In Children with Provoked Venous Thromboembolism, Increasing Plasma Coagulability during the First 3 Months Postdiagnosis is Prognostic of Recurrence.

Prognostic factors for venous thromboembolism (VTE) recurrence following provoked VTE are largely unknown. Using the Clot Formation and Lysis (CloFAL) assay, single institutional research has shown overall improvement in acute hypercoagulability during the first 3 months postpediatric VTE, yet a rise in plasma coagulability in a subgroup of patients. We sought to define the incidence of rise in coagulability during the first 3 months post-provoked VTE, to investigate its relationship with elevated D-dimer, and to test the hypothesis that a marked rise in coagulability is independently prognostic of VTE recurrence. CloFAL and D-dimer assays were performed on plasma at 4 to 6 weeks and 3 months post-VTE in the Johns Hopkins pediatric VTE cohort and National Institutes of Health-sponsored Kids-DOTT trial. Associations of VTE recurrence with D-dimer and CloFAL assay measures were evaluated via logistic regression. Eighty-seven patients were included. Median follow-up was 1 year. Complete veno-occlusion was determined in 12% at 6 weeks. During the first 3 months post-VTE, a marked rise in coagulability was observed by CloFAL assay in 17% of patients, while D-dimer was elevated in 21%. Recurrent VTE occurred in 10% of patients. CloFAL assay, but not D-dimer, was associated with recurrence (odds ratio [OR] 5.87, 95% confidence interval [95% CI], 1.34-25.8]). After adjustment for veno-occlusion, patients with a marked rise in coagulability by CloFAL assay had a 10-fold increased risk of recurrent VTE (OR 10.33 [95% CI, 1.83-58.19]). Future work should seek to elucidate the mechanisms underlying a rise in plasma coagulability following provoked VTE and to substantiate its prognostic utility for recurrent VTE.

Gut immunity in a protochordate involves a secreted immunoglobulin-type mediator binding host chitin and bacteria.

Protochordate variable region-containing chitin-binding proteins (VCBPs) consist of immunoglobulin-type V domains and a chitin-binding domain (CBD). VCBP V domains facilitate phagocytosis of bacteria by granulocytic amoebocytes; the function of the CBD is not understood. Here we show that the gut mucosa of Ciona intestinalis contains an extensive matrix of chitin fibrils to which VCBPs bind early in gut development, before feeding. Later in development, VCBPs and bacteria colocalize to chitin-rich mucus along the intestinal wall. VCBP-C influences biofilm formation in vitro and, collectively, the findings of this study suggest that VCBP-C may influence the overall settlement and colonization of bacteria in the Ciona gut. Basic relationships between soluble immunoglobulin-type molecules, endogenous chitin and bacteria arose early in chordate evolution and are integral to the overall function of the gut barrier.

The gut of geographically disparate Ciona intestinalis harbors a core microbiota.

It is now widely understood that all animals engage in complex interactions with bacteria (or microbes) throughout their various life stages. This ancient exchange can involve cooperation and has resulted in a wide range of evolved host-microbial interdependencies, including those observed in the gut. Ciona intestinalis, a filter-feeding basal chordate and classic developmental model that can be experimentally manipulated, is being employed to help define these relationships. Ciona larvae are first exposed internally to microbes upon the initiation of feeding in metamorphosed individuals; however, whether or not these microbes subsequently colonize the gut and whether or not Ciona forms relationships with specific bacteria in the gut remains unknown. In this report, we show that the Ciona gut not only is colonized by a complex community of bacteria, but also that samples from three geographically isolated populations reveal striking similarity in abundant operational taxonomic units (OTUs) consistent with the selection of a core community by the gut ecosystem.

A Basal chordate model for studies of gut microbial immune interactions.

Complex symbiotic interactions at the surface of host epithelia govern most encounters between host and microbe. The epithelium of the gut is a physiologically ancient structure that is comprised of a single layer of cells and is thought to possess fully developed immunological capabilities. Ciona intestinalis (sea squirt), which is a descendant of the last common ancestor of all vertebrates, is a potentially valuable model for studying barrier defenses and gut microbial immune interactions. A variety of innate immunological phenomena have been well characterized in Ciona, of which many are active in the gut tissues. Interactions with gut microbiota likely involve surface epithelium, secreted immune molecules including variable region-containing chitin-binding proteins, and hemocytes from a densely populated laminar tissue space. The microbial composition of representative gut luminal contents has been characterized by molecular screening and a potentially relevant, reproducible, dysbiosis can be induced via starvation. The dialog between host and microbe in the gut can be investigated in Ciona against the background of a competent innate immune system and in the absence of the integral elements and processes that are characteristic of vertebrate adaptive immunity.

Genomic and functional characterization of the diverse immunoglobulin domain-containing protein (DICP) family.

A heretofore-unrecognized multigene family encoding diverse immunoglobulin (Ig) domain-containing proteins (DICPs) was identified in the zebrafish genome. Twenty-nine distinct loci mapping to three chromosomal regions encode receptor-type structures possessing two classes of Ig ectodomains (D1 and D2). The sequence and number of Ig domains, transmembrane regions and signaling motifs vary between DICPs. Interindividual polymorphism and alternative RNA processing contribute to DICP diversity. Molecular models indicate that most D1 domains are of the variable (V) type; D2 domains are Ig-like. Sequence differences between D1 domains are concentrated in hypervariable regions on the front sheet strands of the Ig fold. Recombinant DICP Ig domains bind lipids, a property shared by mammalian CD300 and TREM family members. These findings suggest that novel multigene families encoding diversified immune receptors have arisen in different vertebrate lineages and affect parallel patterns of ligand recognition that potentially impact species-specific advantages.

Transfection-based genomic readout for identifying rare transcriptional splice variants.

Understanding the transcriptome, defined as the complete transcriptional component of the genome, is far more complex than originally considered. Even with the near fully resolved human and mouse genomes, for which extensive databases of transcribed sequence data (e.g., expressed sequence tags) are available, it is presently not possible to experimentally recover or computationally predict the full range of transcription products that derive from multiexon genes. Many genes are tightly regulated, which could include alternative processing of RNA, and lead to significant underrepresentation of many transcripts. A multitude of factors in addition to cell lineage- and developmental stage-specific expression as well as shortcomings in computational methods result in a less than complete understanding of transcriptional complexity. Here, we describe an approach to predict and evaluate a more complete repertoire of transcriptional products that derive from specific genetic loci with attention toward analysis of immune receptor genes. This approach is particularly useful in identifying gene products, including alternative splice forms, that originate from complex multigene families.

The basis for haplotype complexity in VCBPs, an immune-type receptor in amphioxus.

Innate immune gene repertoires are restricted primarily to germline variation. Adaptive immunity, by comparison, relies on somatic variation of germline-encoded genes to generate extraordinarily large numbers of non-heritable antigen recognition motifs. Invertebrates lack the key features of vertebrate adaptive immunity, but have evolved a variety of alternative mechanisms to successfully protect the integrity of "self"; in many cases, these appear to be taxon-specific innovations. In the protochordate Branchiostoma floridae (amphioxus), the variable region-containing chitin-binding proteins (VCBPs) constitute a multigene family (comprised of VCBPs 1-5), which possesses features that are consistent with innate immune-type function. A large number of VCBP alleles and haplotypes are shown to exhibit levels of polymorphism exceeding the elevated overall levels determined for the whole amphioxus genome (JGI). VCBP genes of the 2 and 5 types are distinguished further by a highly polymorphic segment (exon 2) in the N-terminal immunoglobulin domain, defined previously as a "hypervariable region" or a "hotspot." Genomic deoxyribonucleic acid (DNA) and complementary DNA (cDNA) sequences from large numbers of animals representing different populations reveal further significant differences in sequence complexity within and across VCBP2/5 haplotypes that arise through overlapping mechanisms of genetic exchange, gene copy number variation as well as mutation and give rise to distinct allelic lineages. The collective observations suggest that mechanisms were in place at the time of divergence of the cephalochordates that could selectively hyperdiversify immune-type receptors within a multigene family.

Genomic complexity of the variable region-containing chitin-binding proteins in amphioxus.

BACKGROUND: The variable region-containing chitin-binding proteins (VCBPs) are found in protochordates and consist of two tandem immunoglobulin variable (V)-type domains and a chitin-binding domain. We previously have shown that these polymorphic genes, which primarily are expressed in the gut, exhibit characteristics of immune genes. In this report, we describe VCBP genomic organization and characterize adjacent and intervening genetic features which may influence both their polymorphism and complex transcriptional repertoire. RESULTS: VCBP genes 1, 2, 4, and 5 are encoded in a single contiguous gene-rich chromosomal region and VCBP3 is encoded in a separate locus. The VCBPs exhibit extensive haplotype variation, including copy number variation (CNV), indel polymorphism and a markedly elevated variation in repeat type and density. In at least one haplotype, inverted repeats occur more frequently than elsewhere in the genome. Multi-animal cDNA screening, as well as transcriptional profilingusing a novel transfection system, suggests that haplotype-specific transcriptional variants may contribute to VCBP genetic diversity. CONCLUSION: The availability of the Branchiostoma floridae genome (Joint Genome Institute, Brafl1), along with BAC and PAC screening and sequencing described here, reveal that the relatively limited number of VCBP genes present in the amphioxus genome exhibit exceptionally high haplotype variation. These VCBP haplotypes contribute a diverse pool of allelic variants, which includes gene copy number variation, pseudogenes, and other polymorphisms, while contributing secondary effects on gene transcription as well.

Novel genes dramatically alter regulatory network topology in amphioxus.

BACKGROUND: Regulation in protein networks often utilizes specialized domains that 'join' (or 'connect') the network through specific protein-protein interactions. The innate immune system, which provides a first and, in many species, the only line of defense against microbial and viral pathogens, is regulated in this way. Amphioxus (Branchiostoma floridae), whose genome was recently sequenced, occupies a unique position in the evolution of innate immunity, having diverged within the chordate lineage prior to the emergence of the adaptive immune system in vertebrates. RESULTS: The repertoire of several families of innate immunity proteins is expanded in amphioxus compared to both vertebrates and protostome invertebrates. Part of this expansion consists of genes encoding proteins with unusual domain architectures, which often contain both upstream receptor and downstream activator domains, suggesting a potential role for direct connections (shortcuts) that bypass usual signal transduction pathways. CONCLUSION: Domain rearrangements can potentially alter the topology of protein-protein interaction (and regulatory) networks. The extent of such arrangements in the innate immune network of amphioxus suggests that domain shuffling, which is an important mechanism in the evolution of multidomain proteins, has also shaped the development of immune systems.

Ancient divergence of a complex family of immune-type receptor genes.

Multigene families of activating/inhibitory receptors belonging to the immunoglobulin superfamily (IgSF) regulate immunological and other cell-cell interactions. A new family of such genes, termed modular domain immune-type receptors (MDIRs), has been identified in the clearnose skate (Raja eglanteria), a phylogenetically ancient vertebrate. At least five different major forms of predicted MDIR proteins are comprised of four different subfamilies of IgSF ectodomains of the intermediate (I)- or C2-set. The predicted number of individual IgSF ectodomains in MDIRs varies from one to six. MDIR1 contains a positively charged transmembrane residue and MDIR2 and MDIR3 each possesses at least one immunoreceptor tyrosine-based inhibitory motif in their cytoplasmic regions. MDIR4 and MDIR5 lack characteristic activating/inhibitory signalling motifs. MDIRs are encoded in a particularly large and complex multigene family. MDIR domains exhibit distant sequence similarity to mammalian CMRF-35-like molecules, polymeric immunoglobulin receptors, triggering receptors expressed on myeloid cells (TREMs), TREM-like transcripts, NKp44 and FcR homologs, as well as to sequences identified in several different vertebrate genomes. Phylogenetic analyses suggest that MDIRs are representative members of an extended family of IgSF genes that diverged before or very early in evolution of the vertebrates and subsequently came to occupy multiple, fully independent distributions in the present day.

Variable domains and a VpreB-like molecule are present in a jawless vertebrate.

Immunoglobulins (Igs) and T cell antigen receptors (TCRs) that undergo somatic diversification have not been identified in the two extant orders of jawless vertebrates, which occupy essential positions in terms of understanding the evolution of the emergence of adaptive immunity. Using a single motif-dependent PCR-based approach coupled with a vector that allows selection of cDNAs encoding secretion signal sequences, four different genes encoding Ig V-type domains were identified in the sea lamprey (Petromyzon marinus). One of the predicted proteins encoded by these genes shares structural characteristics with mammalian VpreB molecules, including the absence of a recognizable transmembrane region, a relatively high proportion of charged amino acids in its C-terminal tail and distinctive features of its secretion signal peptide. This is the first indication of a molecule related to the B cell receptor (BCR) complex in a species that diverged prior to the jawed vertebrates in which RAG-mediated adaptive immunity is first encountered.

Resolution of the novel immune-type receptor gene cluster in zebrafish.

The novel immune-type receptor (NITR) genes encode a unique multigene family of leukocyte regulatory receptors, which possess an extracellular Ig variable (V) domain and may function in innate immunity. Artificial chromosomes that encode zebrafish NITRs have been assembled into a contig spanning approximately 350 kb. Resolution of the complete NITR gene cluster has led to the identification of eight previously undescribed families of NITRs and has revealed the presence of C-type lectins within the locus. A maximum haplotype of 36 NITR genes (138 gene sequences in total) can be grouped into 12 distinct families, including inhibitory and activating receptors. An extreme level of interindividual heterozygosity is reflected in allelic polymorphisms, haplotype variation, and family-specific isoform complexity. In addition, the exceptional diversity of NITR sequences among species suggests divergent evolution of this multigene family with a birth-and-death process of member genes. High-confidence modeling of Nitr V-domain structures reveals a significant shift in the spatial orientation of the Ig fold, in the region of highest interfamily variation, compared with Ig V domains. These studies resolve a complete immune gene cluster in zebrafish and indicate that the NITRs represent the most complex family of activating/inhibitory surface receptors thus far described.

Cloning novel immune-type inhibitory receptors from the rainbow trout, Oncorhynchus mykiss.

Novel immune-type receptor ( NITR) genes that encode two extracellular immunoglobulin domains and cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs) have been described previously in three lineages of bony fish. In the current study, four ITIM-containing NITR cDNAs are identified in the rainbow trout ( Oncorhynchus mykiss), and their expression patterns and genomic complexity are characterized. The ITIM-containing NITR2 gene maps 1.3 cM from an ITIM-containing C-type lectin receptor ( TCL-2) on linkage group XXI. A comprehensive, phylogenetic analysis of NITRs from rainbow trout and three other major lineages of bony fish defines conserved families of NITRs and suggests an ancient lineage of distinct groups of genes. Several probable scenarios that explain the origins of variant forms of NITRs are described.

BIVM, a novel gene widely distributed among deuterostomes, shares a core sequence with an unusual gene in Giardia lamblia.

A novel gene, BIVM (for basic, immunoglobulin-like variable motif-containing), has been identified using an electronic search based on the conservation of short sequence motifs within the variable region of immunoglobulin (Ig) genes. BIVM maps to human chromosome 13q32-q33 and is predicted to encode a 503-amino-acid protein with a pI of 9.1. The 5' untranslated region of BIVM is encoded in two exons; the coding portion is encoded in nine exons. BIVM is tightly linked (41 bp) and in the opposite transcriptional orientation to MGC5302 (also known as KDEL1 and EP58) in human. The ubiquitous expression of BIVM in normal tissues and the presence of a 5' CpG island suggest that BIVM is a housekeeping gene. Characterization of BIVM in representative species demonstrates significant conservation throughout deuterostomes; no sequence with significant identity to BIVM has been detected in proteostomes. However, an unusual gene has been identified in the protozoan pathogen Giardia lamblia that is similar to the core sequence of BIVM, suggesting the possibility of a horizontal gene transfer.