Computer-Aided detection of tuberculosis from X-ray images using CNN and PatternNet classifier.

BACKGROUND: Tuberculosis (TB) is a highly infectious disease that mainly affects the human lungs. The gold standard for TB diagnosis is Xpert Mycobacterium tuberculosis/ resistance to rifampicin (MTB/RIF) testing. X-ray, a relatively inexpensive and widely used imaging modality, can be employed as an alternative for early diagnosis of the disease. Computer-aided techniques can be used to assist radiologists in interpreting X-ray images, which can improve the ease and accuracy of diagnosis. OBJECTIVE: To develop a computer-aided technique for the diagnosis of TB from X-ray images using deep learning techniques. METHODS: This research paper presents a novel approach for TB diagnosis from X-ray using deep learning methods. The proposed method uses an ensemble of two pre-trained neural networks, namely EfficientnetB0 and Densenet201, for feature extraction. The features extracted using two CNNs are expected to generate more accurate and representative features than a single CNN. A custom-built artificial neural network (ANN) called PatternNet with two hidden layers is utilized to classify the extracted features. RESULTS: The effectiveness of the proposed method was assessed on two publicly accessible datasets, namely the Montgomery and Shenzhen datasets. The Montgomery dataset comprises 138 X-ray images, while the Shenzhen dataset has 662 X-ray images. The method was further evaluated after combining both datasets. The method performed exceptionally well on all three datasets, achieving high Area Under the Curve (AUC) scores of 0.9978, 0.9836, and 0.9914, respectively, using a 10-fold cross-validation technique. CONCLUSION: The experiments performed in this study prove the effectiveness of features extracted using EfficientnetB0 and Densenet201 in combination with PatternNet classifier in the diagnosis of tuberculosis from X-ray images.

Genetic dissection of TLR9 reveals complex regulatory and cryptic proinflammatory roles in mouse lupus.

In lupus, Toll-like receptor 7 (TLR7) and TLR9 mediate loss of tolerance to RNA and DNA, respectively. Yet, TLR7 promotes disease, while TLR9 protects from disease, implying differences in signaling. To dissect this 'TLR paradox', we generated two TLR9 point mutants (lacking either ligand (TLR9K51E) or MyD88 (TLR9P915H) binding) in lupus-prone MRL/lpr mice. Ameliorated disease of Tlr9K51E mice compared to Tlr9-/- controls revealed a TLR9 'scaffold' protective function that is ligand and MyD88 independent. Unexpectedly, Tlr9P915H mice were more protected than both Tlr9K51E and Tlr9WT mice, suggesting that TLR9 also possesses ligand-dependent, but MyD88-independent, regulatory signaling and MyD88-mediated proinflammatory signaling. Triple-mixed bone marrow chimeras showed that TLR9-MyD88-independent regulatory roles were B cell intrinsic and restrained differentiation into pathogenic age-associated B cells and plasmablasts. These studies reveal MyD88-independent regulatory roles of TLR9, shedding light on the biology of endosomal TLRs.

Engineered RBCs Encapsulating Antigen Induce Multi-Modal Antigen-Specific Tolerance and Protect Against Type 1 Diabetes.

Antigen-specific therapies that suppress autoreactive T cells without inducing systemic immunosuppression are a much-needed treatment for autoimmune diseases, yet effective strategies remain elusive. We describe a microfluidic Cell Squeeze® technology to engineer red blood cells (RBCs) encapsulating antigens to generate tolerizing antigen carriers (TACs). TACs exploit the natural route of RBC clearance enabling tolerogenic presentation of antigens. TAC treatment led to antigen-specific T cell tolerance towards exogenous and autoantigens in immunization and adoptive transfer mouse models of type 1 diabetes (T1D), respectively. Notably, in several accelerated models of T1D, TACs prevented hyperglycemia by blunting effector functions of pathogenic T cells, particularly in the pancreas. Mechanistically, TACs led to impaired trafficking of diabetogenic T cells to the pancreas, induced deletion of autoreactive CD8 T cells and expanded antigen specific Tregs that exerted bystander suppression. Our results highlight TACs as a novel approach for reinstating immune tolerance in CD4 and CD8 mediated autoimmune diseases.

Pre-existing immunity to cytomegalovirus in macaques influences human CMV vaccine responses in preclinical models.

Development of a human cytomegalovirus (HCMV) vaccine is a Tier 1 priority by the National Institutes of Medicine, as HCMV is the most common congenital infection globally and most frequent infectious complication in transplant patients. Relevant preclinical non-human primate models used for testing HCMV vaccine immunogenicity are rhesus and cynomolgous monkeys. However, a complication in using these models is that species-specific CMV variants are endemic in non-human primate breeding colonies. We hypothesize that natural immunity to species-specific CMV in rhesus and cynomolgous monkeys impacts HCMV vaccine immunogenicity and may interfere with our ability to fully interpret vaccine immunogenicity. A modified mRNA vaccine encoding HCMV glycoprotein (gB) and the pentameric complex (PC) packaged in lipid nanoparticles (LNP) was delivered intramuscularly to groups of cynomolgous (n = 16, CyCMV-seropositive) and rhesus macaques (n = 24, RhCMV-seropositive). High pre-vaccination IgG binding responses to HCMV gB were present in both species, but pre-vaccination binding responses to PC were mostly present in rhesus macaques. Yet, at least a log increase in both PC and gB-specific plasma IgG levels was detected post-second HCMV mRNA vaccination in both species. Both species responded with high epithelial cell neutralizing antibody responses at 4 weeks post second HCMV mRNA vaccination, but limited fibroblast neutralizing antibodies. HCMV gB + PC mRNA/LNP vaccine also elicited IgG binding responses to cell-associated gB, an identified immune correlate of protection, in both species after the second vaccination, and there was a moderately strong direct correlation between this pre- and post-vaccination response in rhesus macaques. Based on the correlation between pre-existing and post-vaccine gB-specific binding responses in rhesus macaques, we conclude that species-specific CMV variant-specific antibody responses contribute to antibody responses to HCMV vaccination in primate models, indicating that pre-existing immunity must be taken into account in non-human primate preclinical models and will impact immunogenicity of HCMV vaccines seropositive human vaccinees.

B cell-intrinsic TLR9 expression is protective in murine lupus.

Toll-like receptor 9 (TLR9) is a regulator of disease pathogenesis in systemic lupus erythematosus (SLE). Why TLR9 represses disease while TLR7 and MyD88 have the opposite effect remains undefined. To begin to address this question, we created 2 alleles to manipulate TLR9 expression, allowing for either selective deletion or overexpression. We used these to test cell type-specific effects of Tlr9 expression on the regulation of SLE pathogenesis. Notably, Tlr9 deficiency in B cells was sufficient to exacerbate nephritis while extinguishing anti-nucleosome antibodies, whereas Tlr9 deficiency in dendritic cells (DCs), plasmacytoid DCs, and neutrophils had no discernable effect on disease. Thus, B cell-specific Tlr9 deficiency unlinked disease from autoantibody production. Critically, B cell-specific Tlr9 overexpression resulted in ameliorated nephritis, opposite of the effect of deleting Tlr9. Our findings highlight the nonredundant role of B cell-expressed TLR9 in regulating lupus and suggest therapeutic potential in modulating and perhaps even enhancing TLR9 signals in B cells.

Corrigendum: Induction of Robust B Cell Responses After Influenza mRNA Vaccination Is Accompanied by Circulating Hemagglutinin-Specific ICOS+ PD-1+ CXCR3+ T Follicular Helper Cells.

[This corrects the article DOI: 10.3389/fimmu.2017.01539.].

Multi-antigenic human cytomegalovirus mRNA vaccines that elicit potent humoral and cell-mediated immunity.

A cytomegalovirus (CMV) vaccine that is effective at preventing congenital infection and reducing CMV disease in transplant patients remains a high priority as no approved vaccines exist. While the precise correlates of protection are unknown, neutralizing antibodies and antigen-specific T cells have been implicated in controlling infection. We demonstrate that the immunization of mice and nonhuman primates (NHPs) with lipid nanoparticles (LNP) encapsulating modified mRNA encoding CMV glycoproteins gB and pentameric complex (PC) elicit potent and durable neutralizing antibody titers. Since the protective correlates in pregnant women and transplant recipients may differ, we developed an additional mRNA vaccine expressing the immunodominant CMV T cell antigen pp65. Administration of pp65 vaccine with PC and gB elicited robust multi-antigenic T cell responses in mice. Our data demonstrate that mRNA/LNP is a versatile platform that enables the development of vaccination strategies that could prevent CMV infection and consequent disease in different target populations.

Tissue-Resident Macrophages Are Locally Programmed for Silent Clearance of Apoptotic Cells.

Although apoptotic cells (ACs) contain nucleic acids that can be recognized by Toll-like receptors (TLRs), engulfment of ACs does not initiate inflammation in healthy organisms. Here we identified macrophage populations that continually engulf ACs in distinct tissues and found that these macrophages share characteristics compatible with immunologically silent clearance of ACs; such characteristics include high expression of AC recognition receptors, low expression of TLR9, and reduced TLR responsiveness to nucleic acids. Removal of the macrophages from tissues resulted in loss of many of these characteristics and the ability to generate inflammatory responses to AC-derived nucleic acids, suggesting that cues from the tissue microenvironment program macrophages for silent AC clearance. The transcription factors KLF2 and KLF4 control the expression of many genes within this AC clearance program. The coordinated expression of AC receptors with genes that limit responses to nucleic acids might ensure maintenance of homeostasis and thus represent a central feature of tissue macrophages.

Induction of Robust B Cell Responses after Influenza mRNA Vaccination Is Accompanied by Circulating Hemagglutinin-Specific ICOS+ PD-1+ CXCR3+ T Follicular Helper Cells.

Modified mRNA vaccines have developed into an effective and well-tolerated vaccine platform that offers scalable and precise antigen production. Nevertheless, the immunological events leading to strong antibody responses elicited by mRNA vaccines are largely unknown. In this study, we demonstrate that protective levels of antibodies to hemagglutinin were induced after two immunizations of modified non-replicating mRNA encoding influenza H10 encapsulated in lipid nanoparticles (LNP) in non-human primates. While both intradermal (ID) and intramuscular (IM) administration induced protective titers, ID delivery generated this response more rapidly. Circulating H10-specific memory B cells expanded after each immunization, along with a transient appearance of plasmablasts. The memory B cell pool waned over time but remained detectable throughout the 25-week study. Following prime immunization, H10-specific plasma cells were found in the bone marrow and persisted over time. Germinal centers were formed in vaccine-draining lymph nodes along with an increase in circulating H10-specific ICOS+ PD-1+ CXCR3+ T follicular helper cells, a population shown to correlate with high avidity antibody responses after seasonal influenza vaccination in humans. Collectively, this study demonstrates that mRNA/LNP vaccines potently induce an immunological repertoire associated with the generation of high magnitude and quality antibodies.

Efficient Targeting and Activation of Antigen-Presenting Cells In Vivo after Modified mRNA Vaccine Administration in Rhesus Macaques.

mRNA vaccines are rapidly emerging as a powerful platform for infectious diseases because they are well tolerated, immunogenic, and scalable and are built on precise but adaptable antigen design. We show that two immunizations of modified non-replicating mRNA encoding influenza H10 hemagglutinin (HA) and encapsulated in lipid nanoparticles (LNP) induce protective HA inhibition titers and H10-specific CD4+ T cell responses after intramuscular or intradermal delivery in rhesus macaques. Administration of LNP/mRNA induced rapid and local infiltration of neutrophils, monocytes, and dendritic cells (DCs) to the site of administration and the draining lymph nodes (LNs). While these cells efficiently internalized LNP, mainly monocytes and DCs translated the mRNA and upregulated key co-stimulatory receptors (CD80 and CD86). This coincided with upregulation of type I IFN-inducible genes, including MX1 and CXCL10. The innate immune activation was transient and resulted in priming of H10-specific CD4+ T cells exclusively in the vaccine-draining LNs. Collectively, this demonstrates that mRNA-based vaccines induce type-I IFN-polarized innate immunity and, when combined with antigen production by antigen-presenting cells, lead to generation of potent vaccine-specific responses.

Sequential Activation of Two Pathogen-Sensing Pathways Required for Type I Interferon Expression and Resistance to an Acute DNA Virus Infection.

Toll-like receptor 9 (TLR9), its adaptor MyD88, the downstream transcription factor interferon regulatory factor 7 (IRF7), and type I interferons (IFN-I) are all required for resistance to infection with ectromelia virus (ECTV). However, it is not known how or in which cells these effectors function to promote survival. Here, we showed that after infection with ECTV, the TLR9-MyD88-IRF7 pathway was necessary in CD11c(+) cells for the expression of proinflammatory cytokines and the recruitment of inflammatory monocytes (iMos) to the draining lymph node (dLN). In the dLN, the major producers of IFN-I were infected iMos, which used the DNA sensor-adaptor STING to activate IRF7 and nuclear factor κB (NF-κB) signaling to induce the expression of IFN-α and IFN-ß, respectively. Thus, in vivo, two pathways of DNA pathogen sensing act sequentially in two distinct cell types to orchestrate resistance to a viral disease.

Requirement for Transcription Factor Ets1 in B Cell Tolerance to Self-Antigens.

The differentiation and survival of autoreactive B cells is normally limited by a variety of self-tolerance mechanisms, including clonal deletion, anergy, and clonal ignorance. The transcription factor c-ets-1 (encoded by the Ets1 gene) has B cell-intrinsic roles in regulating formation of Ab-secreting cells by controlling the activity of Blimp1 and Pax5 and may be required for B cell tolerance to self-antigen. To test this, we crossed Ets1(-/-) mice to two different transgenic models of B cell self-reactivity, the anti-hen egg lysozyme BCR transgenic strain and the AM14 rheumatoid factor transgenic strain. BCR transgenic Ets1(-/-) mice were subsequently crossed to mice either carrying or lacking relevant autoantigens. We found that B cells lacking c-ets-1 are generally hyperresponsive in terms of Ab secretion and form large numbers of Ab-secreting cells even in the absence of cognate Ags. When in the presence of cognate Ag, different responses were noted depending on the physical characteristics of the Ag. We found that clonal deletion of highly autoreactive B cells in the bone marrow was intact in the absence of c-ets-1. However, peripheral B cells lacking c-ets-1 failed to become tolerant in response to stimuli that normally induce B cell anergy or B cell clonal ignorance. Interestingly, high-affinity soluble self-antigen did cause B cells to adopt many of the classical features of anergic B cells, although such cells still secreted Ab. Therefore, maintenance of appropriate c-ets-1 levels is essential to prevent loss of self-tolerance in the B cell compartment.

Genetic Interaction between Lyn, Ets1, and Btk in the Control of Antibody Levels.

Tight control of B cell differentiation into plasma cells (PCs) is critical for proper immune responses and the prevention of autoimmunity. The Ets1 transcription factor acts in B cells to prevent PC differentiation. Ets1(-/-) mice accumulate PCs and produce autoantibodies. Ets1 expression is downregulated upon B cell activation through the BCR and TLRs and is maintained by the inhibitory signaling pathway mediated by Lyn, CD22 and SiglecG, and SHP-1. In the absence of these inhibitory components, Ets1 levels are reduced in B cells in a Btk-dependent manner. This leads to increased PCs, autoantibodies, and an autoimmune phenotype similar to that of Ets1(-/-) mice. Defects in inhibitory signaling molecules, including Lyn and Ets1, are associated with human lupus, although the effects are more subtle than the complete deficiency that occurs in knockout mice. In this study, we explore the effect of partial disruption of the Lyn/Ets1 pathway on B cell tolerance and find that Lyn(+/-)Ets1(+/-) mice demonstrate greater and earlier production of IgM, but not IgG, autoantibodies compared with Lyn(+/-) or Ets1(+/-) mice. We also show that Btk-dependent downregulation of Ets1 is important for normal PC homeostasis when inhibitory signaling is intact. Ets1 deficiency restores the decrease in steady state PCs and Ab levels observed in Btk(-/-) mice. Thus, depending on the balance of activating and inhibitory signals to Ets1, there is a continuum of effects on autoantibody production and PC maintenance. This ranges from full-blown autoimmunity with complete loss of Ets1-maintaining signals to reduced PC and Ab levels with impaired Ets1 downregulation.

Transcription factor Ets1, but not the closely related factor Ets2, inhibits antibody-secreting cell differentiation.

B cell differentiation into antibody-secreting cells (ASCs) is a tightly regulated process under the control of multiple transcription factors. One such transcription factor, Ets1, blocks the transition of B cells to ASCs via two separate activities: (i) stimulating the expression of target genes that promote B cell identity and (ii) interfering with the functional activity of the transcription factor Blimp1. Ets1 is a member of a multigene family, several members of which are expressed within the B cell lineage, including the closely related protein Ets2. In this report, we demonstrate that Ets1, but not Ets2, can block ASC formation despite the fact that Ets1 and Ets2 bind to apparently identical DNA sequence motifs and are thought to regulate overlapping sets of target genes. The DNA binding domain of Ets1 is required, but not sufficient by itself, to block ASC formation. In addition, less conserved regions within the N terminus of Ets1 play an important role in inhibiting B cell differentiation. Differences between the N termini of Ets1 and Ets2, rather than differences in the DNA binding domains, determine whether the proteins are capable of blocking ASC formation or not.

Blimp1: a conserved transcriptional repressor critical for differentiation of many tissues.

B lymphocyte induced maturation protein 1 (Blimp1) is a zinc finger transcriptional repressor whose function as a master regulator of terminal differentiation of B cells into plasma cells has long been studied and is well established. Recent studies have identified novel roles for Blimp1 including homeostasis of effector T cells, specification of primordial germ cells in mouse, specification of muscle fiber type in zebrafish and as a tumor suppressor gene in germinal center derived B cells. Blimp1 associates with a multitude of chromatin modifying enzymes inducing epigenetic changes at specific targets to regulate these diverse cell fates. In this review, we focus on the novel and emerging roles of Blimp1 in multiple tissues, on mechanisms of transcriptional repression by Blimp1 and on the activity of Blimp1 as a tumor suppressor.

IL-17 and the Th17 lineage in systemic lupus erythematosus.

PURPOSE OF REVIEW: Systemic lupus erythematosus etiology includes both genetic and environmental factors. Evidence suggests that many genetic loci in humans and mouse models contribute to the occurrence and clinical presentation of lupus. This large array of different genes affects many aspects of immune cell function, including the activation and functional differentiation of B cells, T cells, dendritic cells and other immune cells. In particular, the T-cell components that contribute to systemic lupus erythematosus pathogenesis are incompletely defined. RECENT FINDINGS: A major paradigm shift in understanding how CD4+ T cells contribute to autoimmunity recently occurred with the discovery of a new T-cell population that produces the cytokine IL-17 (IL-17A), termed 'Th17'. Although Th17 cells contribute to autoimmune disease in rheumatoid arthritis and Crohn's disease, their role in systemic lupus erythematosus is far less clear. SUMMARY: In this review, we focus on an emerging role for the cytokine IL-17 and the cells that produce it in contributing to lupus in particular based on recent findings in animal models.

Ets-1 regulates plasma cell differentiation by interfering with the activity of the transcription factor Blimp-1.

Development of immunoglobulin-secreting plasma cells from B cells is a tightly regulated process controlled by the action of a number of transcription factors. In particular, the transcription factor Blimp-1 is a key positive regulator of plasmacytic differentiation via its ability to suppress expression of genes involved in the mature B cell program. The transcription factor Ets-1 is a negative regulator of plasmacytic differentiation, as indicated by the development of increased numbers of IgM-secreting plasma cells in Ets-1 knock-out mice. We have previously shown that Ets-1-deficient B cells undergo enhanced differentiation into IgM-secreting plasma cells in response to Toll-like receptor 9 (TLR9) signaling. We now explore the mechanism by which Ets-1 limits differentiation downstream of TLR9. Our results indicate that Ets-1 physically interacts with Blimp-1, which leads to a block in Blimp-1 DNA binding activity and a reduction in the ability of Blimp-1 to repress target genes without interfering with Blimp-1 protein levels. In addition, we show that Ets-1 induces the expression of several target genes that are repressed by Blimp-1, including Pax-5. These results reveal a previously unknown mechanism for the control of Blimp-1 activity by Ets-1 and suggest that expression of Ets-1 must be down-regulated before plasmacytic differentiation can occur.

Impaired generation of CD8+ thymocytes in Ets-1-deficient mice.

The Ets family of transcription factors function as key regulators of multiple aspects of immune cell development and function. To date, Ets-1 has been implicated in regulating early stages of thymic maturation and lymphocyte function and homeostasis. This report describes a novel role for Ets-1 in supporting later stages of thymic selection, in that positive selection of MHC class I-restricted CD4+CD8+ double-positive thymocytes is markedly inhibited in mice expressing a hypomorphic allele of Ets-1. This effect is thymocyte intrinsic, as Ets-1 mutant thymocytes fail to efficiently generate CD8+ single-positive thymocytes in mixed bone marrow chimeric backgrounds. Although peripheral CD8+ T cells are present in Ets-1 mutant mice, both CD4+ and CD8+ subsets contain an elevated proportion of cells with an effector memory (CD62L-CD44+) phenotype. In addition, while thymic expression of Thy1 is relatively normal, peripheral T cells isolated from Ets-1 mutant mice display a striking loss of Thy1 expression. These data identify Ets-1 as a key transcription factor regulating thymocyte positive selection and lineage commitment of MHC class I-restricted thymocytes.

Ets-1 deficiency leads to altered B cell differentiation, hyperresponsiveness to TLR9 and autoimmune disease.

It has been shown that mice with a targeted mutation in the Ets-1 gene exhibit increased B cell terminal differentiation to IgM-secreting plasma cells. Here, we show that mice, formerly described to lack Ets-1 protein, actually express low levels of an internally deleted Ets-1 protein. Mice harboring this Ets-1 hypomorphic allele possess very few marginal zone B cells and have increased expression of activation markers on follicular B cells. Adoptive transfer experiments indicate that this activated phenotype can be reversed upon transfer of Ets-1-deficient B cells to a wild-type host, suggesting a role for B cell-extrinsic factors in regulating the activated state. Supporting this observation, the reverse transfer experiment of wild-type B cells into an Ets-1-deficient host resulted in increased expression of activation markers on the transferred B cells. However, there are also cell-intrinsic changes in Ets-1-deficient B cells as demonstrated by their increased differentiation to plasma cells in vitro in response to stimulation with cytosine-phosphate-guanine DNA sequence-containing oligodeoxynucleotide [CpG DNA, a Toll-like receptor (TLR) 9 ligand]. Consistent with the activated phenotype and increased terminal differentiation of Ets-1-deficient B cells, Ets-1 mutant mice develop autoimmune disease. Hence, our studies establish Ets-1 as an important regulator of peripheral B cell differentiation and B cell responses to TLR9 activation.

Multiple strains of the parasitic dinoflagellate Amoebophrya exist in Chesapeake Bay.

Small subunit rRNA sequences were amplified from Amoebophrya strains infecting Karlodinium micrum, Gymnodinium instriatum and an unidentified Scrippsiella species in Chesapeake Bay. The alignable parts of the sequences differed from each other and from the previously reported rRNA sequence of the Amoebophrya strain infecting Akashiwo sanguinea in Chesapeake Bay by 4 to 10%. This is a greater degree of difference than sometimes found between sequences from separate genera of free-living dinoflagellates. These sequence differences indicate that the Amoebophrya strains parasitizing dinoflagellates in Chesapeake Bay do not all belong to the same species. In spite of their relative dissimilarity, the sequences do group together into a single clade with high bootstrap support in phylogenetic trees constructed from the sequences.