Gouania willdenowi is a teleost fish without immunoglobulin genes.

Immunoglobulin (Ig) genes encode antibodies in jawed vertebrates. They are essential elements of the adaptive immune response. Ig exists in soluble form or as part of the B cell membrane antigen receptor (BCR). Studies of Ig genes in fish genomes reveal the absence of Ig genes in Gouania willdenowi by deletion of the entire Ig locus from the canonical chromosomal region. The genes coding for integral BCR proteins, CD79a and CD79b, are also absent. Genes exist for T α/ß lymphocyte receptors but not for the T γ/δ receptors. The results of the genomic analysis are independently corroborated with RNA-Seq transcriptomes from other Gobiesocidae species. From the transcriptome studies, Ig is also absent from these other Gobiesocidae species, Acyrtus sp. and Tomicodon sp. Present evidence suggests that Ig is missing from all species of the Gobiesocidae family.

Insights into the evolution of IG genes in Amphibians and reptiles.

Immunoglobulins are essential proteins of the immune system to neutralize pathogens. Gene encoding B cell receptors and antibodies (Ig genes) first appeared with the emergence of early vertebrates having a jaw, and are now present in all extant jawed vertebrates, or Gnathostomata. The genes have undergone evolutionary changes. In particular, genomic structural changes corresponding to genes of the adaptive immune system were coincident or in parallel with the adaptation of vertebrates from the sea to land. In cartilaginous fish exist IgM, IgD/W, and IgNAR and in bony fish IgM, IgT, IgD. Amphibians and reptiles witnessed significant modifications both in the structure and orientation of IG genes. In particular, for these amphibians and Amniota that adapted to land, IgM and IgD genes were retained, but other isotypes arose, including genes for IgA(X)1, IgA(X)2, and IgY. Recent progress in high throughput genome sequencing is helping to uncover the IG gene structure of all jawed vertebrates. In this work, we review the work and present knowledge of immunoglobulin genes in genomes of amphibians and reptiles.

Immunoglobulin T genes in Actinopterygii.

In teleost fishes, there are three immunoglobulin isotypes named immunoglobulin M (IgM), D (IgD), and T (IgT). IgT was the last to be described in teleost fishes, and it is specific to them. From recent fish genomes, we identified and studied the immunoglobulin heavy chain genes in Actinopterygii. For this analysis, a custom bioinformatics and machine learning pipeline, we call CHfinder, was developed that identifies the exons coding for the CH domains of fish immunoglobulins. Some IgT in teleost and holostean fish found from this systematic study have not been previously described. Phylogenetic analysis of the deduced amino acid sequences of the IgT CH1 exons reveals they are similar to the CH1 of IgM. This analysis also shows that the other three domains (CH2, CH3, and CH4) were not the result of recent IgM duplication processes in Actinopterygii, demonstrating that it is an immunoglobulin of earlier origin. The bioinformatics program, CHfinder, is publicly available at https://github.com/compimmuno/CHfinder.

Vortex revivals and Fermi-Pasta-Ulam-Tsingou recurrence.

We study the self-trapped vortex-ring eigenstates of the two-dimensional Schrödinger equation with focusing Poisson and cubic nonlinearities. For each value of the topological charge l, there is a family of solutions depending on a parameter that can be understood as the relative importance of the cubic term. We analyze the perturbative stability of the solutions and simulate the fate of the unstable ones. For l=1 and l=2, there is an interval of the family of eigenstates for which the initial profile breaks apart but subsequently reconstructs itself, a process that can be interpreted as a nontrivial nonlinear oscillation between the vortex and an azimuthon. This revival provides a compelling realization of a recurrence of the Fermi-Pasta-Ulam-Tsingou type. Outside this interval, the vortices can be stable (for small cubic terms) or unstable and nonrecurrent (for large cubic terms). We argue that there is a crossover between these regimes that resembles a strong stochasticity threshold. For l≥3, all solutions are unstable and nonrecurrent. Finally, we comment on the possible experimental implementation of this phenomenon in the context of nonlinear laser beam propagation in thermo-optical media.

Iterative Variable Gene Discovery from Whole Genome Sequencing with a Bootstrapped Multiresolution Algorithm.

In jawed vertebrates, variable (V) genes code for antigen-binding regions of B and T lymphocyte receptors, which generate a specific response to foreign pathogens. Obtaining the detailed repertoire of these genes across the jawed vertebrate kingdom would help to understand their evolution and function. However, annotations of V-genes are known for only a few model species since their extraction is not amenable to standard gene finding algorithms. Also, the more distant evolution of a taxon is from such model species, and there is less homology between their V-gene sequences. Here, we present an iterative supervised machine learning algorithm that begins by training a small set of known and verified V-gene sequences. The algorithm successively discovers homologous unaligned V-exons from a larger set of whole genome shotgun (WGS) datasets from many taxa. Upon each iteration, newly uncovered V-genes are added to the training set for the next predictions. This iterative learning/discovery process terminates when the number of new sequences discovered is negligible. This process is akin to "online" or reinforcement learning and is proven to be useful for discovering homologous V-genes from successively more distant taxa from the original set. Results are demonstrated for 14 primate WGS datasets and validated against Ensembl annotations. This algorithm is implemented in the Python programming language and is freely available at http://vgenerepertoire.org.

Immunoglobulin genes in Primates.

Five classes of immunoglobulins are known to exist in mammals. The number of isotypes of classes G, E and A varies among species for unknown reasons. Here, a study of the presence of immunoglobulin genes in Primates was carried out from the genomes and transcriptomes deposited in the NCBI repository. For this, a machine learning application based upon neural networks was implemented that scans the genomes and identifies the exon sequences that encode the immunoglobulin CH domains. From these exons, the immunoglobulins that each species possess can be inferred. Also, the presence of sequences outside the IGHC locus was found which were produced by retrotranscription of RNA that are probably not viable. From this study, the distribution of immunoglobulin genes across primate orders is described in detail. In Prosimians, IgD genes are not found; in Platyrrhines, a gene is identified for each of the immunoglobulin classes but the IgD gene does not have the CH2 exon; in the Cercopithecidae family, a gene is detected for each class in the Colobinae family, while in Cercopithecidae the genes for IgG have been duplicated several times. In hominids, a greater number of duplications that include the genes that code for IgA and IgE are observed. These results indicate that from the appearance of the Cercopithecidae, there is an evolutionary instability in the Ig locus.

Immunoglobulin and T cell receptor genes in Chinese crocodile lizard Shinisaurus crocodilurus.

Squamata are reptiles that diverged from mammals 300 million years ago. During this period, the immunoglobulin (IG) and T lymphocyte receptor (TCR) genes evolved parallel to mammals. However, unlike mammals whose IG/TCR locus has retained a constant structure throughout evolution, Squamata have witnessed duplications, losses, and/or gains in the domains of their immunoglobulin genes. The recent genome sequencing of Shinisaurus crocodilurus, a representative species of the oldest reptiles, provides an opportunity to contrast the structure of IG and TCR genes from previously studied Squamata. This study revealed ten immunoglobulin genes: five genes for immunoglobulin M (IgM), two for immunoglobulin D (IgD), one for immunoglobulin D2 (IgD2), and two for immunoglobulin Y (IgY). As in other Squamata, there are genes for the λ light chain (IGLV) but not for the κ chain (IGKV). Here, the data shows that in some IgM genes, the cysteine needed to bind the λ chains does not exist, but we present evidence for possible non-covalent binding to the light chain. With respect to TCR, one gene is detected for the α constant chain (TRAC) and two genes for the ß constant chain (TRBC); one of which is located in the locus of the variable regions of the heavy chain. As in the rest of the Squamata, genes for the γ/δ T cell receptor were not found. The V gene repertoire is found to be consistent with all other Squamata with few V genes for beta chain of TCR.

Clinical and hematological data to group different chronic kidney disease patients: A practical approach to establish different groups of patients.

BACKGROUND: Chronic kidney disease (CKD) is the convergent point of several pathological processes, and its evolution is insidious and characterized by a progressive and irreversible loss of kidney function. This impaired function induces the accumulation of uremic toxins and individuals with terminal CKD often have altered physiological responses, including a persistent state of immuno-suppression and development of diseases. A better characterization and stratification of these patients with CKD in different immuno-compromised groups would contribute to more effective and personalized treatments. The focus of this study was to use two parameters to stratify patients with CKD into four separate groups that are representative of different immunological status. METHODS: Patients with CKD were chosen randomly and stratified into four separate groups according to the period of time receiving dialysis treatment and leukocyte blood counts. The amount of apoptotic CD4 T cells were measured in each group of patients, and clinical/hematological parameters were correlated by multivariate analysis with each group. RESULTS: Observations reveal that one of the four groups of patients with CKD (group 3) had more apoptotic CD4 T cells than the other group; this group also had an increased malnutrition inflammation score (MIS), an elevated Kt/V, and a higher incidence of smoking. CONCLUSION: A simple two-parameter-based stratification strategy could be used to design effective immunological therapies that differentiate the degrees of immuno-suppression across groups of patients with CKD.

The role of clinical pharmacists in treatment adherence: fast impact in suppression of chronic myeloid leukemia development and symptoms.

PURPOSE: Chronic myeloid leukemia (CML) is a clonal myeloproliferative disease, accounting for 15 to 20% of leukemias, with an incidence of one to two cases/100,000 inhabitants. In Brazil, the estimated incidence of leukemia is six cases/100,000 men and 4.28 cases/100,000 women. CML is characterized by the presence of the Philadelphia chromosome. At present, three types of tyrosine kinase inhibitors (TKI) are administered to treat CML patients in the Brazilian public national health system (NHS), called the Unified Health System (in Portuguese, "Sistema Único de Saúde", SUS). Such treatments are only effective if patients adhere to strict dosage regimens; protocol improvements that increase patient adherence to treatment would have economic and health benefits for overburdened health care systems. Here, pharmacist-monitored treatment is assessed. METHODS: In our study, we applied two questionnaires, one to assess the adherence to pharmacological treatment and another to assess the quality of life. All patients studied (n = 23) were diagnosed with CML at a local hospital in "Espírito Santo" State, the "Hospital Evangélico Vila Velha" (HEVV). RESULTS: Treatment adherence was significantly higher in pharmacist-monitored patients than in nonmonitored patients (p = 0.0135). The quality of life of CML patients was also analyzed, indicating that monitored patients had a lower number of symptoms/complaints during treatment periods than nonmonitored patients. Finally, improved treatment adherence also translated into better clinical conditions, particularly during the early stage of treatment (e.g., the first 4 months). CONCLUSIONS: The intervention of a clinical pharmacist is significant to obtain positive clinical results. Therefore, it is recommended that this protocol be included in the standard NHS treatment protocol CML patient outcomes to reduce the indirect and recurring costs to the health care system caused by nonadherence.

Genomic structure and expression of immunoglobulins in Squamata.

The Squamata order represents a major evolutionary reptile lineage, yet the structure and expression of immunoglobulins in this order has been scarcely studied in detail. From the genome sequences of four Squamata species (Gekko japonicus, Ophisaurus gracilis, Pogona vitticeps and Ophiophagus hannah) and RNA-seq datasets from 18 other Squamata species, we identified the immunoglobulins present in these animals as well as the tissues in which they are found. All Squamata have at least three immunoglobulin classes; namely, the immunoglobulins M, D, and Y. Unlike mammals, however, we provide evidence that some Squamata lineages possess more than one Cµ gene which is located downstream from the Cδ gene. The existence of two evolutionary lineages of immunoglobulin Y is shown. Additionally, it is demonstrated that while all Squamata species possess the λ light chain, only Iguanidae species possess the κ light chain.

Stabilizing 3D in vivo intravital microscopy images with an iteratively refined soft-tissue model for immunology experiments.

We describe a set of new algorithms and a software tool, StabiTissue, for stabilizing in vivo intravital microscopy images that suffer from soft-tissue background movement. Because these images lack predetermined anchors and are dominated by noise, we use a pixel weighted image alignment together with a correction for nonlinear tissue deformations. We call this correction a poor man׳s diffeomorphic map since it ascertains the nonlinear regions of the image without resorting to a full integral equation method. To determine the quality of the image stabilization, we developed an ensemble sampling method that quantifies the coincidence between image pairs from randomly distributed image regions. We obtain global stabilization alignment through an iterative constrained simulated annealing optimization procedure. To show the accuracy of our algorithm with existing software, we measured the misalignment error rate in datasets taken from two different organs and compared the results to a similar and popular open-source solution. Present open-source stabilization software tools perform poorly because they do not treat the specific needs of the IV-2pM datasets with soft-tissue deformation, speckle noise, full 5D inter- and intra-stack motion error correction, and undefined anchors. In contrast, the results of our tests demonstrate that our method is more immune to noise and provides better performance for datasets' possessing nonlinear tissue deformations. As a practical application of our software, we show how our stabilization improves cell tracking, where the presence of background movement would degrade track information. We also provide a qualitative comparison of our software with other open-source libraries/applications. Our software is freely available at the open source repository http://sourceforge.net/projects/stabitissue/.

Evolution of V genes from the TRV loci of mammals.

Information concerning the evolution of T lymphocyte receptors (TCR) can be deciphered from that part of the molecule that recognizes antigen presented by major histocompatibility complex (MHC), namely the variable (V) regions. The genes that code for these variable regions are found within the TCR loci. Here, we describe a study of the evolutionary origin of V genes that code for the α and ß chains of the TCR loci of mammals. In particular, we demonstrate that most of the 35 TRAV and 25 TRBV conserved genes found in Primates are also found in other Eutheria, while in Marsupials, Monotremes, and Reptiles, these genes diversified in a different manner. We also show that in mammals, all TRAV genes are derived from five ancestral genes, while all TRBV genes originate from four such genes. In Reptiles, the five TRAV and three out of the four TRBV ancestral genes exist, as well as other V genes not found in mammals. We also studied the TRGV and TRDV loci from all mammals, and we show a relationship of the TRDV to the TRAV locus throughout evolutionary time.

Automatic recognition and scoring of olympic rhythmic gymnastic movements.

We describe a conceptually simple algorithm for assigning judgement scores to rhythmic gymnastic movements, which could improve scoring objectivity and reduce judgemental bias during competitions. Our method, implemented as a real-time computer vision software, takes a video shot or a live performance video stream as input and extracts detailed velocity field information from body movements, transforming them into specialized spatio-temporal image templates. The collection of such images over time, when projected into a velocity covariance eigenspace, trace out unique but similar trajectories for a particular gymnastic movement type. By comparing separate executions of the same atomic gymnastic routine, our method assigns a quality judgement score that is related to the distance between the respective spatio-temporal trajectories. For several standard gymnastic movements, the method accurately assigns scores that are comparable to those assigned by expert judges. We also describe our rhythmic gymnastic video shot database, which we have made freely available to the human movement research community. The database can be obtained at http://www.milegroup.net/apps/gymdb/.

Intravital microscopy technique to study parasite dynamics in the labyrinth layer of the mouse placenta.

Intravital imaging techniques are the best approach to investigate in situ cellular behavior under physiological conditions. Many techniques have emerged during these last few years for this purpose. We recently described an intravital imaging technique that allows for the observation of placenta physiological responses at the labyrinth layer of this tissue. This technique will be very useful to study many placental opportunistic infections and in this article we reinforce its usefulness by analyzing placental physiological entrapment of beads and parasites. In particular, our results show that small beads (1.0 µm) or Plasmodium chabaudi-GFP-infected-Red Blood Cells (Pc-GFP-iRBCs) cannot get trapped inside small or large blood vessels of popliteal lymph nodes (PLNs). Inside the placenta, clusters of beads could only be found inside the maternal blood vessels. However, Pc-GFP-iRBCs were found inside and outside the maternal blood vessels. We observed that trophoblasts can ingest infected-Red Blood Cells (iRBCs) in vitro and immunofluorescence of placenta revealed Pc-GFP-iRBCs inside and outside the maternal blood vessels. Taken together, we conclude that fast deposition of particles inside blood vessels seems to be an intrinsic characteristic of placenta blood flow, but iRBCs could be internalized by trophoblast cells. Thus these results represent one of the many possible uses of our intravital imaging technique to address important questions inside the parasitological field.

Software tool for 3D extraction of germinal centers.

BACKGROUND: Germinal Centers (GC) are short-lived micro-anatomical structures, within lymphoid organs, where affinity maturation is initiated. Theoretical modeling of the dynamics of the GC reaction including follicular CD4+ T helper and the recently described follicular regulatory CD4+ T cell populations, predicts that the intensity and life span of such reactions is driven by both types of T cells, yet controlled primarily by follicular regulatory CD4+ T cells. In order to calibrate GC models, it is necessary to properly analyze the kinetics of GC sizes. Presently, the estimation of spleen GC volumes relies upon confocal microscopy images from 20-30 slices spanning a depth of ~ 20 - 50 µm, whose GC areas are analyzed, slice-by-slice, for subsequent 3D reconstruction and quantification. The quantity of data to be analyzed from such images taken for kinetics experiments is usually prohibitively large to extract semi-manually with existing software. As a result, the entire procedure is highly time-consuming, and inaccurate, thereby motivating the need for a new software tool that can automatically identify and calculate the 3D spot volumes from GC multidimensional images. RESULTS: We have developed pyBioImage, an open source cross platform image analysis software application, written in python with C extensions that is specifically tailored to the needs of immunologic research involving 4D imaging of GCs. The software provides 1) support for importing many multi-image formats, 2) basic image processing and analysis, and 3) the ExtractGC module, that allows for automatic analysis and visualization of extracted GC volumes from multidimensional confocal microscopy images. We present concrete examples of different microscopy image data sets of GC that have been used in experimental and theoretical studies of mouse model GC dynamics. CONCLUSIONS: The pyBioImage software framework seeks to be a general purpose image application for immunological research based on 4D imaging. The ExtractGC module uses a novel clustering algorithm for automatically extracting quantitative spatial information of a large number of GCs from a collection of confocal microscopy images. In addition, the software provides 3D visualization of the GCs reconstructed from the image stacks. The application is available for public use at http://sourceforge.net/projects/pybioimage/.

Intravital placenta imaging reveals microcirculatory dynamics impact on sequestration and phagocytosis of Plasmodium-infected erythrocytes.

Malaria in pregnancy is exquisitely aggressive, causing a range of adverse maternal and fetal outcomes prominently linked to Plasmodium-infected erythrocyte cytoadherence to fetal trophoblast. To elucidate the physiopathology of infected erythrocytes (IE) sequestration in the placenta we devised an experimental system for intravital placental examination of P. berghei-infected mice. BALB/c females were mated to C57Bl/6 CFP+ male mice and infected with GFP+ P. berghei IE, and at gestational day 18, placentas were exposed for time-lapse imaging acquisition under two-photon microscopy. Real-time images and quantitative measurements revealed that trophoblast conformational changes transiently restrain blood flow in the mouse placental labyrinth. The complex dynamics of placental microcirculation promotes IE accumulation in maternal blood spaces with low blood flow and allows the establishment of stable IE-trophoblast contacts. Further, we show that the fate of sequestered IE includes engulfment by both macrophagic and trophoblastic fetal-derived cells. These findings reinforce the current paradigm that IE interact with the trophoblast and provide definitive evidence on two novel pathogenesis mechanisms: (1) trophoblast layer controls placental microcirculation promoting IE sequestration; and (2) fetal-derived placental cells engulf sequestered IE.

Techniques for visualization of cell-cell contact at the fetal-maternal interface.

Historically, several in vitro/ex vivo microscopy imaging techniques have been used to study cellular interactions within the uterus and the placenta. As these experimental methods have revealed compelling facts about the biologic phenomena of cell-cell contacts in these organs, they cannot be used to study complex dynamic behavior of living cells inside their physiologic environment. For this, recent advances in intravital imaging techniques, together with two-photon microscopy, offer an exciting opportunity to study such dynamic immunologic processes at the cellular level in the complex uterine and placental tissues. In this article, we review experimental imaging techniques that have been used for studying the uterus and placenta. In particular, we describe the advantages of intravital techniques and discuss novel procedures that can be used in reproductive immunology. We also describe several technical details involved in image sequence post-processing required to extract useful data. Finally, we conclude by discussing how the reproductive immunology field may benefit from the broad use of these intravital techniques.

In vivo multiphoton microscopy technique to reveal the physiology of the mouse uterus.

PROBLEM: Pregnancy is a challenge to the maternal immune system as it allows the growing of a semiallogeneic fetus within the uterus. Such tolerance suggests a set of complex cellular distributions and interactions inside the organ. Until now, direct observation of such processes was absent because proper intravital imaging techniques were not available. METHOD: We developed a new two-photon microscope stage together with a set of surgical procedures to provide direct observation of immune cell within the mouse uterus. RESULTS: Using our technique, we observed an accumulation of dendritic cells (DCs) in the uterus during the estrus phase of the estrus cycle. Some of the observed DC clusters were located near the lumen of the uterus or small blood vessels, each situated on the antimesometrium side. CONCLUSION: While two-photon microscopy has become a widely used technology for intravital imaging, new advances in the development of staging and experimental protocols can still push the limits of this technique for exploring new biology. As proof of this, we demonstrated that with specially designed staging and surgical protocols, we observed the formation of DC clusters in the uterus; structures that may play a role in the complex immunology of the uterus-fetal interface.

Early skin immunological disturbance after Plasmodium-infected mosquito bites.

Although the role of regulatory T cells (Tregs) during malaria infection has been studied extensively, such studies have focused exclusively on the role of Treg during the blood stage of infection; little is known about the detailed mechanisms of Tregs and sporozoite deposition in the dermis by mosquito bites. In this paper we show that sporozoites introduced into the skin by mosquito bites increase the mobility of skin Tregs and dendritic cells (DCs). We also show differences in MHC class II and/or CD86 expression on skin-resident dendritic cell subtypes and macrophages. From the observed decrease of the number of APCs into draining lymph nodes, suppression of CD28 expression in conventional CD4 T cells, and a low homeostatic proliferation of skin-migrated CD4 T found in nude mice indicate that Tregs may play a fundamental role during the initial phase of malaria parasite inoculation into the mammalian host.