T Cell-Intrinsic CX3CR1 Marks the Most Differentiated Effector CD4+ T Cells, but Is Largely Dispensable for CD4+ T Cell Responses during Chronic Viral Infection.

CD4+ T cells play critical roles during chronic viral infections, but the factors that regulate these responses remain incompletely defined. During chronic infection of mice with lymphocytic choriomeningitis virus clone 13 (LCMV13), the TNFR family member GITR plays a critical CD4+ T cell-intrinsic role in allowing T cell accumulation and viral control. Previously, RNA sequencing of GITR+/+ and GITR-/- T cells sorted from the spleen of mice at day 3 of LCMV13 infection identified the chemokine receptor CX3CR1 as increased by GITR signaling in CD4+ T cells. In this study, we evaluated the role of CX3CR1 on CD4+ T cells during LCMV13 infection. CX3CR1 expression is induced on Ag-specific CD4+ T cells upon Ag stimulation, and GITR signaling further increases the level of CX3CR1 expression. CX3CR1 marks the most differentiated T-bethi, Th1 effector population. Adoptively transferred CX3CR1-/- SMARTA cells had slightly reduced expression of T-bet and IFN-γ per cell compared with their CX3CR1+/+ counterparts but showed no deficit in accumulation in the spleen, lung, or liver. In mixed-radiation chimeras reconstituted with CX3CR1+/+ and CX3CR1-/- bone marrow, CX3CR1+/+ CD4+ T cells showed a marginal deficit in tissue-resident memory T cell numbers compared with the CX3CR1-/- T cells. CX3CR1 may limit acquisition of the tissue-resident memory T cell phenotype because of its effects on increasing T-bet expression, albeit these small effects are unlikely to be of major biological significance. Taken together, these studies show that CX3CR1 marks the most highly differentiated CD4+ Th1 effector population but is largely dispensable for CD4+ T cell responses during chronic viral infection.

Deep learning and SURF for automated classification and detection of calcaneus fractures in CT images.

BACKGROUND AND OBJECTIVES: The calcaneus is the most fracture-prone tarsal bone and injuries to the surrounding tissue are some of the most difficult to treat. Currently there is a lack of consensus on treatment or interpretation of computed tomography (CT) images for calcaneus fractures. This study proposes a novel computer-assisted method for automated classification and detection of fracture locations in calcaneus CT images using a deep learning algorithm. METHODS: Two types of Convolutional Neural Network (CNN) architectures with different network depths, a Residual network (ResNet) and a Visual geometry group (VGG), were evaluated and compared for the classification performance of CT scans into fracture and non-fracture categories based on coronal, sagittal, and transverse views. The bone fracture detection algorithm incorporated fracture area matching using the speeded-up robust features (SURF) method, Canny edge detection, and contour tracing. RESULTS: Results showed that ResNet was comparable in accuracy (98%) to the VGG network for bone fracture classification but achieved better performance for involving a deeper neural network architecture. ResNet classification results were used as the input for detecting the location and type of bone fracture using SURF algorithm. CONCLUSIONS: Results from real patient fracture data sets demonstrate the feasibility using deep CNN and SURF for computer-aided classification and detection of the location of calcaneus fractures in CT images.

GITRL on inflammatory antigen presenting cells in the lung parenchyma provides signal 4 for T-cell accumulation and tissue-resident memory T-cell formation.

T-cell responses in the lung are critical for protection against respiratory pathogens. TNFR superfamily members play important roles in providing survival signals to T cells during respiratory infections. However, whether these signals take place mainly during priming in the secondary lymphoid organs and/or in the peripheral tissues remains unknown. Here we show that under conditions of competition, GITR provides a T-cell intrinsic advantage to both CD4 and CD8 effector T cells in the lung tissue, as well as for the formation of CD4 and CD8 tissue-resident memory T cells during respiratory influenza infection in mice. In contrast, under non-competitive conditions, GITR has a preferential effect on CD8 over CD4 T cells. The nucleoprotein-specific CD8 T-cell response partially compensated for GITR deficiency by expansion of higher affinity T cells; whereas, the polymerase-specific response was less flexible and more GITR dependent. Following influenza infection, GITR is expressed on lung T cells and GITRL is preferentially expressed on lung monocyte-derived inflammatory antigen presenting cells. Accordingly, we show that GITR+/+ T cells in the lung parenchyma express more phosphorylated-ribosomal protein S6 than their GITR-/- counterparts. Thus, GITR signaling within the lung tissue critically regulates effector and tissue-resident memory T-cell accumulation.

Dichotomous Expression of TNF Superfamily Ligands on Antigen-Presenting Cells Controls Post-priming Anti-viral CD4+ T Cell Immunity.

T cell antigen-presenting cell (APC) interactions early during chronic viral infection are crucial for determining viral set point and disease outcome, but how and when different APC subtypes contribute to these outcomes is unclear. The TNF receptor superfamily (TNFRSF) member GITR is important for CD4+ T cell accumulation and control of chronic lymphocytic choriomeningitis virus (LCMV). We found that type I interferon (IFN-I) induced TNFSF ligands GITRL, 4-1BBL, OX40L, and CD70 predominantly on monocyte-derived APCs and CD80 and CD86 predominantly on classical dendritic cells (cDCs). Mice with hypofunctional GITRL in Lyz2+ cells had decreased LCMV-specific CD4+ T cell accumulation and increased viral load. GITR signals in CD4+ T cells occurred after priming to upregulate OX40, CD25, and chemokine receptor CX3CR1. Thus IFN-I (signal 3) induced a post-priming checkpoint (signal 4) for CD4+ T cell accumulation, revealing a division of labor between cDCs and monocyte-derived APCs in regulating T cell expansion.

Constitutive interaction between 4-1BB and 4-1BBL on murine LPS-activated bone marrow dendritic cells masks detection of 4-1BBL by TKS-1 but not 19H3 antibody.

4-1BB is a TNFR family member associated with NF-κB mediated survival signaling. 4-1BB is widely expressed on activated cells of the immune system, including activated T cells, NK cells and dendritic cells. Its ligand, 4-1BBL, is transiently expressed on activated antigen presenting cells and at low levels on activated T cells. Although 4-1BBL-deficient mice clearly demonstrate a role for 4-1BBL in CD8 T cell responses to viruses such as influenza, 4-1BBL can be difficult to detect following infection of mice. Here we provide evidence for a constitutive interaction between endogenous 4-1BB and 4-1BBL on LPS activated bone marrow-derived murine dendritic cells that can mask its detection, with implications for measurement of 4-1BBL expression. The masking of 4-1BBL by its receptor results in loss of reactivity to the anti-4-1BBL antibody TKS-1, whereas the 19H3 antibody binds to 4-1BBL in the presence or absence of 4-1BB. Moreover, 4-1BB/4-1BBL interaction can occur in trans between 4-1BB+/+ and 4-1BB-/- dendritic cells in culture. These data suggest that 19H3 is the preferable antibody to use to detect 4-1BBL in the presence of its receptor.

Evidence-based outcomes on diagnostic accuracy of quantitative ultrasound for assessment of pediatric osteoporosis - a systematic review.

BACKGROUND: Dual-energy absorptiometry (DXA) is the current reference standard for assessing pediatric osteoporosis; however due to its areal nature, it has limitations. Thus, quantitative ultrasound (QUS), a modality free of ionizing radiation, has been proposed as a potential surrogate for DXA. OBJECTIVE: To semi-quantitatively assess the diagnostic accuracy of QUS for evaluating pediatric osteoporosis according to the U.S. Preventive Services Task Force guidelines. MATERIALS AND METHODS: We retrieved articles on the diagnostic accuracy of quantitative US for assessing abnormal bone quality or quantity in patients of mean age ≤19 years from MEDLINE, EMBASE and Cochrane Library CCTR databases. Evidences were analyzed for reliability, construct and criterion validity, and responsiveness of quantitative US, according to the following questions: (1) How reliable is the acquisition of QUS measurements? (2) Is QUS diagnostically accurate to characterize bone strength and quality in osteoporotic children? (3) Is QUS sensitive to detect changes in bone status over time? (4) Is QUS able to predict future skeletal fractures/degeneration? Three reviewers independently evaluated the quality of reporting and methodological quality using the Standards for Reporting of Diagnostic Accuracy (STARD) and the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tools. RESULTS: Out of 262 retrieved references (215 unique), we included 28 studies (1,963 patients; 807 reported boys and 761 girls, others unspecified; reported mean age, 0-19 years). The mean quality of reporting score was "excellent" in 24/28 (86%) studies; 11/28 (39%) studies had "adequate" research design quality. CONCLUSION: There is no evidence of the diagnostic value of QUS at the present time despite the overall excellent and adequate research design quality of primary studies. Although QUS can produce reliable measurements, insufficient evidence has been reported to support other clinimetric properties of this technique.

Mutations in Ralstonia solanacearum loci involved in lipopolysaccharide biogenesis, phospholipid trafficking and peptidoglycan recycling render bacteriophage infection.

Ralstonia solanacearum causes deadly wilting on many crops worldwide. However, the information on its components important for cell integrity and interactions with phages is limited. By systematically characterizing mutants resistant to a T7-like phage, we showed that the biosynthesis of rough lipopolysaccharides (R-LPS) was crucial for maintaining the membrane integrity, while the production of smooth LPS (S-LPS) was required for the resistance to polymyxin B and phage adsorption. Furthermore, RSc0154/ampG disruption did not affect LPS production and phage adsorption but may have caused aberrant release of peptidoglycan fragments, thus hindering phage DNA injection into or virion release from the cell. Mutations in the RSc2958-RSc2962/mla cluster, although not affecting LPS production, may have caused elevated phospholipid level in the outer leaflet of the outer membrane, consequently sheltering the mutants from phage adsorption on the O-antigen. These results specify important roles of the biogenesis and homeogenesis of envelope components for R. solanacearum-phage interaction.

Roles of different forms of lipopolysaccharides in Ralstonia solanacearum pathogenesis.

Lipopolysaccharides (LPS) are critical components for the fitness of most gram-negative bacteria. Ralstonia solanacearum causes a deadly wilting disease in many crops; however, the pathogenic roles of different forms of LPS and their pathways of biogenesis remain unknown. By screening for phage-resistant mutants of R. solanacearum Pss4, whose genome sequence is unavailable, mutants with various types of structural defects in LPS were isolated. Pathogenesis assays of the mutants revealed that production of rough LPS (R-LPS), which does not contain O-polysaccharides, was sufficient to cause necrosis on Nicotiana benthamiana and induce the hypersensitive response on N. tabacum. However, biosynthesis of smooth LPS (S-LPS), which contains O-polysaccharides, was required for bacterial proliferation at infection sites on N. benthamiana leaves and for proliferation and causing wilt on tomato. Complementation tests confirmed the involvement of the previously unidentified cluster RSc2201 to RSc2204 in the formation of R. solanacearum S-LPS. With these data and the availability of the annotated genomic sequence of strain GMI1000, certain loci involved in key steps of R. solanacearum LPS biosynthesis were identified. The strategy of this work could be useful for similar studies in other bacteria without available genome sequences.