymphocytovirus. The virus is an exclusively human pathogen and thus also termed as human herpesvirus 4 (HHV4). It was the first oncogenic virus recognized and has been incriminated in the causation of tumors of both lymphatic and epithelial nature. It was reported in some previous studies that 95% of the population worldwide are serologically positive to the virus. Clinically, EBV primary infection is almost silent, persisting as a life-long asymptomatic latent infection in B cells although it may be responsible for a transient clinical syndrome called infectious mononucleosis. Following reactivation of the virus from latency due to immunocompromised status, EBV was found to be associated with several tumors. EBV linked to oncogenesis as detected in lymphoid tumors such as Burkitt's lymphoma (BL), Hodgkin's disease (HD), post-transplant lymphoproliferative disorders (PTLD) and T-cell lymphomas (e.g. Peripheral T-cell lymphomas; PTCL and Anaplastic large cell lymphomas; ALCL). It is also linked to epithelial tumors such as nasopharyngeal carcinoma (NPC), gastric carcinomas and oral hairy leukoplakia (OHL). In vitro, EBV many studies have demonstrated its ability to transform B cells into lymphoblastoid cell lines (LCLs). Despite these malignancies showing different clinical and epidemiological patterns when studied, genetic studies have suggested that these EBV- associated transformations were characterized generally by low level of virus gene expression with only the latent virus proteins (LVPs) upregulated in both tumors and LCLs. In this review, we summarize some clinical and epidemiological features of EBV- associated tumors. We also discuss how EBV latent genes may lead to oncogenesis in the different clinical malignancies

Neurokinin-B transcription in erythroid cells - Direct activation by the hematopoietic transcription factor GATA-1

The GATA family of transcription factors establishes genetic networks that control developmental processes including hematopoiesis, vasculogenesis, and cardiogenesis. We found that GATA-1 strongly activates transcription of the Tac-2 gene, which encodes proneurokinin-B, a precursor of neurokinin-B (NK-B). Neurokinins function through G protein-coupled transmembrane receptors to mediate diverse physiological responses including pain perception and the control of vascular tone. Whereas an elevated level of NK-B was implicated in pregnancy-associated pre-eclampsia ( Page, N. M., Woods, R. J., Gardiner, S. M., Lomthaisong, K., Gladwell, R. T., Butlin, D. J., Manyonda, I. T., and Lowry, P. J. ( 2000) Nature 405, 797 - 800), the regulation of NK-B synthesis and function are poorly understood. Tac-2 was expressed in normal murine erythroid cells and was induced upon ex vivo erythropoiesis. An estrogen receptor fusion to GATA-1 (ER-GATA-1) and endogenous GATA-1 both occupied a region of Tac-2 intron-7, which contains two conserved GATA motifs. Genetic complementation analysis in GATA-1-null G1E cells revealed that endogenous GATA-2 occupied the same region of intron-7, and expression of ER-GATA-1 displaced GATA-2 and activated Tac-2 transcription. Erythroid cells did not express neurokinin receptors, whereas aortic and yolk sac endothelial cells differentially expressed neurokinin receptor subtypes. Since NK-B induced cAMP accumulation in yolk sac endothelial cells, these results suggest a new mode of vascular regulation in which GATA-1 controls NK-B synthesis in erythroid cells.

Peroxynitrite-modified collagen-II induces p38/ERK and NF-kappa B-dependent synthesis of prostaglandin E-2 and nitric oxide in chondrogenically differentiated mesenchyrnal progenitor cells

Objective: Peroxynitrite (ONOO-) is formed in the inflamed and degenerating human joint. Peroxynitrite-modified collagen-II (PMC-II) was recently discovered in the serum of patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Therefore we investigated the cellular effects of PMC-II on human mesenchymal progenitor cells (MPCs) as a model of cartilage and cartilage repair cells in the inflamed and degenerating joint. Design: MPCs were isolated from the trabecular bone of patients undergoing reconstructive surgery and were differentiated into a chondrogenic lineage. Cells were exposed to PMC-II and levels of the proinflammatory mediators nitric oxide (NO) and prostaglandin E-2 (PGE(2)) measured. Levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), phosphorylated mitogen activated protein kinases (MAPKs) and nuclear factor kappa B (NF-kappa B) activation were measured by enzyme linked immunosorbent assay (ELISA) together with specific MAPK and NF-kappa B inhibitors. Results: PMC-II induced NO and PGE(2) synthesis through upregulation of iNOS and COX-2 proteins. PMC-II also lead to the phosphorylation of MAPKs, extracellularly regulated kinase 1/2 (ERK1/2) and p38 [but not c-Jun NH2-terminal kinase (JNK1/2)] and the activation of proinflammatory transcription factor NF-kappa B. Inhibitors of p38, ERK1/2 and NF-kappa B prevented PMC-II induced NO and PGE(2) synthesis, NOS and COX-2 protein expression and NF-kappa B activation. Conclusion: iNOS, COX-2, NF-KB and MAPK are known to be activated in the joints of patients with OA and RA. PMC-II induced iNOS and COX-2 synthesis through p38, ERK1/2 and NF-KB dependent pathways suggesting a previously unidentified pathway for the synthesis of the proinflammatory mediators, NO and PGE(2), further suggesting that inhibitors of these pathways may be therapeutic in the inflamed and degenerating human joint. (c) 2005 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Differences in cell morphology, lipid and apo B secretory capacity in caco-2 cells following long term treatment with saturated and monounsaturated fatty acids

The suitability of the caco-2 cell line as a model for studying the long term impact of dietary fatty acids on intestinal lipid handling and chylomicron production was examined. Chronic supplementation of caco-2 cells with palmitic acid (PA) resulted in a lower triacylglycerol secretion than oleic acid (OA). This was coupled with a detrimental effect of PA, but not OA, on transepithelial electrical resistance (TER) measurements, suggesting a loss of structural integrity across the cell monolayer. Addition of OA reversed the adverse effects of PA and stearic acid on TER and increased the ability of cells to synthesise and accumulate lipid, but did not normalise the secretion of lipids by caco-2 cells. Increasing amounts of OA and decreasing amounts of PA in the incubation media markedly improved the ability of cells to synthesise apolipoprotein B and secrete lipids. Real time RT-PCR revealed a down regulation of genes involved in lipoprotein synthesis following PA than OA. Electron microscopy showed adverse effects of PA on cellular morphology consistent with immature enterocytes such as stunted microvilli and poor tight junction formation. In conclusion, previously reported differences in lipoprotein secretion by caco-2 cells supplemented with saturated fatty acids (SFA) and OA may partly reflect early cytotoxic effects of SFA on cellular integrity and function. (C) 2007 Elsevier B.V. All rights reserved.