Antibody deficiency due to a missense mutation in CD19 demonstrates the importance of the conserved tryptophan 41 in immunoglobulin superfamily domain formation.

Immunoglobulin superfamily (IgSF) domains are conserved structures present in many proteins in eukaryotes and prokaryotes. These domains are well-capable of facilitating sequence variation, which is most clearly illustrated by the variable regions in immunoglobulins (Igs) and T cell receptors (TRs). We studied an antibody-deficient patient suffering from recurrent respiratory infections and with impaired antibody responses to vaccinations. Patient's B cells showed impaired Ca(2+) influx upon stimulation with anti-IgM and lacked detectable CD19 membrane expression. CD19 sequence analysis revealed a homozygous missense mutation resulting in a tryptophan to cystein (W52C) amino acid change. The affected tryptophan is CONSERVED-TRP 41 located on the C-strand of the first extracellular IgSF domain of CD19 and was found to be highly conserved, not only in mammalian CD19 proteins, but in nearly all characterized IgSF domains. Furthermore, the tryptophan is present in all variable domains in Ig and TR and was not mutated in 117 Ig class-switched transcripts of B cells from controls, despite an overall 10% amino acid change frequency. In vitro complementation studies and CD19 western blotting of patient's B cells demonstrated that the mutated protein remained immaturely glycosylated. This first missense mutation resulting in a CD19 deficiency demonstrates the crucial role of a highly conserved tryptophan in proper folding or stability of IgSF domains.

La molecola IRTA1 (immunoglobulin superfamily receptor trans location associated 1) risulta selettivamente espressa nei linfomi non Hodgkin B della zona marginale

La diagnosi di linfoma non Hodgkin B della zona marginale si basa su criteri morfologici e sulla sostanziale negatività per marcatori immunoistochimici espressi in altri sottotipi di linfoma B. L’ obiettivo di questo lavoro è stato, quindi, quello di ricercare una molecola specifica associata ai linfomi della zona marginale. Materiali e Metodi. Sono stati esaminati 2.104 linfomi periferici di entità nosologia eterogenea mediante un anticorpo monoclonale, diretto contro la molecola IRTA1, che riconosce la zona marginale nei tessuti linfoidi umani. Risultati. Si è riscontrata espressione di IRTA1 nel 93% dei linfomi della zona marginale ad insorgenza extranodale e nel 74% di quelli primitivi linfonodali suggerendo la possibilità che questi linfomi possano originare dalle cellule perifollicolari o monocitoidi IRTA1+ riscontrabili nei linfonodi reattivi. La valutazione immunoistochimica mediante doppia colorazione (IRTA1/bcl6), ha inoltre dimostrato come vi sia una modulazione fenotipica nelle cellule marginali neoplastiche nel momento in cui esse colonizzano i follicoli linfoidi e durante la loro circolazione nei centri germinativi. Le cellule marginali neoplastiche che differenziano in senso plasmacellulare perdono l’ espressione di IRTA1 Discussione. In conclusione, tali evidenze hanno permesso di ampliare la conoscenza sulla biologia dei linfomi marginali e sottolineano come IRTA1 sia il primo marcatore diagnostico positivo per queste neoplasie.

The platelet collagen receptor glycoprotein VI is a member of the immunoglobulin superfamily closely related to FcalphaR and the natural killer receptors

We have cloned the platelet collagen receptor glycoprotein (GP) VI from a human bone marrow cDNA library using rapid amplification of cDNA ends with platelet mRNA to complete the 5' end sequence. GPVI was isolated from platelets using affinity chromatography on the snake C-type lectin, convulxin, as a critical step. Internal peptide sequences were obtained, and degenerate primers were designed to amplify a fragment of the GPVI cDNA, which was then used as a probe to screen the library. Purified GPVI, as well as Fab fragments of polyclonal antibodies made against the receptor, inhibited collagen-induced platelet aggregation. The GPVI receptor cDNA has an open reading frame of 1017 base pairs coding for a protein of 339 amino acids including a putative 23-amino acid signal sequence and a 19-amino acid transmembrane domain between residues 247 and 265. GPVI belongs to the immunoglobulin superfamily, and its sequence is closely related to FcalphaR and to the natural killer receptors. Its extracellular chain has two Ig-C2-like domains formed by disulfide bridges. An arginine residue is found in position 3 of the transmembrane portion, which should permit association with Fcgamma and its immunoreceptor tyrosine-based activation motif via a salt bridge. With 51 amino acids, the cytoplasmic tail is relatively long and shows little homology to the C-terminal part of the other family members. The ability of the cloned GPVI cDNA to code for a functional platelet collagen receptor was demonstrated in the megakaryocytic cell line Dami. Dami cells transfected with GPVI cDNA mobilized intracellular Ca(2+) in response to collagen, unlike the nontransfected or mock transfected Dami cells, which do not respond to collagen.

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.

Serum levels of soluble receptor for advanced glycation end products and of S100 proteins are associated with inflammatory, autoantibody, and classical risk markers of joint and vascular damage in rheumatoid arthritis

Introduction: The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface receptor molecules. High concentrations of three of its putative proinflammatory ligands, S100A8/A9 complex (calprotectin), S100A8, and S100A12, are found in rheumatoid arthritis (RA) serum and synovial fluid. In contrast, soluble RAGE (sRAGE) may prevent proinflammatory effects by acting as a decoy. This study evaluated the serum levels of S100A9, S100A8, S100A12 and sRAGE in RA patients, to determine their relationship to inflammation and joint and vascular damage. Methods: Serum sRAGE, S100A9, S100A8 and S100A12 levels from 138 patients with established RA and 44 healthy controls were measured by ELISA and compared by unpaired t test. In RA patients, associations with disease activity and severity variables were analyzed by simple and multiple linear regressions. Results: Serum S100A9, S100A8 and S100A12 levels were correlated in RA patients. S100A9 levels were associated with body mass index (BMI), and with serum levels of S100A8 and S100A12. S100A8 levels were associated with serum levels of S100A9, presence of anti-citrullinated peptide antibodies (ACPA), and rheumatoid factor (RF). S100A12 levels were associated with presence of ACPA, history of diabetes, and serum S100A9 levels. sRAGE levels were negatively associated with serum levels of C-reactive protein (CRP) and high-density lipoprotein (HDL), history of vasculitis, and the presence of the RAGE 82Ser polymorphism. Conclusions: sRAGE and S100 proteins were associated not just with RA inflammation and autoantibody production, but also with classical vascular risk factors for end-organ damage. Consistent with its role as a RAGE decoy molecule, sRAGE had the opposite effects to S100 proteins in that S100 proteins were associated with autoantibodies and vascular risk, whereas sRAGE was associated with protection against joint and vascular damage. These data suggest that RAGE activity influences co-development of joint and vascular disease in rheumatoid arthritis patients.

Novel conditional mouse models for targeted kidney research : Emphasis on the analysis of the biological function of nephrin

Nephrin is a transmembrane protein belonging to the immunoglobulin superfamily and is expressed primarily in the podocytes, which are highly differentiated epithelial cells needed for primary urine formation in the kidney. Mutations leading to nephrin loss abrogate podocyte morphology, and result in massive protein loss into urine and consequent early death in humans carrying specific mutations in this gene. The disease phenotype is closely replicated in respective mouse models. The purpose of this thesis was to generate novel inducible mouse-lines, which allow targeted gene deletion in a time and tissue-specific manner. A proof of principle model for succesful gene therapy for this disease was generated, which allowed podocyte specific transgene replacement to rescue gene deficient mice from perinatal lethality. Furthermore, the phenotypic consequences of nephrin restoration in the kidney and nephrin deficiency in the testis, brain and pancreas in rescued mice were investigated. A novel podocyte-specific construct was achieved by using standard cloning techniques to provide an inducible tool for in vitro and in vivo gene targeting. Using modified constructs and microinjection procedures two novel transgenic mouse-lines were generated. First, a mouse-line with doxycycline inducible expression of Cre recombinase that allows podocyte-specific gene deletion was generated. Second, a mouse-line with doxycycline inducible expression of rat nephrin, which allows podocyte-specific nephrin over-expression was made. Furthermore, it was possible to rescue nephrin deficient mice from perinatal lethality by cross-breeding them with a mouse-line with inducible rat nephrin expression that restored the missing endogenous nephrin only in the kidney after doxycycline treatment. The rescued mice were smaller, infertile, showed genital malformations and developed distinct histological abnormalities in the kidney with an altered molecular composition of the podocytes. Histological changes were also found in the testis, cerebellum and pancreas. The expression of another molecule with limited tissue expression, densin, was localized to the plasma membranes of Sertoli cells in the testis by immunofluorescence staining. Densin may be an essential adherens junction protein between Sertoli cells and developing germ cells and these junctions share similar protein assembly with kidney podocytes. This single, binary conditional construct serves as a cost- and time-efficient tool to increase the understanding of podocyte-specific key proteins in health and disease. The results verified a tightly controlled inducible podocyte-specific transgene expression in vitro and in vivo as expected. These novel mouse-lines with doxycycline inducible Cre recombinase and with rat nephrin expression will be useful for conditional gene targeting of essential podocyte proteins and to study in detail their functions in the adult mice. This is important for future diagnostic and pharmacologic development platforms.

The neuronal cell adhesion molecule ICAM-5

The neuronal cell adhesion molecule ICAM-5 ICAM-5 (telencephalin) belongs to the intercellular adhesion molecule (ICAM)-subgroup of the immunoglobulin superfamily (IgSF). ICAMs participate in leukocyte adhesion and adhesion-dependent functions in the central nervous system (CNS) through interacting with the leukocyte-specific b2 integrins. ICAM-5 is found in the mammalian forebrain, appears at the time of birth, and is located at the cell soma and neuronal dendrites. Recent studies also show that it is important for the regulation of immune functions in the brain and for the development and maturation of neuronal synapses. The clinical importance of ICAM-5 is still under investigation; it may have a role in the development of Alzheimer s disease (AD). In this study, the role of ICAM-5 in neuronal differentiation and its associations with a-actinin and N-methyl-D-aspartic acid (NMDA) receptors were examined. NMDA receptors (NMDARs) are known to be involved in many neuronal functions, including the passage of information from one neuron to another one, and thus it was thought important to study their role related to ICAM-5. The results suggested that ICAM-5 was able to induce dendritic outgrowth through homophilic adhesion (ICAM-5 monomer binds to another ICAM-5 monomer in the same or neighbouring cell), and the homophilic binding activity appeared to be regulated by monomer/multimer transition. Moreover, ICAM-5 binding to a-actinin was shown to be important for neuritic outgrowth. It was examined whether matrix metalloproteinases (MMPs) are the main enzymes involved in ICAM-5 ectodomain cleavage. The results showed that stimulation of NMDARs leads to MMP activation, cleavage of ICAM-5 and it is accompanied by dendritic spine maturation. These findings also indicated that ICAM-5 and NMDA receptor subunit 1 (NR1) compete for binding to a-actinin, and ICAM-5 may regulate the NR1 association with the actin cytoskeleton. Thus, it is concluded that ICAM-5 is a crucial cell adhesion molecule involved in the development of neuronal synapses, especially in the regulation of dendritic spine development, and its functions may also be involved with memory formation and learning.

Two levels of MCT1 and CD147 expression in the equine red blood cells and muscle

Monocarboxylate transporters (MCTs), especially the isoforms MCT1 - MCT4, cotransport lactate and protons across the cell membranes. They are thus essential for pH regulation and homeostasis in glycolytic cells such as red blood cells (RBCs), and skeletal muscle cells during intense exercise. In 70% of the Standardbred horses the lactate transport activity (TA) in RBCs is high and transport is mediated mainly by MCTs. In the rest 30% of the Standardbreds MCT mediated transport route is not active and the TA is low. MCTs need an ancillary protein for their proper localization and functioning in the plasma membrane. The ancillary protein for MCT1 and MCT4 is a member of immunoglobulin superfamily, CD147. Here we determined the expression of MCT isoforms and CD147 in equine RBCs and gluteal muscle. We sequenced the cDNA of horse MCT1 and CD147 to achieve horse-specific antibodies and to reveal sequence variations that may affect the TA of RBCs. The amount of MCT1 and CD147 mRNA in muscle were also studied. ---- In all, 73 horses representing different breeds were used. Blood samples were drawn from the jugular vein and muscle samples were taken either from gluteal muscle using biopsy needle or during castration from expendable cremaster muscle. The TA of RBCs was studied using radiolabeled lactate and the amount of MCT isoforms and CD147 in the plasma membranes using Western blotting. The level of mRNA in muscle cells was determined using qPCR. Isoforms MCT1 and MCT2 were found in the RBCs and isoforms MCT1 and MCT4 in the muscle cells of horses. The TA of RBCs was dependent on the expression of CD147 and MCT1 in the plasma membrane. Sequence variations were found in the cDNA of both MCT1 and CD147, but they did not explain the inactivity of MCT1 mediated transport route. The single nucleotide polymorphism (SNP) Met125Val in CD147 that existed parallel with an SNP in 3´-untranslated region explained, however, attenuation in CD147 expression in Standardbreds. A single mutation Ile51Val also decreased the expression of CD147 in one Warmblood. The MCT1 and CD147 mRNA concentrations in the gluteal muscle were higher in horses with higher MCT1 and CD147 expression in RBCs and lower in horses with minor expression of CD147 and MCT1. This suggests that the bimodal distribution of TA is due to differences in transcriptional regulation that is functioning in parallel in MCT1 and CD147 gene.

The expression and regulation of pregnancy-specific glycoproteins in the mouse

Pregnancy-Specific Glycoproteins (PSG) are the most abundant fetally expressed proteins in the maternal bloodstream at term. This multigene family are immunoglobulin superfamily members and are predominantly expressed in the syncytiotrophoblast of human placenta and in giant cells and spongiotrophoblast of rodent placenta. PSGs are encoded by seventeen genes in the mouse and ten genes in the human. Little is known about the function of this gene family, although they have been implicated in immune modulation and angiogenesis through the induction of cytokines such as IL-10 and TGFβ1 in monocytes, and more recently, have been shown to inhibit the platelet-fibrinogen interaction. I provide new information concerning the evolution of the murine Psg genomic locus structure and organisation, through the discovery of a recent gene inversion event of Psg22 within the major murine Psg cluster. In addition to this, I have performed an examination of the expression patterns of individual Psg genes in placental and non-placental tissues. This study centres on Psg22, which is the most abundant murine Psg transcript detected in the first half of pregnancy. A novel alternative splice variant transcript of Psg22 lacking the protein N1-domain was discovered, and similar to the full length isoform induces TGFβ1 in macrophage and monocytic cell lines. The identification of a bidirectional antisense long non-coding RNA transcript directly adjacent to Psg22 and its associated active local chromatin conformation, suggests an interesting epigenetic gene-specific regulatory mechanism that may be responsible for the high level of Psg22 expression relative to the other Psg family members upon trophoblast giant cell differentiation

The mouse and human IGSF6 (DORA) genes map to the inflammatory bowel disease 1 locus and are embedded in an intron of a gene of unknown function.

We have previously characterized IGSF6 (DORA), a novel member of the immunoglobulin superfamily (IGSF) from human and rat expressed in dendritic and myeloid cells. Using a probe from the open reading frame of the rat cDNA, we isolated a cosmid which contains the entire mouse gene. By comparative analysis and reverse transcriptase polymerase chain reaction, we defined the intron/exon structure and the mRNA of the mouse gene and, with respect to human BAC clones, the human gene. The genes span 10 kb (mouse) and 12 kb (human), with six exons arranged in a manner similar to other members of the IGSF. All intron/exon boundaries follow the GT-AG rule. Expression of the mouse Igsf6 gene is restricted to cells of the immune system, particularly macrophages. Northern blot revealed a single mRNA of 2.5 kb, in contrast to the human gene which is expressed as two mRNAs of 1 and 2.5 kb. The human and mouse genes were localized to a locus associated with inflammatory bowel disease. Analysis of the flanking regions of the Igsf6 gene revealed the presence of an unrelated gene, transcribed from the opposite strand of the DNA and oriented such that the Igsf6 gene is encoded entirely within an intron. An identical organization is seen in human. This gene of unknown function is transcribed and processed, contains homologues in Caenorhabditis elegans and prokaryotes, and is expressed in most organs in the mouse.

Evolution of immune systems: specificity and autoreactivity

Multicellularity evolved well before 600 million years ago, and all multicellular animals have evolved since then with the need to protect against pathogens. There is no reason to expect their immune systems to be any less sophisticated than ours. The vertebrate system, based on rearranging immunoglobulin-superfamily domains, appears to have evolved partly as a result of chance insertion of RAG genes by horizontal transfer. Remarkably sophisticated systems for expansion of immunological repertoire have evolved in parallel in many groups of organisms. Vaccination of invertebrates against commercially important pathogens has been empirically successful, and suggests that the definition of an adaptive and innate immune system should no longer depend on the presence of memory and specificity, since these terms are hard to define in themselves. The evolution of randomly-created immunological repertoire also carries with it the potential for generating autoreactive specificities and consequent autoimmune damage.While invertebrates may use systems analogous to ours to control autoreactive specificities, they may have evolved alternative mechanisms which operate either at the level of individuals-within-populations rather than cells-within-individuals, by linking self-reactive specificities to regulatory pathways and non-self-reactive to effector pathways.

The evolutionary basis for differences between the immune systems of man, mouse, pig and ruminants

Studying the pathogenesis of an infectious disease like colibacillosis requires an understanding of the responses of target hosts to the organism both as a pathogen and as a commensal. The mucosal immune system constitutes the primary line of defence against luminal micro-organisms. The immunoglobulin-superfamily-based adaptive immune system evolved in the earliest jawed vertebrates, and the adaptive and innate immune system of humans, mice, pigs and ruminants co-evolved in common ancestors for approximately 300 million years. The divergence occurred only 100 mya and, as a consequence, most of the fundamental immunological mechanisms are very similar. However, since pressure on the immune system comes from rapidly evolving pathogens, immune systems must also evolve rapidly to maintain the ability of the host to survive and reproduce. As a consequence, there are a number of areas of detail where mammalian immune systems have diverged markedly from each other, such that results obtained in one species are not always immediately transferable to another. Thus, animal models of specific diseases need to be selected carefully, and the results interpreted with caution. Selection is made simpler where specific host species like cattle and pigs can be both target species and reservoirs for human disease, as in infections with Escherichia coli.

Identification of natural killer cell receptor genes in the genome of the marsupial Tasmanian devil (Sarcophilus harrisii)

Within the mammalian immune system, natural killer (NK) cells contribute to the first line of defence against infectious agents and tumours. Their activity is regulated, in part, by cell surface NK cell receptors. NK receptors can be divided into two unrelated, but functionally analogous superfamilies based on the structure of their extracellular ligand-binding domains. Receptors belonging to the C-type lectin superfamily are predominantly encoded in the natural killer complex (NKC), while receptors belonging to the immunoglobulin superfamily are predominantly encoded in the leukocyte receptor complex (LRC). Natural killer cell receptors are emerging as a rapidly evolving gene family which can display significant intra- and interspecific variation. To date, most studies have focused on eutherian mammals, with significantly less known about the evolution of these receptors in marsupials. Here, we describe the identification of 43 immunoglobulin domain-containing LRC genes in the genome of the Tasmanian devil (Sarcophilus harrisii), the largest remaining marsupial carnivore and only the second marsupial species to be studied. We also identify orthologs of NKC genesKLRK1, CD69, CLEC4E, CLEC1B, CLEC1A and an ortholog of an opossum NKC receptor. Characterisation of these regions in a second, distantly related marsupial provides new insights into the dynamic evolutionary histories of these receptors in mammals. Understanding the functional role of these genes is also important for the development of therapeutic agents against Devil Facial Tumour Disease, a contagious cancer that threatens the Tasmanian devil with extinction.

CD80 and CD86 polymorphisms in populations of various ancestries: 5 new CD80 promoter alleles

CD80 and CD86 are closely linked genes on chromosome 3 that code for glycoproteins of the immunoglobulin superfamily, expressed on the surface of antigen-presenting cells. These costimulatory molecules play essential roles for stimulation and inhibition of T cells through binding to CD28 and CTLA-4 receptors. In this study, CD80 promoter and CD86 exon 8 polymorphisms were analyzed to investigate the genetic diversity and microevolution of the 2 genes. We genotyped 1,124 individuals, including Brazilians of predominantly European, mixed African and European, and Japanese ancestry, 5 Amerindian populations, and an African sample. All variants were observed in Africans, which suggests their origin in Africa before the human migrations out of that continent. Five new CD80 promoter alleles were identified and confirmed by cloning and sequencing, and promoter 2 is most likely the ancestral allele. Nucleotide -79 is monomorphic in 4 Amerindian populations, where the presence of the -79 G allele is probably the result of gene flow from non-Amerindians. (C) 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Innate Immunity in Insects, Function and Regulation of Hemolin from Hyalophora cecropia

Insects are useful models for the study of innate immune reactions and development. The distinction between recognition mechanisms preceding the breakdown of apoptotic cells during metamorphosis, and the breakdown of cells in response to infections, is unclear. Hemolin, a Lepidopteran member of the immunoglobulin superfamily, is a candidate molecule in self/nonself recognition. This thesis investigates hemolin function and hemolin gene regulation at a molecular level. We investigated the binding and cell adhesion properties of hemolin from H. cecropia and demonstrated that the proteins could homodimerize in presence of calcium. Moreover, a higher molecular weight membrane form of hemolin was present on hemocytes. These results, taken together with an earlier finding that soluble hemolin inhibits hemocyte adhesion, indicated that the secreted hemolin could modulate hemocyte aggregation in a competitive manner in the blood. In addition, hemolin was expressed in different tissues and at different developmental stages. Since hemolin is expressed both during development and during the immune response, its different regulatory factors must act in concert. We found that the third intron contains an enhancer, through which Dif, C/EBP and HMGI synergistically activate a reporter construct in vitro. We concluded that the enhancer is used during infection, since the κB-site is crucial for an immune response. Interestingly, we also found that the active form of the steroid hormone, ecdysone, induces the hemolin gene transcription in vivo, and in addition, acts synergistically during bacterial infection. Preliminary in vivo results indicate a secondary effect of ecdysone and the importance of hormone receptor elements in the upstream promoter region of hemolin. To explore the use of Drosophila as a genetic tool for understanding hemolin function and regulation, we sought to isolate the functional homologue in this species. A fly cDNA library in yeast was screened using H. cecropia hemolin as bait. The screen was not successful. However, it did lead to the discovery of a Drosophila protein with true binding specificity for hemolin. Subsequent characterization revealed a new, highly conserved gene, which we named yippee. Yippee is distantly related to zinc finger proteins and represents a novel family of proteins present in numerous eukaryotes, including fungi, plants and humans. Notably, when the Drosophila genome sequence was revealed, no hemolin orthologue could be detected. Finally, an extensive Drosophila genome chip analysis was initiated. The goal was to investigate the Drosophila immune response, and, in contrast to earlier studies of artificially injected flies, to examine a set of natural microbes, orally and externally applied. In parallel experiments viruses, bacteria, fungi and parasites were compared to unchallenged controls. We obtained a unique set of genes that were up-regulated in the response to the parasite Octosporea muscadomesticae and to the fungus Beauveria bassiana. We expect both down-regulated and up-regulated genes to serve as a source for the discovery of new effector molecules, in particular those that are active against parasites and fungi.