B cell profiles, antibody repertoire and reactivity reveal dysregulated responses with autoimmune features in melanoma.

B cells are known to contribute to the anti-tumor immune response, especially in immunogenic tumors such as melanoma, yet humoral immunity has not been characterized in these cancers to detail. Here we show comprehensive phenotyping in samples of circulating and tumor-resident B cells as well as serum antibodies in melanoma patients. Memory B cells are enriched in tumors compared to blood in paired samples and feature distinct antibody repertoires, linked to specific isotypes. Tumor-associated B cells undergo clonal expansion, class switch recombination, somatic hypermutation and receptor revision. Compared with blood, tumor-associated B cells produce antibodies with proportionally higher levels of unproductive sequences and distinct complementarity determining region 3 properties. The observed features are signs of affinity maturation and polyreactivity and suggest an active and aberrant autoimmune-like reaction in the tumor microenvironment. Consistent with this, tumor-derived antibodies are polyreactive and characterized by autoantigen recognition. Serum antibodies show reactivity to antigens attributed to autoimmune diseases and cancer, and their levels are higher in patients with active disease compared to post-resection state. Our findings thus reveal B cell lineage dysregulation with distinct antibody repertoire and specificity, alongside clonally-expanded tumor-infiltrating B cells with autoimmune-like features, shaping the humoral immune response in melanoma.

Detection of Critical Spinal Epidural Lesions on CT Using Machine Learning.

BACKGROUND: Critical spinal epidural pathologies can cause paralysis or death if untreated. Although magnetic resonance imaging is the preferred modality for visualizing these pathologies, computed tomography (CT) occurs far more commonly than magnetic resonance imaging in the clinical setting. OBJECTIVE: A machine learning model was developed to screen for critical epidural lesions on CT images at a large-scale teleradiology practice. This model has utility for both worklist prioritization of emergent studies and identifying missed findings. MATERIALS AND METHODS: There were 153 studies with epidural lesions available for training. These lesions were segmented and used to train a machine learning model. A test data set was also created using previously missed epidural lesions. The trained model was then integrated into a teleradiology workflow for 90 days. Studies were sent to secondary manual review if the model detected an epidural lesion but none was mentioned in the clinical report. RESULTS: The model correctly identified 50.0% of epidural lesions in the test data set with 99.0% specificity. For prospective data, the model correctly prioritized 66.7% of the 18 epidural lesions diagnosed on the initial read with 98.9% specificity. There were 2.0 studies flagged for potential missed findings per day, and 17 missed epidural lesions were found during a 90-day time period. These results suggest almost half of critical spinal epidural lesions visible on CT imaging are being missed on initial diagnosis. CONCLUSION: A machine learning model for identifying spinal epidural hematomas and abscesses on CT can be implemented in a clinical workflow.

Enriched circulating and tumor-resident TGF-ß+ regulatory B cells in patients with melanoma promote FOXP3+ Tregs.

B cells are emerging as key players of anti-tumor adaptive immune responses. We investigated regulatory and pro-inflammatory cytokine-expressing B cells in patients with melanoma by flow cytometric intracellular cytokine, CyTOF, transcriptomic, immunofluorescence, single-cell RNA-seq, and B:T cell co-culture analyses. We found enhanced circulating regulatory (TGF-ß+ and PD-L1+) and reduced pro-inflammatory TNF-α+ B cell populations in patients compared with healthy volunteers (HVs), including lower IFN-γ+:IL-4+ and higher TGF-ß+:TNF-α+ B cell ratios in patients. TGF-ß-expressing B cells in the melanoma tumor microenvironment assembled in clusters and interacted with T cells via lymphoid recruitment (SELL, CXCL13, CCL4, CD74) signals and with Tregs via CD47:SIRP-γ, and FOXP3-promoting Galectin-9:CD44. While reduced in tumors compared to blood, TNF-α-expressing B cells engaged in crosstalk with Tregs via TNF-α signaling and the ICOS/ICOSL axis. Patient-derived B cells promoted FOXP3+ Treg differentiation in a TGF-ß-dependent manner, while sustaining expression of IFN-γ and TNF-α by autologous T-helper cells and promoting T-helper cell proliferation ex vivo, an effect further enhanced with anti-PD-1 checkpoint blockade. Our findings reveal cytokine-expressing B cell compartments skewed toward regulatory phenotypes in patient circulation and melanoma lesions, intratumor spatial localization, and bidirectional crosstalk between B and T cell subsets with immunosuppressive attributes.

Innate stimulation of B cells ex vivo enhances antibody secretion and identifies tumour-reactive antibodies from cancer patients.

Human B cells and their expressed antibodies are crucial in conferring immune protection. Identifying pathogen-specific antibodies following infection is possible due to enhanced humoral immunity against well-described molecules on the pathogen surface. However, screening for cancer-reactive antibodies remains challenging since target antigens are often not identified a priori and the frequency of circulating B cells recognizing cancer cells is likely very low. We investigated whether combined ex vivo culture of human B cells with three innate stimuli, interleukin-17 (IL-17), B-cell activation factor (BAFF), and the toll-like receptor 9 (TLR-9) agonist DNA motif CpG ODN 2006 (CpG), each known to activate B cells through different signalling pathways, promote cell activation, proliferation, and antibody production. Combined IL-17+BAFF+CpG prolonged B-cell survival and increased proliferation compared with single stimuli. IL-17+BAFF+CpG triggered higher IgG secretion, likely by activating differentiated, memory and class-switched CD19+CD20+CD27+IgD- B cells. Regardless of anti-FOLR antibody seropositive status, IL-17+BAFF+CpG combined with a monovalent tumour-associated antigen (folate receptor alpha [FOLR]) led to secreted antibodies recognizing the antigen and the antigen-expressing IGROV1 cancer cells. In a seropositive individual, FOLR stimulation favoured class-switched memory B-cell precursors (CD27-CD38-IgD-), class-switched memory B cells and anti-FOLR antibody production, while IL-17+BAFF+CpG combined with FOLR, promoted class-switched memory B-cell precursors and antibody-secreting (CD138+IgD-) plasma cells. Furthermore, IL-17+BAFF+CpG stimulation of peripheral blood B cells from patients with melanoma revealed tumour cell-reactive antibodies in culture supernatants. These findings suggest that innate signals stimulate B-cell survival and antibody production and may help identify low-frequency antigen-reactive humoral responses.

Tensor-Network Codes.

We introduce tensor-network stabilizer codes which come with a natural tensor-network decoder. These codes can correspond to any geometry, but, as a special case, we generalize holographic codes beyond those constructed from perfect or block-perfect isometries, and we give an example that corresponds to neither. Using the tensor-network decoder, we find a threshold of 18.8% for this code under depolarizing noise. We show that, for holographic codes, the exact tensor-network decoder (with no bond-dimension truncation) has polynomial complexity in the number of physical qubits, even for locally correlated noise, making this the first efficient decoder for holographic codes against Pauli noise and, also, a rare example of a decoder that is both efficient and exact.

Tumor-Infiltrating B Lymphocyte Profiling Identifies IgG-Biased, Clonally Expanded Prognostic Phenotypes in Triple-Negative Breast Cancer.

In breast cancer, humoral immune responses may contribute to clinical outcomes, especially in more immunogenic subtypes. Here, we investigated B lymphocyte subsets, immunoglobulin expression, and clonal features in breast tumors, focusing on aggressive triple-negative breast cancers (TNBC). In samples from patients with TNBC and healthy volunteers, circulating and tumor-infiltrating B lymphocytes (TIL-B) were evaluated. CD20+CD27+IgD- isotype-switched B lymphocytes were increased in tumors, compared with matched blood. TIL-B frequently formed stromal clusters with T lymphocytes and engaged in bidirectional functional cross-talk, consistent with gene signatures associated with lymphoid assembly, costimulation, cytokine-cytokine receptor interactions, cytotoxic T-cell activation, and T-cell-dependent B-cell activation. TIL-B-upregulated B-cell receptor (BCR) pathway molecules FOS and JUN, germinal center chemokine regulator RGS1, activation marker CD69, and TNFα signal transduction via NFκB, suggesting BCR-immune complex formation. Expression of genes associated with B lymphocyte recruitment and lymphoid assembly, including CXCL13, CXCR4, and DC-LAMP, was elevated in TNBC compared with other subtypes and normal breast. TIL-B-rich tumors showed expansion of IgG but not IgA isotypes, and IgG isotype switching positively associated with survival outcomes in TNBC. Clonal expansion was biased toward IgG, showing expansive clonal families with specific variable region gene combinations and narrow repertoires. Stronger positive selection pressure was present in the complementarity determining regions of IgG compared with their clonally related IgA in tumor samples. Overall, class-switched B lymphocyte lineage traits were conspicuous in TNBC, associated with improved clinical outcomes, and conferred IgG-biased, clonally expanded, and likely antigen-driven humoral responses. SIGNIFICANCE: Tumor-infiltrating B lymphocytes assemble in clusters, undergoing B-cell receptor-driven activation, proliferation, and isotype switching. Clonally expanded, IgG isotype-biased humoral immunity associates with favorable prognosis primarily in triple-negative breast cancers.

Measurement of Endotracheal Tube Positioning on Chest X-Ray Using Object Detection.

Patients who are intubated with endotracheal tubes often receive chest x-ray (CXR) imaging to determine whether the tube is correctly positioned. When these CXRs are interpreted by a radiologist, they evaluate whether the tube needs to be repositioned and typically provide a measurement in centimeters between the endotracheal tube tip and carina. In this project, a large dataset of endotracheal tube and carina bounding boxes was annotated on CXRs, and a machine-learning model was trained to generate these boxes on new CXRs and to calculate a distance measurement between the tube and carina. This model was applied to a gold standard annotated dataset, as well as to all prospective data passing through our radiology system for two weeks. Inter-radiologist variability was also measured on a test dataset. The distance measurements for both the gold standard dataset (mean error = 0.70 cm) and prospective dataset (mean error = 0.68 cm) were noninferior to inter-radiologist variability (mean error = 0.70 cm) within an equivalence bound of 0.1 cm. This suggests that this model performs at an accuracy similar to human measurements, and these distance calculations can be used for clinical report auto-population and/or worklist prioritization of severely malpositioned tubes.

3D-M3: high-spatial-resolution spectroscopy with extreme AO and 3D-printed micro-lenslets.

By combining integral field spectroscopy with extreme adaptive optics, we are now able to resolve objects close to the diffraction limit of large telescopes, exploring new science cases. We introduce an integral field unit designed to couple light with a minimal plate scale from the SCExAO facility at NIR wavelengths to a single-mode spectrograph. The integral field unit has a 3D-printed micro-lens array on top of a custom single-mode multi-core fiber, to optimize the coupling of light into the fiber cores. We demonstrate the potential of the instrument via initial results from the first on-sky runs at the 8.2 m Subaru Telescope with a spectrograph using off-the-shelf optics, allowing for rapid development with low cost.

Antiviral antibody responses to systemic administration of an oncolytic RNA virus: the impact of standard concomitant anticancer chemotherapies.

BACKGROUND: Oncolytic reovirus therapy for cancer induces a typical antiviral response to this RNA virus, including neutralizing antibodies. Concomitant treatment with cytotoxic chemotherapies has been hypothesized to improve the therapeutic potential of the virus. Chemotherapy side effects can include immunosuppression, which may slow the rate of the antiviral antibody response, as well as potentially make the patient more vulnerable to viral infection. METHOD: Reovirus neutralizing antibody data were aggregated from separate phase I clinical trials of reovirus administered as a single agent or in combination with gemcitabine, docetaxel, carboplatin and paclitaxel doublet or cyclophosphamide. In addition, the kinetics of individual antibody isotypes were profiled in sera collected in these trials. RESULTS: These data demonstrate preserved antiviral antibody responses, with only moderately reduced kinetics with some drugs, most notably gemcitabine. All patients ultimately produced an effective neutralizing antibody response. CONCLUSION: Patients' responses to infection by reovirus are largely unaffected by the concomitant drug treatments tested, providing confidence that RNA viral treatment or infection is compatible with standard of care treatments.

Kinetics and Mechanism of the Gold-Catalyzed Hydroamination of 1,1-Dimethylallene with N-Methylaniline.

The mechanism of the intermolecular hydroamination of 3-methylbuta-1,2-diene (1) with N-methylaniline (2) catalyzed by (IPr)AuOTf has been studied by employing a combination of kinetic analysis, deuterium labelling studies, and in situ spectral analysis of catalytically active mixtures. The results of these and additional experiments are consistent with a mechanism for hydroamination involving reversible, endergonic displacement of N-methylaniline from [(IPr)Au(NHMePh)]+ (4) by allene to form the cationic gold π-C1,C2-allene complex [(IPr)Au(η2 -H2 C=C=CMe2 )]+ (I), which is in rapid, endergonic equilibrium with the regioisomeric π-C2,C3-allene complex [(IPr)Au(η2 -Me2 C=C=CH2 )]+ (I'). Rapid and reversible outer-sphere addition of 2 to the terminal allene carbon atom of I' to form gold vinyl complex (IPr)Au[C(=CH2 )CMe2 NMePh] (II) is superimposed on the slower addition of 2 to the terminal allene carbon atom of I to form gold vinyl complex (IPr)Au[C(=CMe2 )CH2 NMePh] (III). Selective protodeauration of III releases N-methyl-N-(3-methylbut-2-en-1-yl)aniline (3 a) with regeneration of 4. At high conversion, gold vinyl complex II is competitively trapped by an (IPr)Au+ fragment to form the cationic bis(gold) vinyl complex {[(IPr)Au]2 [C(=CH2 )CMe2 NMePh]}+ (6).

Combined anti-PD-1 and anti-CTLA-4 checkpoint blockade: Treatment of melanoma and immune mechanisms of action.

Cytotoxic T-lymphocyte associated protein-4 (CTLA-4) and the Programmed Death Receptor 1 (PD-1) are immune checkpoint molecules that are well-established targets of antibody immunotherapies for the management of malignant melanoma. The monoclonal antibodies, Ipilimumab, Pembrolizumab, and Nivolumab, designed to interfere with T cell inhibitory signals to activate immune responses against tumors, were originally approved as monotherapy. Treatment with a combination of immune checkpoint inhibitors may improve outcomes compared to monotherapy in certain patient groups and these clinical benefits may be derived from unique immune mechanisms of action. However, treatment with checkpoint inhibitor combinations also present significant clinical challenges and increased rates of immune-related adverse events. In this review, we discuss the potential mechanisms attributed to single and combined checkpoint inhibitor immunotherapies and clinical experience with their use.

Four annular structures in a protostellar disk less than 500,000 years old.

Annular structures (rings and gaps) in disks around pre-main-sequence stars have been detected in abundance towards class II protostellar objects that are approximately 1,000,000 years old1. These structures are often interpreted as evidence of planet formation1-3, with planetary-mass bodies carving rings and gaps in the disk4. This implies that planet formation may already be underway in even younger disks in the class I phase, when the protostar is still embedded in a larger-scale dense envelope of gas and dust5. Only within the past decade have detailed properties of disks in the earliest star-forming phases been observed6,7. Here we report 1.3-millimetre dust emission observations with a resolution of five astronomical units that show four annular substructures in the disk of the young (less than 500,000 years old)8 protostar IRS 63. IRS 63 is a single class I source located in the nearby Ophiuchus molecular cloud at a distance of 144 parsecs9, and is one of the brightest class I protostars at millimetre wavelengths. IRS 63 also has a relatively large disk compared to other young disks (greater than 50 astronomical units)10. Multiple annular substructures observed towards disks at young ages can act as an early foothold for dust-grain growth, which is a prerequisite of planet formation. Whether or not planets already exist in the disk of IRS 63, it is clear that the planet-formation process begins in the initial protostellar phases, earlier than predicted by current planet-formation theories11.

The Mechanism of Rhodium-Catalyzed Allylic C-H Amination.

Herein, the mechanism of catalytic allylic C-H amination reactions promoted by Cp*Rh complexes is reported. Reaction kinetics experiments, stoichiometric studies, and DFT calculations demonstrate that the allylic C-H activation to generate a Cp*Rh(π-allyl) complex is viable under mild reaction conditions. The role of external oxidants in the catalytic cycle is elucidated. Quantum mechanical calculations, stoichiometric reactions, and cyclic voltammetry experiments concomitantly support an oxidatively induced reductive elimination process of the allyl fragment with an acetate ligand proceeding through a Rh(IV) intermediate. Stoichiometric oxidation and bulk electrolysis of the proposed π-allyl intermediate are also reported to support these analyses. Lastly, evidence supporting the amination of an allylic acetate intermediate is presented. We show that Cp*Rh(III)2+ behaves as a Lewis acid catalyst to complete the allylic amination reaction.

B Cells in Patients With Melanoma: Implications for Treatment With Checkpoint Inhibitor Antibodies.

The contributions of the humoral immune response to melanoma are now widely recognized, with reports of positive prognostic value ascribed to tumor-infiltrating B cells (TIL-B) and increasing evidence of B cells as key predictors of patient response to treatment. There are disparate views as to the pro- and anti-tumor roles of B cells. B cells appear to play an integral role in forming tumor-associated tertiary lymphoid structures (TLSs) which can further modulate T cell activation. Expressed antibodies may distinctly influence tumor regulation in the tumor microenvironment, with some isotypes associated with strong anti-tumor immune response and others with progressive disease. Recently, B cells have been evaluated in the context of cancer immunotherapy. Checkpoint inhibitors (CPIs), targeting T cell effector functions, have revolutionized the management of melanoma for many patients; however, there remains a need to accurately predict treatment responders. Increasing evidence suggests that B cells may not be simple bystanders to CPI immunotherapy. Mature and differentiated B cell phenotypes are key positive correlates of CPI response. Recent evidence also points to an enrichment in activatory B cell phenotypes, and the contribution of B cells to TLS formation may facilitate induction of T cell phenotypes required for response to CPI. Contrastingly, specific B cell subsets often correlate with immune-related adverse events (irAEs) in CPI. With increased appreciation of the multifaceted role of B cell immunity, novel therapeutic strategies and biomarkers can be explored and translated into the clinic to optimize CPI immunotherapy in melanoma.

Classification of Aortic Dissection and Rupture on Post-contrast CT Images Using a Convolutional Neural Network.

Aortic dissections and ruptures are life-threatening injuries that must be immediately treated. Our national radiology practice receives dozens of these cases each month, but no automated process is currently available to check for critical pathologies before the images are opened by a radiologist. In this project, we developed a convolutional neural network model trained on aortic dissection and rupture data to assess the likelihood of these pathologies being present in prospective patients. This aortic injury model was used for study prioritization over the course of 4 weeks and model results were compared with clinicians' reports to determine accuracy metrics. The model obtained a sensitivity and specificity of 87.8% and 96.0% for aortic dissection and 100% and 96.0% for aortic rupture. We observed a median reduction of 395 s in the time between study intake and radiologist review for studies that were prioritized by this model. False-positive and false-negative data were also collected for retraining to provide further improvements in subsequent versions of the model. The methodology described here can be applied to a number of modalities and pathologies moving forward.

Immune mediator expression signatures are associated with improved outcome in ovarian carcinoma.

Immune and inflammatory cascades may play multiple roles in ovarian cancer. We aimed to identify relationships between expression of immune and inflammatory mediators and patient outcomes. We interrogated differential gene expression of 44 markers and marker combinations (n = 1,978) in 1,656 ovarian carcinoma patient tumors, alongside matched 5-year overall survival (OS) data in silico. Using machine learning methods, we investigated whether genomic expression of these 44 mediators can discriminate between malignant and non-malignant tissues in 839 ovarian cancer and 115 non-malignant ovary samples. We furthermore assessed inflammation markers in 289 ovarian cancer patients' sera in the Swedish Apolipoprotein MOrtality-related RISk (AMORIS) cohort. Expression of the 44 mediators could discriminate between malignant and non-malignant tissues with at least 96% accuracy. Higher expression of classical Th1, Th2, Th17, anti-parasitic/infection and M1 macrophage mediator signatures were associated with better OS. Contrastingly, inflammatory and angiogenic mediators, CXCL-12, C-reactive protein (CRP) and platelet-derived growth factor subunit A (PDGFA) were negatively associated with OS. Of the serum inflammatory markers in the AMORIS cohort, women with ovarian cancer who had elevated levels of haptoglobin (≥1.4 g/L) had a higher risk of dying from ovarian cancer compared to those with haptoglobin levels <1.4 g/L (HR = 2.09, 95% CI:1.38-3.16). Our findings indicate that elevated "classical" immune mediators, associated with response to pathogen antigen challenge, may confer immunological advantage in ovarian cancer, while inflammatory markers appear to have negative prognostic value. These highlight associations between immune protection, inflammation and clinical outcomes, and offer opportunities for patient stratification based on secretome markers.

Combining Immune Checkpoint Inhibitors: Established and Emerging Targets and Strategies to Improve Outcomes in Melanoma.

The immune system employs several checkpoint pathways to regulate responses, maintain homeostasis and prevent self-reactivity and autoimmunity. Tumor cells can hijack these protective mechanisms to enable immune escape, cancer survival and proliferation. Blocking antibodies, designed to interfere with checkpoint molecules CTLA-4 and PD-1/PD-L1 and counteract these immune suppressive mechanisms, have shown significant success in promoting immune responses against cancer and can result in tumor regression in many patients. While inhibitors to CTLA-4 and the PD-1/PD-L1 axis are well-established for the clinical management of melanoma, many patients do not respond or develop resistance to these interventions. Concerted efforts have focused on combinations of approved therapies aiming to further augment positive outcomes and survival. While CTLA-4 and PD-1 are the most-extensively researched targets, results from pre-clinical studies and clinical trials indicate that novel agents, specific for checkpoints such as A2AR, LAG-3, IDO and others, may further contribute to the improvement of patient outcomes, most likely in combinations with anti-CTLA-4 or anti-PD-1 blockade. This review discusses the rationale for, and results to date of, the development of inhibitory immune checkpoint blockade combination therapies in melanoma. The clinical potential of new pipeline therapeutics, and possible future therapy design and directions that hold promise to significantly improve clinical prognosis compared with monotherapy, are discussed.

Selective middle cerebral artery occlusion in the rabbit: Technique and characterization with pathologic findings and multimodal MRI.

BACKGROUND: A reliable animal model of ischemic stroke is vital for pre-clinical evaluation of stroke therapies. We describe a reproducible middle cerebral artery (MCA) embolic occlusion in the French Lop rabbit characterized with multimodal MRI and histopathologic tissue analysis. NEW METHOD: Fluoroscopic-guided microcatheter placement was performed in five consecutive subjects with angiographic confirmation of MCA occlusion with autologous clot. Multimodal MRI was obtained prior to occlusion and up to six hours post after which repeat angiography confirmed sustained occlusion. The brain was harvested for histopathologic examination. RESULTS: Angiography confirmed successful MCA catheterization and durable (>6 h) MCA occlusion in all animals. There was increase of ADC volume over time and variable final core volume presumably related to individual variation in collateral flow. FLAIR hyperintensity indicative of cytotoxic edema and parenchymal contrast enhancement reflective of blood brain barrier disruption was observed over time. Tissue staining of the ischemic brain showed edema and structural alterations consistent with infarction. COMPARISON WITH EXISTING METHODS: This study describes a technique of selective catheterization and embolic occlusion of the MCA in the rabbit with MRI characterization of evolution of ischemia in the model. CONCLUSIONS: We demonstrate the feasibility of a rabbit model of embolic MCA occlusion with angiographic documentation. Serial MR imaging demonstrated changes comparable to those observed in human ischemic stroke, confirmed histopathologically.

Inroads through the bacterial cell envelope: seeing is believing.

A singular feature of all prokaryotic cells is the presence of a cell envelope composed of a cytoplasmic membrane and a cell wall. The introduction of bacterial cell fractionation techniques in the 1950s and 1960s along with developments in procedures for electron microscopy opened the window towards an understanding of the chemical composition and architecture of the cell envelope. This review traces the contribution of Terry Beveridge in these endeavours, beginning with his doctoral studies in the 1970s on the structure of paracrystalline surface arrays (S-layers), followed by an exploration of cryogenic methods for preserving bacteria for ultrastructural analyses. His insights are reflected in a current example of the contribution of cryo-electron microscopy to S-layer studies - the structure and assembly of the surface array of Caulobacter crescentus. The review then focuses on Terry's contributions to imaging the ultrastructure of bacterial cell envelopes and to the development of cryo-electron microscopy techniques, including the use of CEMOVIS (Cryo-electron Microscopy of Vitreous Sections) to "see" the ultrastructure of the Gram-positive cell envelope - his last scientific endeavour.