Excitotoxicity in the lung: N-methyl-D-aspartate-induced, nitric oxide-dependent, pulmonary edema is attenuated by vasoactive intestinal peptide and by inhibitors of poly(ADP-ribose) polymerase.

Excitatory amino acid toxicity, resulting from overactivation of N-methyl-D-aspartate (NMDA) glutamate receptors, is a major mechanism of neuronal cell death in acute and chronic neurological diseases. We have investigated whether excitotoxicity may occur in peripheral organs, causing tissue injury, and report that NMDA receptor activation in perfused, ventilated rat lungs triggered acute injury, marked by increased pressures needed to ventilate and perfuse the lung, and by high-permeability edema. The injury was prevented by competitive NMDA receptor antagonists or by channel-blocker MK-801, and was reduced in the presence of Mg2+. As with NMDA toxicity to central neurons, the lung injury was nitric oxide (NO) dependent: it required L-arginine, was associated with increased production of NO, and was attenuated by either of two NO synthase inhibitors. The neuropeptide vasoactive intestinal peptide and inhibitors of poly(ADP-ribose) polymerase also prevented this injury, but without inhibiting NO synthesis, both acting by inhibiting a toxic action of NO that is critical to tissue injury. The findings indicate that: (i) NMDA receptors exist in the lung (and probably elsewhere outside the central nervous system), (ii) excessive activation of these receptors may provoke acute edematous lung injury as seen in the "adult respiratory distress syndrome," and (iii) this injury can be modulated by blockade of one of three critical steps: NMDA receptor binding, inhibition of NO synthesis, or activation of poly(ADP-ribose) polymerase.

Vascular endothelial growth factor-related protein: a ligand and specific activator of the tyrosine kinase receptor Flt4.

The tyrosine kinases Flt4, Flt1, and Flk1 (or KDR) constitute a family of endothelial cell-specific receptors with seven immunoglobulin-like domains and a split kinase domain. Flt1 and Flk1 have been shown to play key roles in vascular development; these two receptors bind and are activated by vascular endothelial growth factor (VEGF). No ligand has been identified for Flt4, whose expression becomes restricted during development to the lymphatic endothelium. We have identified cDNA clones from a human glioma cell line that encode a secreted protein with 32% amino acid identity to VEGF. This protein, designated VEGF-related protein (VRP), specifically binds to the extracellular domain of Flt4, stimulates the tyrosine phosphorylation of Flt4 expressed in mammalian cells, and promotes the mitogenesis of human lung endothelial cells. VRP fails to bind appreciably to the extracellular domain of Flt1 or Flk1. The protein contains a C-terminal, cysteine-rich region of about 180 amino acids that is not found in VEGF. A 2.4-kb VRP mRNA is found in several human tissues including adult heart, placenta, ovary, and small intestine and in fetal lung and kidney.

Cloning and comparative analysis of the human pre-T-cell receptor alpha-chain gene.

In immature T cells the T-cell receptor (TCR) beta-chain gene is rearranged and expressed before the TCR alpha-chain gene. At this stage TCR beta chain can form disulfide-linked heterodimers with the pre-T-cell receptor alpha chain (pTalpha). Using the recently isolated murine pTalpha cDNA as a probe, we have isolated the human pTalpha cDNA. The complete nucleotide sequence predicts a mature protein of 282 aa consisting of an extracellular immunoglobulin-like domain, a connecting peptide, a transmembrane region, and a long cytoplasmic tail. Amino acid sequence comparison of human pTalpha with the mouse pTalpha molecule reveals high sequence homology in the extracellular as well as the transmembrane region. In contrast, the cytoplasmic region differs in amino acid composition and in length from the murine homologue. The human pTalpha gene is expressed in immature but not mature T cells and is located at the p21.2-p12 region of the short arm of chromosome 6.

Determinants of the phagocytic signal mediated by the type IIIA Fc gamma receptor, Fc gamma RIIIA: sequence requirements and interaction with protein-tyrosine kinases.

The Fc gamma receptor-associated gamma and zeta subunits contain a conserved cytoplasmic motif, termed the immunoglobulin gene tyrosine activation motif, which contains a pair of YXXL sequences. The tyrosine residues within these YXXL sequences have been shown to be required for transduction of a phagocytic signal. We have previously reported that the gamma subunit of the type IIIA Fc gamma receptor (Fc gamma RIIIA) is approximately 6 times more efficient in mediating phagocytosis than the zeta subunit of Fc gamma RIIIA. By exchanging regions of the cytoplasmic domains of the homologous gamma and zeta chains, we observed that the cytoplasmic area of the gamma chain bearing a pair of the conserved YXXL sequences is important in phagocytic signaling. Further specificity of phagocytic signaling is largely determined by the two internal XX amino acids in the YXXL sequences. In contrast, the flanking amino acids of the YXXL sequences including the seven intervening amino acids between the two YXXL sequences do not significantly affect the phagocytic signal. Furthermore, the protein-tyrosine kinase Syk, but not the related kinase ZAP-70, stimulated Fc gamma RIIIA-mediated phagocytosis. ZAP-70, however, increased phagocytosis when coexpressed with the Src family kinase Fyn. These data demonstrate the importance of the two specific amino acids within the gamma subunit YXXL cytoplasmic sequences in phagocytic signaling and explain the difference in phagocytic efficiency of the gamma and zeta chains. These results indicate the importance of Syk in Fc gamma RIIIA-mediated phagocytosis and demonstrate that ZAP-70 and syk differ in their requirement for a Src-related kinase in signal transduction.

The Lutheran blood group glycoprotein, another member of the immunoglobulin superfamily, is widely expressed in human tissues and is developmentally regulated in human liver.

Glycoproteins expressing the Lutheran blood group antigens were isolated from human erythrocyte membranes and from human fetal liver. Amino acid sequence analyses allowed the design of redundant oligonucleotides that were used to generate a 459-bp, sequence-specific probe by PCR. A cDNA clone of 2400 bp was isolated from a human placental lambda gt 11 library and sequenced, and the deduced amino acid sequence was studied. The predicted mature protein is a type I membrane protein of 597 amino acids with five potential N-glycosylation sites. There are five disulfide-bonded, extracellular, immunoglobulin superfamily domains (two variable-region set and three constant-region set), a single hydrophobic, membrane-spanning domain, and a cytoplasmic domain of 59 residues. The overall structure is similar to that of the human tumor marker MUC 18 and the chicken neural adhesion molecule SC1. The extracellular domains and cytoplasmic domain contain consensus motifs for the binding of integrin and Src homology 3 domains, respectively, suggesting possible receptor and signal-transduction function. Immunostaining of human tissues demonstrated a wide distribution and provided evidence that the glycoprotein is under developmental control in liver and may also be regulated during differentiation in other tissues.

Regulation of CD45 engagement by the B-cell receptor CD22.

The B-cell receptor CD22 binds sialic acid linked alpha-2-6 to terminal galactose residues on N-linked oligosaccharides associated with several cell-surface glycoproteins. The first of these sialoglycoproteins to be identified was the receptor-linked phosphotyrosine phosphatase CD45, which is required for antigen/CD3-induced T-cell activation. In the present work, we examine the effect of interaction between the extracellular domain of CD45 and CD22 on T-cell activation. Using soluble CD22-immunoglobulin fusion proteins and T cells expressing wild-type and chimeric CD45 forms, we show that engagement of CD45 by soluble CD22 can modulate early T-cell signals in antigen receptor/CD3-mediated stimulation. We also show that addition of sialic acid by beta-galactoside alpha-2,6-sialyltransferase to the CD22 molecule abrogates interactions between CD22 and its ligands. Together, these observations provide direct evidence for a functional role of the interaction between the extracellular domain of CD45 and a natural ligand and suggest another regulatory mechanism for CD22-mediated ligand engagement.

The activating role of phospho-(Tyr)-calmodulin on the epidermal growth factor receptor

The activity of calmodulin (CaM) is modulated not only by oscillations in the cytosolic concentration of free Ca2+, but also by its phosphorylation status. In the present study, the role of tyrosine-phosphorylated CaM [P-(Tyr)-CaM] on the regulation of the epidermal growth factor receptor (EGFR) has been examined using in vitro assay systems. We show that phosphorylation of CaM by rat liver solubilized EGFR leads to a dramatic increase in the subsequent phosphorylation of poly-L-(Glu:Tyr) (PGT) by the receptor in the presence of ligand, both in the absence and in the presence of Ca2+. This occurred in contrast with assays where P-(Tyr)-CaM accumulation was prevented by the presence of Ca2+, absence of a basic cofactor required for CaM phosphorylation and/or absence of CaM itself. Moreover, an antibody against CaM, which inhibits its phosphorylation, prevented the extra ligand-dependent EGFR activation. Addition of purified P-(Tyr)-CaM, phosphorylated by recombinant c-Src (cellular sarcoma kinase) and free of non-phosphorylated CaM, obtained by affinity-chromatography using an immobilized anti-phospho-(Tyr)-antibody, also increased the ligand-dependent tyrosine kinase activity of the isolated EGFR toward PGT. Also a CaM(Y99D/Y138D) mutant mimicked the effect of P-(Tyr)-CaM on ligand-dependent EGFR activation. Finally, we demonstrate that P-(Tyr)-CaM binds to the same site (645R-R-R-H-I-V-R-K-R-T-L-R-R-L-L-Q660) as non-phosphorylated CaM, located at the cytosolic juxtamembrane region of the EGFR. These results show that P-(Tyr)-CaM is an activator of the EGFR and suggest that it could contribute to the CaM-mediated ligand-dependent activation of the receptor that we previously reported in living cells.

N-terminal fusion of a toll-like receptor 2-ligand to a Neospora caninum chimeric antigen efficiently modifies the properties of the specific immune response

Immunoprophylactic products against neosporosis during pregnancy should induce an appropriately balanced immune response. In this respect, OprI, a bacterial lipoprotein targeting toll like receptor (TLR)2, provides promising adjuvant properties. We report on the manipulation of the innate and the T-cell immune response through the fusion of OprI with the Neospora caninum chimeric protein Mic3-1-R. In contrast to Mic3-1-R, OprI-MIC3-1-R significantly activated bone-marrow dendritic cells from naïve mice. Mice immunized with OprI-Mic3-1-R induced an immune response with mixed T helper (Th)1 and Th2 properties (high levels of both immunoglobulin (Ig)G1 and IgG2a and of interleukin (IL)-10, IL-12(p70) and interferon-γ responses) whereas Mic3-1-R+saponin induced a clear Th2-biased response (low IgG2a and high IL-4 and IL-10). After mating and challenge with N. caninum, increased expression of interferon-γ was only found in placentas from OprI-Mic3-1-R immunized dams. However, no protection against vertical transmission and neonatal mortality was observed in either of the two groups. These results indicated that more exhaustive studies must be done to elucidate the immune mechanisms associated with transplacental transmission. Antigen linkage to TLR2-ligands, such as OprI, is a useful tool to investigate this enigma by reorienting the innate and adaptive immune responses against other candidate antigens in future studies.

Vascular cell responsiveness to toll-like receptor ligands in carotid atheroma

Background Atherosclerosis is potentiated by stimulation of Toll-like receptors (TLRs), which serve to detect pathogen associated molecular patterns (PAMPs). However little is known of which PAMPs may be present in atheroma, or capable of stimulating inflammatory signalling in vascular cells. Materials and Methods DNA extracted from human carotid atheroma samples was amplified and sequenced using broad-range 16S gene specific primers to establish historical exposure to bacterial PAMPs. Responsiveness of primary human arterial and venous endothelial and smooth muscle cells to PAMPs specific for each of the TLRs was assessed by measurement of interleukin-8 secretion and E-selectin expression. Results Extracts of atheromatous tissue stimulated little or no signalling in TLR-transfected HEK-293 cells. However, sequencing of bacterial DNA amplified from carotid atheroma revealed the presence of DNA from 17 different bacterial genera, suggesting historical exposure to bacterial lipopeptide, lipopolysaccharide and flagellin. All cells examined were responsive to the ligands of TLR3 and TLR4, poly inosine:cytosine and lipopolysaccharide. Arterial cells were responsive to a wider range of PAMPs than venous cells, being additionally responsive to bacterial flagellin and unmethylated cytosine-phosphate-guanosine DNA motifs, the ligands of TLR5 and TLR9, respectively. Cells were generally unresponsive towards the ligands of human TLR7 and TLR8, loxoribine and single stranded RNA. Only coronary artery endothelial cells expressed TLR2 mRNA and responded to the TLR2 ligand Pam3CSK4. Conclusions Vascular cells are responsive to a relatively diverse range of TLR ligands and may be exposed, at least transiently, to ligands of TLR2, TLR4, TLR5 and TLR9 during the development of carotid atheroma.

Oxidized phospholipid inhibition of toll-like receptor (TLR) signaling is restricted to TLR2 and TLR4 roles for cd14, lps-binding protein, and md2 as targets for specificity of inhibition

The generation of reactive oxygen species is a central feature of inflammation that results in the oxidation of host phospholipids. Oxidized phospholipids, such as 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC), have been shown to inhibit signaling induced by bacterial lipopeptide or lipopolysac-charide (LPS), yet the mechanisms responsible for the inhibition of Toll-like receptor (TLR) signaling by OxPAPC remain incompletely understood. Here, we examined the mechanisms by which OxPAPC inhibits TLR signaling induced by diverse ligands in macrophages, smooth muscle cells, and epithelial cells. OxPAPC inhibited tumor necrosis factor- production, IB degradation, p38 MAPK phosphorylation, and NF-B-dependent reporter activation induced by stimulants of TLR2 and TLR4 (Pam3CSK4 and LPS) but not by stimulants of other TLRs (poly(I·C), flagellin, loxoribine, single-stranded RNA, or CpG DNA) in macrophages and HEK-293 cells transfected with respective TLRs and significantly reduced inflammatory responses in mice injected subcutaneously or intraperitoneally with Pam3CSK4. Serum proteins, including CD14 and LPS-binding protein, were identified as key targets for the specificity of TLR inhibition as supplementation with excess serum or recombinant CD14 or LBP reversed TLR2 inhibition by OxPAPC, whereas serum accessory proteins or expression of membrane CD14 potentiated signaling via TLR2 and TLR4 but not other TLRs. Binding experiments and functional assays identified MD2 as a novel additional target of OxPAPC inhibition of LPS signaling. Synthetic phospholipid oxidation products 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine inhibited TLR2 signaling from 30 µM. Taken together, these results suggest that oxidized phospholipid-mediated inhibition of TLR signaling occurs mainly by competitive interaction with accessory proteins that interact directly with bacterial lipids to promote signaling via TLR2 or TLR4.

Interactions among secretory immunoglobulin A, CD71, and transglutaminase-2 affect permeability of intestinal epithelial cells to gliadin peptides

BACKGROUND & AIMS: The transferrin receptor (CD71) is up-regulated in duodenal biopsy samples from patients with active celiac disease and promotes retrotransport of secretory immunolglobulin A (SIgA)-gliadin complexes. We studied intestinal epithelial cell lines that overexpress CD71 to determine how interactions between SIgA and CD71 promote transepithelial transport of gliadin peptides. METHODS: We analyzed duodenal biopsy specimens from 8 adults and 1 child with active celiac disease. Caco-2 and HT29-19A epithelial cell lines were transfected with fluorescence-labeled small interfering RNAs against CD71. Interactions among IgA, CD71, and transglutaminase 2 (Tgase2) were analyzed by flow cytometry, immunoprecipitation, and confocal microscopy. Transcytosis of SIgACD71 complexes and intestinal permeability to the gliadin 3H-p3149 peptide were analyzed in polarized monolayers of Caco-2 cells. RESULTS: Using fluorescence resonance energy transfer and in situ proximity ligation assays, we observed physical interactions between SIgA and CD71 or CD71 and Tgase2 at the apical surface of enterocytes in biopsy samples and monolayers of Caco-2 cells. CD71 and Tgase2 were co-precipitated with SIgA, bound to the surface of Caco-2 cells. SIgACD71 complexes were internalized and localized in early endosomes and recycling compartments but not in lysosomes. In the presence of celiac IgA or SIgA against p3149, transport of intact 3H-p3149 increased significantly across Caco-2 monolayers; this transport was inhibited by soluble CD71 or Tgase2 inhibitors. CONCLUSIONS: Upon binding to apical CD71, SIgA (with or without gliadin peptides) enters a recycling pathway and avoids lysosomal degradation; this process allows apicalbasal transcytosis of bound peptides. This mechanism is facilitated by Tgase2 and might be involved in the pathogenesis of celiac disease.

Improving recombinant human adenosine A2A receptor production in yeast

Over 50% of clinically-marketed drugs target membrane proteins; in particular G protein-coupled receptors (GPCRs). GPCRs are vital to living cells, performing an active role in many processes, making them integral to drug development. In nature, GPCRs are not sufficiently abundant for research and their structural integrity is often lost during extraction from cell membranes. The objectives of this thesis were to increase recombinant yield of the GPCR, human adenosine A2A receptor (hA2AR) by investigating bioprocess conditions in large-scale Pichia pastoris and small-scale Saccharomyces cerevisiae cultivations. Extraction of hA2AR from membranes using novel polymers was also investigated. An increased yield of hA2AR from P. pastoris was achieved by investigating the methanol feeding regime. Slow, exponential feed during induction (μlow) was compared to a faster, exponential feed (μhigh) in 35 L pilot-scale bioreactors. Overall hA2AR yields were increased for the μlow cultivation (536.4pmol g-1) compared to the μhigh148.1 pmol g-1. hA2AR levels were maintained in cytotoxic methanol conditions and unexpectedly, pre-induction levels of hA2AR were detected. Small-scale bioreactor work showed that Design of Experiments (DoE) could be applied to screen for bioprocess conditions to give optimal hA2AR yields. Optimal conditions were retrieved for S. cerevisiae using a d-optimal screen and response surface methodology. The conditions were 22°C, pH 6.0, 30% DO without dimethyl sulphoxide. A polynomial equation was generated to predict hA2AR yields if conditions varied. Regarding the extraction, poly (maleic anhydride-styrene) or PMAS was successful in solubilising hA2AR from P. pastoris membranes compared with dodcecyl-β-D-maltoside (DDM) detergent. Variants of PMAS worked well as solubilising agents with either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or cholesteryl hemisuccinate (CHS). Moreover, esterification of PMAS improved solubilisation, suggesting that increased hydrophobicity stabilises hA2AR during extraction. Overall, hA2AR yields were improved in both, P. pastoris and S. cerevisiae and the use of novel polymers for efficient extraction was achieved.

Molecular characterization of immunoglobulin gene rearrangements in diffuse large B-cell lymphoma: antigen-driven origin and IGHV4-34 as a particular subgroup of the non-GCB subtype.

The pathogenesis of diffuse large B-cell lymphoma (DLBCL) remains partially unknown. The analysis of the B-cell receptor of the malignant cells could contribute to a better understanding of the DLBCL biology. We studied the molecular features of the immunoglobulin heavy chain (IGH) rearrangements in 165 patients diagnosed with DLBCL not otherwise specified. Clonal IGH rearrangements were amplified according to the BIOMED-2 protocol and PCR products were sequenced directly. We also analyzed the criteria for stereotyped patterns in all complete IGHV-IGHD-IGHJ (V-D-J) sequences. Complete V-D-J rearrangements were identified in 130 of 165 patients. Most cases (89%) were highly mutated, but 12 sequences were truly unmutated or minimally mutated. Three genes, IGHV4-34, IGHV3-23, and IGHV4-39, accounted for one third of the whole cohort, including an overrepresentation of IGHV4-34 (15.5% overall). Interestingly, all IGHV4-34 rearrangements and all unmutated sequences belonged to the nongerminal center B-cell-like (non-GCB) subtype. Overall, we found three cases following the current criteria for stereotyped heavy chain VH CDR3 sequences, two of them belonging to subsets previously described in CLL. IGHV gene repertoire is remarkably biased, implying an antigen-driven origin in DLBCL. The particular features in the sequence of the immunoglobulins suggest the existence of particular subgroups within the non-GCB subtype.

Anti-N-methyl-D-Aspartate Receptor Encephalitis with Positive Serum Antithyroid Antibodies, IgM Antibodies Against Mycoplasma Pneumoniae and Human Herpesvirus 7 PCR in the CSF

We report the case of a boy with an encephalopathy associated with extrapyramidal and psychiatric symptoms and anti-N-methyl-D-aspartate receptor antibodies. He had positive serum antithyroid antibodies, IgM antibodies against Mycoplasma pneumoniae and human herpesvirus 7 polymerase chain reaction in the cerebrospinal fluid. He was successfully treated with rituximab, after steroids, intravenous immunoglobulin and plasma exchange. The pathophysiology of this disorder may be post-infectious and autoimmune.