Multiple Alr genes exhibit allorecognition-associated variation in the colonial cnidarian Hydractinia.

The genetics of allorecognition has been studied extensively in inbred lines of Hydractinia symbiolongicarpus, in which genetic control is attributed mainly to the highly polymorphic loci allorecognition 1 (Alr1) and allorecognition 2 (Alr2), located within the Allorecognition Complex (ARC). While allelic variation at Alr1 and Alr2 can predict the phenotypes in inbred lines, these two loci do not entirely predict the allorecognition phenotypes in wild-type colonies and their progeny, suggesting the presence of additional uncharacterized genes that are involved in the regulation of allorecognition in this species. Comparative genomics analyses were used to identify coding sequence differences from assembled chromosomal intervals of the ARC and from genomic scaffold sequences between two incompatible H. symbiolongicarpus siblings from a backcross population. New immunoglobulin superfamily (Igsf) genes are reported for the ARC, where five of these genes are closely related to the Alr1 and Alr2 genes, suggesting the presence of multiple Alr-like genes within this complex. Complementary DNA sequence evidence revealed that the allelic polymorphism of eight Igsf genes is associated with allorecognition phenotypes in a backcross population of H. symbiolongicarpus, yet that association was not found between parental colonies and their offspring. Alternative splicing was found as a mechanism that contributes to the variability of these genes by changing putative activating receptors to inhibitory receptors or generating secreted isoforms of allorecognition proteins. Our findings demonstrate that allorecognition in H. symbiolongicarpus is a multigenic phenomenon controlled by genetic variation in at least eight genes in the ARC complex.

Comparative Genomics of Clinical and Environmental Isolates of Vibrio spp. of Colombia: Implications of Traits Associated with Virulence and Resistance.

There is widespread concern about the increase in cases of human and animal infections caused by pathogenic Vibrio species due to the emergence of epidemic lineages. In Colombia, active surveillance by the National Institute of Health (INS) has confirmed the presence of Vibrio; however, in routine surveillance, these isolates are not genomically characterized. This study focused on the pangenome analysis of six Vibrio species: V. parahaemolyticus, V. vulnificus, V. alginolyticus, V. fluvialis, V. diabolicus and V. furnissii to determine the genetic architectures of potentially virulent and antimicrobial resistance traits. Isolates from environmental and clinical samples were genome sequenced, assembled and annotated. The most important species in public health were further characterized by multilocus sequence typing and phylogenomics. For V. parahaemolyticus, we found the virulent ST3 and ST120 genotypes. For V. vulnificus, we identified isolates belonging to lineages 1 and 2. Virulence gene homologues between species were found even in non-pathogenic species such as V. diabolicus. Annotations related to the mobilome, integrative mobile and conjugative elements and resistance genes were obtained from environmental and clinical isolates. This study contributes genomic information to the intensified surveillance program implemented by the INS to establish potential sources of vibriosis in Colombia.

Structure of the trypanosome transferrin receptor reveals mechanisms of ligand recognition and immune evasion.

To maintain prolonged infection of mammals, African trypanosomes have evolved remarkable surface coats and a system of antigenic variation1. Within these coats are receptors for macromolecular nutrients such as transferrin2,3. These must be accessible to their ligands but must not confer susceptibility to immunoglobulin-mediated attack. Trypanosomes have a wide host range and their receptors must also bind ligands from diverse species. To understand how these requirements are achieved, in the context of transferrin uptake, we determined the structure of a Trypanosoma brucei transferrin receptor in complex with human transferrin, showing how this heterodimeric receptor presents a large asymmetric ligand-binding platform. The trypanosome genome contains a family of around 14 transferrin receptors4, which has been proposed to allow binding to transferrin from different mammalian hosts5,6. However, we find that a single receptor can bind transferrin from a broad range of mammals, indicating that receptor variation is unlikely to be necessary for promiscuity of host infection. In contrast, polymorphic sites and N-linked glycans are preferentially found in exposed positions on the receptor surface, not contacting transferrin, suggesting that transferrin receptor diversification is driven by a need for antigenic variation in the receptor to prolong survival in a host.

A single dose of antibody-drug conjugate cures a stage 1 model of African trypanosomiasis.

Infections of humans and livestock with African trypanosomes are treated with drugs introduced decades ago that are not always fully effective and often have severe side effects. Here, the trypanosome haptoglobin-haemoglobin receptor (HpHbR) has been exploited as a route of uptake for an antibody-drug conjugate (ADC) that is completely effective against Trypanosoma brucei in the standard mouse model of infection. Recombinant human anti-HpHbR monoclonal antibodies were isolated and shown to be internalised in a receptor-dependent manner. Antibodies were conjugated to a pyrrolobenzodiazepine (PBD) toxin and killed T. brucei in vitro at picomolar concentrations. A single therapeutic dose (0.25 mg/kg) of a HpHbR antibody-PBD conjugate completely cured a T. brucei mouse infection within 2 days with no re-emergence of infection over a subsequent time course of 77 days. These experiments provide a demonstration of how ADCs can be exploited to treat protozoal diseases that desperately require new therapeutics.

Colombia, an unknown genetic diversity in the era of Big Data.

BACKGROUND: Latin America harbors some of the most biodiverse countries in the world, including Colombia. Despite the increasing use of cutting-edge technologies in genomics and bioinformatics in several biological science fields around the world, the region has fallen behind in the inclusion of these approaches in biodiversity studies. In this study, we used data mining methods to search in four main public databases of genetic sequences such as: NCBI Nucleotide and BioProject, Pathosystems Resource Integration Center, and Barcode of Life Data Systems databases. We aimed to determine how much of the Colombian biodiversity is contained in genetic data stored in these public databases and how much of this information has been generated by national institutions. Additionally, we compared this data for Colombia with other countries of high biodiversity in Latin America, such as Brazil, Argentina, Costa Rica, Mexico, and Peru. RESULTS: In Nucleotide, we found that 66.84% of total records for Colombia have been published at the national level, and this data represents less than 5% of the total number of species reported for the country. In BioProject, 70.46% of records were generated by national institutions and the great majority of them is represented by microorganisms. In BOLD Systems, 26% of records have been submitted by national institutions, representing 258 species for Colombia. This number of species reported for Colombia span approximately 0.46% of the total biodiversity reported for the country (56,343 species). Finally, in PATRIC database, 13.25% of the reported sequences were contributed by national institutions. Colombia has a better biodiversity representation in public databases in comparison to other Latin American countries, like Costa Rica and Peru. Mexico and Argentina have the highest representation of species at the national level, despite Brazil and Colombia, which actually hold the first and second places in biodiversity worldwide. CONCLUSIONS: Our findings show gaps in the representation of the Colombian biodiversity at the molecular and genetic levels in widely consulted public databases. National funding for high-throughput molecular research, NGS technologies costs, and access to genetic resources are limiting factors. This fact should be taken as an opportunity to foster the development of collaborative projects between research groups in the Latin American region to study the vast biodiversity of these countries using 'omics' technologies.

Phenotypic screening reveals TNFR2 as a promising target for cancer immunotherapy.

Antibodies that target cell-surface molecules on T cells can enhance anti-tumor immune responses, resulting in sustained immune-mediated control of cancer. We set out to find new cancer immunotherapy targets by phenotypic screening on human regulatory T (Treg) cells and report the discovery of novel activators of tumor necrosis factor receptor 2 (TNFR2) and a potential role for this target in immunotherapy. A diverse phage display library was screened to find antibody mimetics with preferential binding to Treg cells, the most Treg-selective of which were all, without exception, found to bind specifically to TNFR2. A subset of these TNFR2 binders were found to agonise the receptor, inducing iκ-B degradation and NF-κB pathway signalling in vitro. TNFR2 was found to be expressed by tumor-infiltrating Treg cells, and to a lesser extent Teff cells, from three lung cancer patients, and a similar pattern was also observed in mice implanted with CT26 syngeneic tumors. In such animals, TNFR2-specific agonists inhibited tumor growth, enhanced tumor infiltration by CD8+ T cells and increased CD8+ T cell IFN-γ synthesis. Together, these data indicate a novel mechanism for TNF-α-independent TNFR2 agonism in cancer immunotherapy, and demonstrate the utility of target-agnostic screening in highlighting important targets during drug discovery.

Phenotypic screening: the future of antibody discovery.

Most antibody therapeutics have been isolated from high throughput target-based screening. However, as the number of validated targets diminishes and the target space becomes increasingly competitive, alternative strategies, such as phenotypic screening, are gaining momentum. Here, we review successful phenotypic screens, including those used to isolate antibodies against cancer and infectious agents. We also consider exciting advances in the expression and phenotypic screening of antibody repertoires in single cell autocrine systems. As technologies continue to develop, we believe that antibody phenotypic screening will increase further in popularity and has the potential to provide the next generation of therapeutic antibodies.

Finding of a novel fungal immunomodulatory protein coding sequence in Ganoderma australe / Hallazgo de una nueva secuencia codificadora para una proteína inmunomoduladora de origen fúngico en Ganoderma australe

Among the most common human diseases with immune system compromise are autoimmune diseases, cancer, and the acquired immunodeficiency syndrome (AIDS). Many of these diseases still have no treatment or their therapies have undesirable side effects. This has aroused a great interest in the search for new natural products with therapeutic potential and scientifically proven effects, showing minimal side effects. Formal clinical and pharmacological investigation in various medicinal fungi of the genus Ganoderma (Ganodermataceae) has shown immunomodulatory effects and tumor growth inhibition in mammals, attributable to the presence of immunomodulatory proteins and other secondary metabolites. To date, six fungal immunomodulatory proteins (FIPs) have been reported in Ganoderma. This paper seeks to advance in the discovery of immunomodulatory proteins present in Ganoderma australe, through mycelium transcriptome 454 Roche® pyrosequencing (RNA-seq) and bioinformatics analyses. The results suggest the presence of gene sequences related to an immunomodulatory protein which has been reported in another fungal species Taiwanofungus camphoratus. The candidate gene sequences found in G. australe exhibit high identity values in their amino acid composition and predicted protein secondary structure with the protein reported for Tai. camphoratus. According to present knowledge about the action mechanisms of these proteins, it is possible to suggest that this is a promising molecule for the treatment and prevention of diseases associated with certain immune deficiencies, cancer, and other diseases with compromised immune systems. Future studies are proposed in order to determine its immunomodulatory potential using in vitro and in vivo assays.

Enfermedades comunes como las autoinmunes, el cáncer y el síndrome de inmunodeficiencia adquirida aún no tienen tratamiento o sus terapias tienen efectos secundarios indeseables. Ello ha suscitado el interés en la investigación de bioproductos con potencial terapéutico, que no impliquen efectos secundarios. Investigaciones farmacológicas y clínicas en algunos hongos medicinales del género Ganoderma (Ganodermataceae) han comprobado efectos inmunomoduladores e inhibidores de crecimiento tumoral en mamíferos, atribuibles a la presencia de proteínas fúngicas inmunomoduladoras (FIPs) y otros metabolitos secundarios. Este trabajo busca avanzar en el descubrimiento de proteínas inmunomoduladoras presentes en Ganoderma australe, mediante la secuenciación del transcriptoma de micelio por tecnología de pirosecuenciación 454 Roche® (RNA-seq) y análisis bioinformáticos. Los resultados sugieren la presencia de secuencias génicas relacionadas con una proteína inmunomoduladora que se ha reportado en la especie de hongos Taiwanofungus camphoratus. Las secuencias génicas candidatas halladas en G. australe exhiben una altos valores de similitud en sus predicciones de composición aminoacídica y estructura secundaria proteica con la proteína reportada para Tai. camphoratus. Los mecanismos de acción de este tipo de proteínas inmunomoduladoras sugieren que se trata de una molécula con potencial promisorio para el tratamiento y prevención de enfermedades con compromiso del sistema inmunológico y el cáncer. Se proponen nuevos estudios que permitan determinar el potencial inmunomodulador de la proteína hipotética hallada mediante estudios in vivo e in vitro.

Tailored amino acid diversity for the evolution of antibody affinity.

Antibodies are a unique class of proteins with the ability to adapt their binding sites for high affinity and high specificity to a multitude of antigens. Many analyses have been performed on antibody sequences and structures to elucidate which amino acids have a predominant role in antibody interactions with antigens. These studies have generally not distinguished between amino acids selected for broad antigen specificity in the primary immune response and those selected for high affinity in the secondary immune response. By studying a large data set of affinity matured antibodies derived from in vitro directed evolution experiments, we were able to specifically highlight a subset of amino acids associated with affinity improvements. In a comparison of affinity maturations using either tailored or full amino acid diversification, the tailored approach was found to be at least as effective at improving affinity while requiring fewer mutagenesis libraries than the traditional method. The resulting sequence data also highlight the potential for further reducing amino acid diversity for high affinity binding interactions.

Interleukin-2 gene variation impairs regulatory T cell function and causes autoimmunity.

Autoimmune diseases are thought to result from imbalances in normal immune physiology and regulation. Here, we show that autoimmune disease susceptibility and resistance alleles on mouse chromosome 3 (Idd3) correlate with differential expression of the key immunoregulatory cytokine interleukin-2 (IL-2). In order to test directly that an approximately twofold reduction in IL-2 underpins the Idd3-linked destabilization of immune homeostasis, we show that engineered haplodeficiency of Il2 gene expression not only reduces T cell IL-2 production by twofold but also mimics the autoimmune dysregulatory effects of the naturally occurring susceptibility alleles of Il2. Reduced IL-2 production achieved by either genetic mechanism correlates with reduced function of CD4(+) CD25(+) regulatory T cells, which are critical for maintaining immune homeostasis.

Type 1 diabetes genes and pathways shared by humans and NOD mice.

The identification of causative genes for the autoimmune disease type 1 diabetes (T1D) in humans and candidate genes in the NOD mouse has made significant progress in recent years. In addition to sharing structural aspects of the MHC class II molecules that confer susceptibility or resistance to T1D, genes and pathways contributing to autoimmune pathogenesis are held in common by the two species. There are data demonstrating a similar need to establish central tolerance to insulin. Gene variants for the interacting molecules IL2 and CD25, members of a pathway that is essential for immune homeostasis, are present in mice and humans, respectively. Variation of two molecules that negatively regulate T cells, CTLA-4 and the tyrosine phosphatase LYP/PEP, are associated with susceptibility to human and NOD T1D. These observations underscore the value of the NOD mouse model for mechanistic studies on human T1D-associated molecular and cellular pathways.

Natural genetic variants influencing type 1 diabetes in humans and in the NOD mouse.

The understanding of the genetic basis of type 1 diabetes and other autoimmune diseases and the application of that knowledge to their treatment, cure and eventual prevention has been a difficult goal to reach. Cumulative progress in both mouse and human are finally giving way to some successes and significant insights have been made in the last few years. Investigators have identified key immune tolerance-associated phenotypes in convincingly reliable ways that are regulated by specific diabetes-associated chromosomal intervals. The combination of positional genetics and functional studies is a powerful approach to the identification of downstream molecular events that are causal in disease aetiology. In the case of type 1 diabetes, the availability of several animal models, especially the NOD mouse, has complemented the efforts to localize human genes causing diabetes and has shown that some of the same genes and pathways are associated with autoimmunity in both species. There is also growing evidence that the initiation or progression of many autoimmune diseases is likely to be influenced by some of the same genes.

Fine mapping, gene content, comparative sequencing, and expression analyses support Ctla4 and Nramp1 as candidates for Idd5.1 and Idd5.2 in the nonobese diabetic mouse.

At least two loci that determine susceptibility to type 1 diabetes in the NOD mouse have been mapped to chromosome 1, Idd5.1 (insulin-dependent diabetes 5.1) and Idd5.2. In this study, using a series of novel NOD.B10 congenic strains, Idd5.1 has been defined to a 2.1-Mb region containing only four genes, Ctla4, Icos, Als2cr19, and Nrp2 (neuropilin-2), thereby excluding a major candidate gene, Cd28. Genomic sequence comparison of the two functional candidate genes, Ctla4 and Icos, from the B6 (resistant at Idd5.1) and the NOD (susceptible at Idd5.1) strains revealed 62 single nucleotide polymorphisms (SNPs), only two of which were in coding regions. One of these coding SNPs, base 77 of Ctla4 exon 2, is a synonymous SNP and has been correlated previously with type 1 diabetes susceptibility and differential expression of a CTLA-4 isoform. Additional expression studies in this work support the hypothesis that this SNP in exon 2 is the genetic variation causing the biological effects of Idd5.1. Analysis of additional congenic strains has also localized Idd5.2 to a small region (1.52 Mb) of chromosome 1, but in contrast to the Idd5.1 interval, Idd5.2 contains at least 45 genes. Notably, the Idd5.2 region still includes the functionally polymorphic Nramp1 gene. Future experiments to test the identity of Idd5.1 and Idd5.2 as Ctla4 and Nramp1, respectively, can now be justified using approaches to specifically alter or mimic the candidate causative SNPs.

A novel instance of class I modification (cim) affecting two of three rat class I RT1-A molecules within one MHC haplotype.

MHC class I expression by rats of the RT1(o), RT1(d), and RT1(m) MHC haplotypes was investigated. Identical, functional cDNAs were obtained from RT1(o) and BDIX (RT1(dv1)) rats for three MHC class I molecules. RT1-A1(o/d) and -A2(o/d) are closely related in sequence to other cloned rat class Ia genes that have been shown to map to the RT1-A region, while RT1-A3 degrees is highly homologous to a class I gene identified by sequencing an RT1-A(n) genomic contig and is named A3(n). Detailed analysis of the three molecules was undertaken using serology with mAbs, two-dimensional gel analysis of immunoprecipitates, and killing assays using cytotoxic T cells. Arguments are presented suggesting that A1 degrees is the principal MHC class Ia (classical) restricting element of this haplotype. A2 degrees, which is highly cross-reactive with A1 degrees, and A3 degrees probably play more minor or distinct roles in Ag presentation. Unexpectedly, cDNAs encoding exactly the same three molecules were cloned from rats of the RT1(m) haplotype, an MHC that until now was thought to possess unique class Ia genes. RT1(m) contains the TAP-B allele of the TAP transporter, and we present evidence that functional polymorphism in rat TAP has an even greater impact on the expression of RT1-A1 degrees and -A2 degrees than it does on RT1-A(a) in the established case of class I modification (cim). Historically, this led to the misclassification of RT1(m) class Ia molecules as separate and distinct.