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Supplementary Figure 1 from CD47 Regulates Bone Mass and Tumor Metastasis to Bone

Supplementary Figure Legends 1-2 from CD47 Regulates Bone Mass and Tumor Metastasis to Bone

Supplementary Figure 1 from Senescent Stromal-Derived Osteopontin Promotes Preneoplastic Cell Growth

Alterations in the tissue microenvironment collaborate with cell autonomous genetic changes to contribute to neoplastic progression. The importance of the microenvironment in neoplastic progression is underscored by studies showing that fibroblasts isolated from a tumor stimulate the growth of preneoplastic and neoplastic cells in xenograft models. Similarly, senescent fibroblasts promote preneoplastic cell growth in vitro and in vivo. Because senescent cells accumulate with age, their presence is hypothesized to facilitate preneoplastic cell growth and tumor formation in older individuals. To identify senescent stromal factors directly responsible for stimulating preneoplastic cell growth, we carried out whole-genome transcriptional profiling and compared senescent fibroblasts with their younger counterparts. We identified osteopontin (OPN) as one of the most highly elevated transcripts in senescent fibroblasts. Importantly, reduction of OPN protein levels by RNA interference did not affect senescence induction in fibroblasts; however, it dramatically reduced the growth-promoting activities of senescent fibroblasts in vitro and in vivo, showing that OPN is necessary for paracrine stimulation of preneoplastic cell growth. In addition, we found that recombinant OPN was sufficient to stimulate preneoplastic cell growth. Finally, we show that OPN is expressed in senescent stroma within preneoplastic lesions that arise following 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate treatment of mice, suggesting that stromal-derived OPN-mediated signaling events affect neoplastic progression. [Cancer Res 2009;69(3):1230–9]

CD47, also called integrin-associated protein, plays a critical role in the innate immune response and is an atypical member of the immunoglobulin superfamily that interacts with and activates β3 integrins. β3 integrin−/− mice have defective platelet and osteoclast function and are protected from bone metastasis. The role of CD47 in skeletal homeostasis and bone metastasis has not been described. CD47−/− mice had increased bone mass and defective osteoclast function in vivo. Although the number of functional osteoclasts formed by differentiating CD47−/− bone marrow macrophages was decreased, high doses of RANKL rescued differentiation and function of CD47−/− osteoclasts ex vivo and rescued the osteoclast defect in CD47−/− mice. Inhibition of nitric oxide (NO) synthase, which is expressed at higher levels in CD47−/− osteoclasts, also rescued the osteoclast defect in CD47−/− cells. We then examined the consequences of this osteoclast defect in bone metastasis. In a model of tumor metastasis to bone, bone tumor burden was decreased in the CD47−/− mice compared with wild-type (WT) controls, with no decrease in s.c. tumor growth in CD47−/− mice. There was decreased tumor-associated bone destruction in the CD47−/− mice compared with WT controls, consistent with a defect in osteoclast function that was not rescued by the presence of tumor. Our data show that CD47 regulates osteoclastogenesis, in part, via regulation of NO production, and its disruption leads to a decrease in tumor bone metastasis. CD47 is a novel therapeutic target to strengthen bone mass and diminish metastatic tumor growth in bone. [Cancer Res 2009;69(7):3196–204]

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Supplementary Figure 2 from Senescent Stromal-Derived Osteopontin Promotes Preneoplastic Cell Growth

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Supplementary Figure 2 from CD47 Regulates Bone Mass and Tumor Metastasis to Bone

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Aims Risk factors for asymptomatic echocardiographic abnormalities that predict symptomatic heart failure (HF) may provide insight into early mechanisms of HF pathogenesis. We examined risk factors associated with asymptomatic echocardiographic structural, systolic, and diastolic abnormalities, separately and in combination, and interactions between risk factors, in the prospective community‐based SCReening Evaluation of the Evolution of New HF (SCREEN‐HF) Study cohort of 3190 participants at increased risk of cardiovascular disease. Methods and results Inclusion criteria were age ≥ 60 years with one or more of hypertension, diabetes, ischaemic heart disease, valvular heart disease, abnormal heart rhythm, cerebrovascular disease, or renal impairment. Exclusion criteria were known HF, ejection fraction mild valve abnormality. Structural, systolic, and diastolic echocardiographic abnormalities were defined according to the Atherosclerosis Risk in Communities study criteria, and risk factors for asymptomatic structural, systolic, and diastolic abnormalities were identified using logistic regression analysis. In multivariable analysis, increased body mass index (BMI), non‐steroidal anti‐inflammatory drug therapy, and alcohol intake were risk factors for isolated structural abnormality, whereas male gender, increased heart rate, atrial fibrillation (AF), angiotensin‐converting enzyme inhibitor therapy, and obstructive sleep apnoea were associated with a lower risk. Moreover, male gender, smoking, increased systolic blood pressure, and physical inactivity were risk factors for isolated systolic abnormality, whereas increased pulse pressure and antihypertensive therapy were associated with a lower risk. Furthermore, increased age, blood pressure, amino‐terminal pro‐B‐type natriuretic peptide level, and warfarin therapy (associated with AF) were risk factors for isolated diastolic abnormality, whereas increased heart rate and triglyceride level (associated with BMI) were associated with a lower risk. The association of increased heart rate with lower risk of structural and diastolic abnormalities was independent of β‐blocker therapy. Interactions between risk factors differed for structural, systolic, and diastolic abnormalities. Conclusions The different risk factors for asymptomatic structural, systolic, and diastolic abnormalities that predict symptomatic HF, and the interactions between risk factors, illustrate how these structural, systolic, and diastolic abnormalities represent unique trajectories that lead to symptomatic HF. Improved understanding of these trajectories may assist in the design of HF prevention strategies.

Purpose Physical exercise is shown to mitigate catecholamine metabolites; however, it is unknown if exercise-induced increases in sympatho-adrenal activity or catecholamine metabolites are influenced by ingestion of specific catechins found within green tea. This study explored the impact of epigallocatechin gallate (EGCG) ingestion on catecholamine metabolism during graded cycle exercise in humans. Methods Eight males (22.4 ± 3.3 years, BMI:25.7 ± 2.4 kg.m2) performed a randomised, placebo-controlled, single-blind, cross-over trial after consumption (1450 mg) of either EGCG or placebo (PLAC) and performed graded cycling to volitional exhaustion. Venous bloods were taken at rest, 2 h post-ingestion and after every 3-min stage. Blood variables were analysed for catecholamines, catecholamine metanephrines and metabolic variables at rest, 2 h post-ingestion (POST-ING), peak rate of lipid oxidation (FATpeak), lactate threshold (LT) and peak rate of oxygen consumption (VO2peak). Data were analysed using SPSS (Version 26). Results Resting catecholamine and metanephrines were similar between trials. Plasma adrenaline (AD) was lower in ECGC treatment group between trials at FATpeak (P P VO2peak (P P P P VO2peak (P −1, P Conclusion Acute EGCG supplementation reduced circulating catecholamines but not; metanephrine, glucose or lactates, response to graded exercise. Lower circulating catecholamines may explain a lower lipid oxidation rate.

In metastatic breast cancer, HER2 activating mutations frequently co-occur with mutations in thePIK3CA,TP53, or E-cadherin genes. Of these co-occurring mutations,HER2andPIK3CAmutations are the most prevalent gene pair, with approximately 40% ofHER2mutated breast cancers also having activating mutations inPIK3CA. To study the effects of co-occurringHER2andPIK3CAmutations, we bred genetically engineered mice with theHER2V777L;PIK3CAH1047Rtransgenes (HP mice) and studied the resulting breast cancers bothin vivoas well asex vivousing cancer organoids. HP breast cancers show accelerated tumor formationin vivoand increased invasion and migration inin vitroassays. HP breast cancers have resistance to the pan-HER tyrosine kinase inhibitor, neratinib, but are effectively treated by neratinib plus trastuzumab deruxtecan. Proteomic and RNA-Seq analysis of HP breast cancers showed increased gene expression of Cyclin D1 and p21WAF1/Cip1 and changes in cell cycle markers. Combining neratinib with CDK4/6 inhibitors was another effective strategy for HP breast cancers with neratinib plus palbociclib showing a statistically significant reduction in mouse HP tumors as compared to either drug alone. We validated both the neratinib plus trastuzumab deruxtecan and neratinib plus palbociclib combinations using a human breast cancer patient-derived xenograft that has very similar HER2 andPIK3CAmutations. Both of these drug combinations are being tested in phase 1 clinical trials and this study provides valuable preclinical evidence for them.

Pulmonary hypertension (PH) is a debilitating and potentially life threatening condition in which increased pressure in the pulmonary arteries may result from a variety of pathological processes. These can include disease primarily involving the pulmonary vasculature, but more commonly PH may result from left-sided heart disease, including valvular heart disease. Chronic thromboembolic pulmonary hypertension (CTEPH) is an important disease to identify because it may be amenable to surgical pulmonary artery endarterectomy or balloon pulmonary angioplasty. Parenchymal lung diseases are also widespread in the community. Any of these disease processes may result in adverse remodeling of the right ventricle and progressive right heart (RH) failure as a common final pathway. Because of the breadth of pathological processes which cause PH, multiple imaging modalities play vital roles in ensuring accurate diagnosis and classification, which will lead to application of the most appropriate therapy. Multimodality imaging may also provide important prognostic information and has a role in the assessment of response to therapies which ultimately dictate clinical outcomes. This review provides an overview of the wide variety of established imaging techniques currently in use, but also examines many of the novel imaging techniques which may be increasingly utilized in the future to guide comprehensive care of patients with PH.

Aims We investigated long-term mortality associated with changes in left ventricular ejection fraction (LVEF) in a large, real-world patient cohort. Methods and results A total of 117 275 adults (63 ± 16 years, 46% women) had LVEF quantified by the same method ≥6 months apart. This included 17 343 cases (66 ± 15 years, 48% women) being initially investigated for heart failure (HF). During 3.3 [interquartile range (IQR) 1.7–6.0] years from first to last echocardiogram, median change in LVEF was −1 (IQR −8 to +5) units from a baseline of 62% (IQR 54–69%). During subsequent 7.6 (IQR 4.3–10.1) years of follow-up, 11 397 (9.7%) and 34 101 (29.1%) cases died from cardiovascular disease and all causes, respectively. Actual 5-year, all-cause mortality increased from 12% to 29% among those with the smallest to the largest decrease in LVEF (from 30 units); the adjusted risk of cardiovascular-related mortality increased two- to eightfold beyond a >10-unit decline in LVEF (vs. minimal change; P 30-unit increase to >30-unit decline in LVEF (vs. minimal change; P

The sugars streptose and dihydrohydroxystreptose (DHHS) are unique to the bacteria Streptomyces griseus and Coxiella burnetii, respectively. Streptose forms the central moiety of the antibiotic streptomycin, while DHHS is found in the O-antigen of the zoonotic pathogen C. burnetii. Biosynthesis of these sugars has been proposed to follow a similar path to that of TDP-rhamnose, catalyzed by the enzymes RmlA, RmlB, RmlC, and RmlD, but the exact mechanism is unclear. Streptose and DHHS biosynthesis unusually requires a ring contraction step that could be performed by orthologs of RmlC or RmlD. Genome sequencing of S. griseus and C. burnetii has identified StrM and CBU1838 proteins as RmlC orthologs in these respective species. Here, we demonstrate that both enzymes can perform the RmlC 3’’,5’’ double epimerization activity necessary to support TDP-rhamnose biosynthesis in vivo. This is consistent with the ring contraction step being performed on a double epimerized substrate. We further demonstrate that proton exchange is faster at the 3’’-position than the 5’’-position, in contrast to a previously studied ortholog. We additionally solved the crystal structures of CBU1838 and StrM in complex with TDP and show that they form an active site highly similar to those of the previously characterized enzymes RmlC, EvaD, and ChmJ. These results support the hypothesis that streptose and DHHS are biosynthesized using the TDP pathway and that an RmlD paralog most likely performs ring contraction following double epimerization. This work will support the elucidation of the full pathways for biosynthesis of these unique sugars.

Purpose To examine: (1) the rate of clinical events precluding cardiac rehabilitation (CR) continuation, (2) CR attendance by component in those without events, and (3) the association between disease severity (eg, tobacco use, diabetes, and depression) and component attendance (eg, exercise, diet, stress management, and tobacco cessation). Methods Retrospective analysis of electronic records of the CR program in London, Ontario, from 1999 to 2017. Patients in the supervised program are offered exercise sessions 2 times/wk with a minimum of 48 prescribed sessions tailored to patient need. Patients attending ≥1 session without major factors that would limit their exercise ability were included. Intervening events were recorded, as was component attendance. Results Of 5508 enrolled, supervised patients, 3696 did not have a condition that could preclude exercise. Of those enrolled, one-sixth (n = 912) had an intervening event; these patients were less likely to work, more likely to have medical risk factors, had more severe angina and depression, and lower functional capacity. The remaining cohort attended a mean of 26.5 ± 21.3 sessions overall (median = 27; 19% attending ≥48 sessions), including 20.5 ± 17.4 exercise sessions (median = 21). After exercise, the most common components attended were individual dietary and psychological counseling. Patients with more severe angina and depressive symptoms as well as tobacco users attended significantly fewer total sessions, but more of some specific components. Conclusions In one-sixth of patients, CR attendance and completion are impacted by clinical factors beyond their control. Many patients are taking advantage of components specific to their risk factors, buttressing the value of individually tailored, menu-based programming.

Rationale: Multiple myeloma (MM) is a multifocal malignancy of bone marrow plasma cells, characterized by vicious cycles of remission and relapse that eventually culminate in death. The disease remains mostly incurable largely due to the complex interactions between the bone microenvironment (BME) and MM cells (MMC). In the “vicious cycle” of bone disease, abnormal activation of osteoclasts (OCs) by MMC causes severe osteolysis, promotes immune evasion, and stimulates the growth of MMC. Disrupting these cancer-stroma interactions would enhance treatment response. Methods: To disrupt this cycle, we orthogonally targeted nanomicelles (NM) loaded with non-therapeutic doses of a photosensitizer, titanocene (TC), to VLA-4 (α4ß1, CD49d/CD29) expressing MMC (MM1.S) and αvß3 (CD51/CD61) expressing OC. Concurrently, a non-lethal dose of a radiopharmaceutical, 18F-fluorodeoxyglucose ([18F]FDG) administered systemically interacted with TC (radionuclide stimulated therapy, RaST) to generate cytotoxic reactive oxygen species (ROS). The in vitro and in vivo effects of RaST were characterized in MM1.S cell line, as well as in xenograft and isograft MM animal models. Results: Our data revealed that RaST induced non-enzymatic hydroperoxidation of cellular lipids culminating in mitochondrial dysfunction, DNA fragmentation, and caspase-dependent apoptosis of MMC using VLA-4 avid TC-NMs. RaST upregulated the expression of BAX, Bcl-2, and p53, highlighting the induction of apoptosis via the BAK-independent pathway. The enhancement of multicopper oxidase enzyme F5 expression, which inhibits lipid hydroperoxidation and Fenton reaction, was not sufficient to overcome RaST-induced increase in the accumulation of irreversible function-perturbing α,ß-aldehydes that exerted significant and long-lasting damage to both DNA and proteins. In vivo, either VLA-4-TC-NM or αvß3-TC-NMs RaST induced a significant therapeutic effect on immunocompromised but not immunocompetent MM-bearing mouse models. Combined treatment with both VLA-4-TC-NM and αvß3-TC-NMs synergistically inhibited osteolysis, reduced tumor burden, and prevented rapid relapse in both in vivo models of MM. Conclusions: By targeting MM and bone cells simultaneously, combination RaST suppressed MM disease progression through a multi-prong action on the vicious cycle of bone cancer. Instead of using the standard multidrug approach, our work reveals a unique photophysical treatment paradigm that uses nontoxic doses of a single light-sensitive drug directed orthogonally to cancer and bone cells, followed by radionuclide-stimulated generation of ROS to inhibit tumor progression and minimize osteolysis in both immunocompetent murine and immunocompromised human MM models.

AIMS We investigated the sex-based risk of mortality across the spectrum of left ventricular ejection fraction (LVEF) in a large cohort of patients in Australia. METHODS AND RESULTS Quantified levels of LVEF from 237 046 women (48.1%) and 256 109 men undergoing first-time, routine echocardiography (2000-2019) were linked to 119 232 deaths (median 5.6 years of follow-up). Overall, 17.6% of men vs. 8.3% of women had an LVEF

Musculoskeletal pain is the greatest cause of disability worldwide. Owing to its increasing prevalence and burden, the importance of affordable treatments has been highlighted. Text message interventions are accessible, low cost, and effective in promoting healthy behaviour and managing chronic diseases. However, little is known about their role in musculoskeletal pain. This systematic review was conducted to appraise the literature on the effects of text messages (as an intervention or a component of an intervention) compared with any control on pain and function in people with musculoskeletal pain (PROSPERO: CRD42018117371). MEDLINE, EMBASE, CINAHL, Cochrane, and PEDro databases were searched from inception to April 2020. Keywords relating to musculoskeletal pain, text messages, and randomised controlled trials were combined. Methodological quality was assessed using the PEDro score. Of the 12,022 studies identified, 11 were included, with a mean PEDro score of 5.4/10 points (SD 1.3). Pooled analyses were not performed because of heterogeneity of interventions and clinical characteristics. When text messages were added to and compared with usual care, some positive effects were found only on treatment adherence. Although small and inconsistent, some positive effects were reported for pain intensity, function, care-seeking behaviour, adherence, and quality of life when text messages were added to multicomponent interventions. Moreover, text message and telephone counselling interventions had similar effects on function. Overall included studies were of limited methodological quality and heterogeneous. However, our results indicate potential benefits of text messages in the treatment of musculoskeletal pain, which need to be confirmed in future trials.

Aim Heart failure (HF) incidence increases markedly with age. We examined age‐associated longitudinal change in cardiac structure and function, and their prediction by age and cardiovascular disease (CVD) risk factors, in a community‐based cohort aged ≥60 years at increased CVD risk but without HF. Methods and results CVD risk factors were recorded in 3065 participants who underwent a baseline echocardiographic examination, of whom 2358 attended a follow‐up examination 3.8 [median, inter‐quartile range (IQR) 3.5, 4.2] years later. Median age was 71 (IQR 67, 76) years and 55% of participants were male. Age was associated with longitudinal increase in left ventricular (LV) mass index (LVMI); decrease in LV volumes; increase in LV ejection fraction; decrease in mitral annular systolic velocity; decrease in diastolic function (decreased mitral early diastolic annular velocity (e′); and increase in left atrial volume index, mitral peak early diastolic flow velocity (E)/e′ ratio, and tricuspid regurgitant velocity (TRVmax) in men and women, except for TRVmax in men). In multivariable analysis, longitudinal increase in LVMI was explained by CVD risk factors alone, whereas age, together with CVD risk factors, independently predicted longitudinal change in all other echocardiographic parameters. CVD risk factors were differentially associated with longitudinal change in different echocardiographic parameters. Conclusions Whereas the increase in LVMI with age was explained by CVD risk factors alone, age, together with risk factors, independently predicted longitudinal change in all other echocardiographic parameters, providing evidence for age‐specific mechanisms of change in cardiac structure and function as people age. Age‐associated change in LVMI, LV volumes, and diastolic function resembled what might be expected for the evolution of HF with preserved ejection fraction. Given the differential association of different CVD risk factors with longitudinal change in different echocardiographic parameters, therapies aimed at attenuation of age‐associated change in cardiac structure and function, and HF evolution, will likely need to address multiple CVD risk factors.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a group of cardiomyopathies associated with ventricular arrhythmias predominantly arising from the right ventricle, sudden cardiac death and right ventricular failure, caused largely due to inherited mutations in proteins of the desmosomal complex. Whilst long recognised as a cause of sudden cardiac death (SCD) during exercise, it has recently been recognised that intense and prolonged exercise can worsen the disease resulting in earlier and more severe phenotypic expression. Changes in cardiac structure and function as a result of exercise training also pose challenges with diagnosis as enlargement of the right ventricle is commonly seen in endurance athletes. Advice regarding restriction of exercise is an important part of patient management, not only of those with established disease, but also in individuals known to carry gene mutations associated with development of ARVC.

Background Bariatric surgery is an effective treatment for morbid obesity and glycaemic dysfunction. Objectives The aim of the work was to examine both the static and dynamic changes of glucose-insulin homeostasis and incretin hormone response following sleeve gastrectomy (SG) in a sample of 55 participants preoperatively and 1 month and 6 months postoperatively. The focus was on a sample of patients with impaired glucose tolerance and type 2 diabetes (T2D). Setting Morriston Hospital, UK. Methods Prospective study comprising of 55 participants with impaired glucose homeostasis and T2D undergoing SG (mean body mass index [BMI] 50.4 kg/m2, mean glycated haemoglobin [A1C] 7.4%). Serial measurements of glucose, insulin, C-peptide, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic hormone (GIP) were performed during oral glucose tolerance testing preoperatively and 1 and 6 months postoperatively. Areas under the curve (AUC) were examined at 30, 60, and 120 min. Results We observed significant improvements in measures of obesity, as well as static and dynamic measures of glucose, insulin, C-peptide and HOMA. Furthermore, significant increases in GLP-1 response as early as 6 months postoperatively were also seen. Conclusions To our knowledge, no study has examined the detailed dynamic changes in glucose and insulin homeostasis in this number of participants undergoing SG in relation to incretin hormones GIP and GLP-1. This current study supports the role of SG for the treatment of obesity-related glucose dysregulation.

In metastatic breast cancer, HER2 activating mutations frequently co-occur with mutations in the PIK3CA, TP53, or E-cadherin genes. Of these co-occurring mutations, HER2 and PIK3CA mutations are the most prevalent gene pair, with approximately 40% of HER2 mutated breast cancers also having activating mutations in PIK3CA. To study the effects of co-occurring HER2 and PIK3CA mutations, we bred genetically engineered mice with the (loxP-STOP-loxP) HER2V777L; PIK3CAH1047R transgenes (HP mice) and studied the resulting breast cancers both in vivo as well as ex vivo using breast cancer organoids. HP mice rapidly developed invasive mammary adenocarcinoma at a median time of 2.1 weeks after adenoviral Cre injection into the mammary gland. Organoids from these breast cancers showed increased number of buddings in branching morphogenesis assay and increased migration and invasion in vitro. In vivo, HP breast cancers are resistance to the pan-HER tyrosine kinase inhibitor, neratinib, but are effectively treated by the combination of neratinib plus trastuzumab deruxtecan (T-DXd). Ex vivo, we found strong synergy between neratinib and T-DXd in HP organoids. Proteomic and RNA-seq analysis of HP breast cancers showed increased gene expression of CCND1 (cyclin D1) and CDKN1A (which encodes p21WAF1/Cip1) and changes in cell cycle markers. An increase in p-p53, p-p27, and p-PDK1 in HP organoids was seen. The GSEA analysis showed that the mTOR pathway and the MYC target signature were significantly upregulated in the HP organoid group. As p21 stabilizes the cyclin D1-CDK4/6 complex to further activate CDK4/6, we found CDK4/6 inhibitors inhibit cell proliferation in HP mice-derived organoids. Combining neratinib with CDK4/6 inhibitors was another effective strategy for HP breast cancers with neratinib plus palbociclib showing a statistically significant reduction in mouse HP tumors as compared to either drug alone. We validated both the neratinib plus T-DXd and neratinib plus palbociclib combinations using a human breast cancer patient-derived xenograft that has HER2 and PIK3CA mutations very similar to our transgenic mouse. This study provides valuable preclinical evidence for these drug combinations, which are being tested in phase 1 clinical trials. Citation Format: Xiaoqing Cheng, Yirui Sun, Maureen Highkin, Nagalaxmi Vemalapally, Xiaohua Jin, Brandon Zhou, Julie L. Prior, Ashley R. Tipton, Shunqiang Li, Anton Iliuk, Samuel Achilefu, Ian S. Hagemann, John Edwards, Ron Bose. Breast cancer mutations HER2V777L and PIK3CAH1047R activate the p21-CDK4/6 -Cyclin D1 axis driving tumorigenesis and drug resistance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5778.

Despite improvement in treatment options for myeloma patients, including targeted immunotherapies, multiple myeloma remains a mostly incurable malignancy. High CS1 (SLAMF7) expression on myeloma cells and limited expression on normal cells makes it a promising target for CAR-T therapy. The CS1 protein has two extracellular domains – the distal Variable (V) domain and the proximal Constant 2 (C2) domain. We generated and tested CS1-CAR-T targeting the V domain of CS1 (Luc90-CS1-CAR-T) and demonstrated anti-myeloma killing in vitro and in vivo using two mouse models. Since fratricide of CD8 + cells occurred during production, we generated fratricide resistant CS1 deficient Luc90- CS1- CAR-T (ΔCS1-Luc90- CS1- CAR-T). This led to protection of CD8 + cells in the CAR-T cultures, but had no impact on efficacy. Our data demonstrate targeting the distal V domain of CS1 could be an effective CAR-T treatment for myeloma patients and deletion of CS1 in clinical production did not provide an added benefit using in vivo immunodeficient NSG preclinical models.

Melioidosis caused by the facultative intracellular pathogen Burkholderia pseudomallei is difficult to treat due to poor intracellular bioavailability of antibiotics and antibiotic resistance. In the absence of novel compounds, polymersome (PM)-encapsulation may increase the efficacy of existing antibiotics and reduce antibiotic resistance by promoting targeted, infection-specific intracellular uptake. In this study we developed PMs composed of widely available polyethylene oxide-polycaprolactone (PEO-PCL) block co-polymers and demonstrated their delivery to intracellular B. thailandensis infection using multispectral imaging flow cytometry (IFC) and coherent anti-Stokes Raman scattering (CARS) microscopy. Antibiotics were tightly sequestered in PMs and did not inhibit the growth of free-living B. thailandensis. However, on uptake of antibiotic-loaded PMs by infected macrophages, IFC demonstrated PM co-localisation with intracellular B. thailandensis and a significant inhibition of their growth. We conclude that PMs are a viable approach for the targeted antibiotic treatment of persistent intracellular Burkholderia infection.

The sugars streptose and dihydrohydroxystreptose (DHHS) are unique to the bacteria Streptomyces griseus and Coxiella burnetii respectively. Streptose forms the central moiety of the antibiotic streptomycin, whilst DHHS is found in the O-antigen of the zoonotic pathogen C. burnetii. Biosynthesis of these sugars has been proposed to follow a similar path to that of TDP-rhamnose, catalysed by the enzymes RmlA/RmlB/RmlC/RmlD. Streptose and DHHS biosynthesis unusually require a ring contraction step that might be performed by the orthologues of RmlC or RmlD. Genome sequencing of S. griseus and C. burnetii proposed the StrM and CBU1838 proteins respectively as RmlC orthologues. Here, we demonstrate through both coupled and direct observation studies that both enzymes can perform the RmlC 3’’,5’’ double epimerisation activity; and that this activity supports TDP-rhamnose biosynthesis in vivo. We demonstrate that proton exchange is faster at the 3’’ position than the 5’’ position, in contrast to a previously studied orthologue. We solved the crystal structures of CBU1838 and StrM in complex with TDP and show that they form an active site highly similar to previously characterised enzymes. These results further support the hypothesis that streptose and DHHS are biosynthesised using the TDP pathway and are consistent with the ring contraction step being performed on a double epimerised substrate, most likely by the RmlD paralogue. This work will support the determination of the full pathways for streptose and DHHS biosynthesis.

Melioidosis is a neglected tropical disease caused by the bacterium Burkholderia pseudomallei. The bacterium is intrinsically resistant to various antibiotics, and melioidosis is therefore difficult to treat successfully without a relapse in infection. B. pseudomallei is an intracellular pathogen and therefore, to eradicate the infection, antimicrobials must be able to access bacteria in an intracellular niche. This study assessed the ability of a panel of monoclonal antibodies (MAbs) to opsonize Burkholderia species and determine the effect that each antibody has on bacterial virulence in vitro. Murine macrophage infection assays demonstrated that monoclonal antibodies against the capsule of B. pseudomallei are opsonizing. Furthermore, one of these monoclonal antibodies reduced bacterial actin tail formation in our in vitro assays, indicating that antibodies could reduce the intracellular spread of Burkholderia thailandensis. The data presented in this paper demonstrate that monoclonal antibodies are opsonizing and can decrease bacterial actin tail formation, thus decreasing their intracellular spread. These data have informed selection of an antibody for development of an antibody-antibiotic conjugate (AAC) for melioidosis. IMPORTANCE Melioidosis is difficult to treat successfully due to the causal bacterium being resistant to many classes of antibiotics, therefore limiting available therapeutic options. New and improved therapies are urgently required to treat this disease. Here, we have investigated the potential of monoclonal antibodies to target this intracellular pathogen. We have demonstrated that monoclonal antibodies can target the bacterium, increase uptake into macrophages, and reduce actin tail formation required by the bacterium for spread between cells. Through targeting the bacterium with antibodies, we hope to disarm the pathogen, reducing the spread of infection. Ultimately, we aim to use an opsonizing antibody to deliver antibiotics intracellularly by developing an antibody-antibiotic conjugate therapeutic for melioidosis.

Congestive Heart Failure (CHF) is an emerging epidemic. Within one generation, the medical community has learned much of CHF syndromes. It has two distinct mechanisms, systolic and diastolic abnormalities, to account for the common CHF presentation. It is complex as it challenges the available health care services, resource, and funding models in providing an equitable service across the health continuum. Despite the improvement in many cardiovascular diseases, some CHF outcomes like readmissions and costs have increased. The reinvigoration of evidence- based medicine, the development of health services models of care, and standardisation of disease processes with taxonomies have also occurred within the same time span. These processes, however, need to be linked with health policy as presented in white papers. In this paper, we explore achieving optimal CHF guideline-recommended outcomes as the science approaches realworld translation.

Producción Científica, Murine dendritic cells, when pulsed with heat-killed Burkholderia pseudomallei and used to immunise naïve mice, have previously been shown to induce protective immunity in vivo. We have now demonstrated the in vitro priming of naïve human T cells against heat-killed B. pseudomallei, by co-culture with syngeneic B. pseudomallei-pulsed dendritic cells. Additionally, we have enriched the DC fraction such that a study of the differential response induced by pulsed DCs of either myeloid or plasmacytoid lineage in syngeneic human T cells was achievable. Whilst both mDCs and pDCs were activated by pulsing, the mDCs contributed the major response to B. pseudomallei with the expression of the migration marker CCR7 and a significantly greater secretion of the proinflammatory TNFα and IL1β. When these DC factions were combined and used to prime syngeneic T cells, a significant proliferation was observed in the CD4+ fraction. Here, we have achieved human T cell priming in vitro with unadjuvanted B. pseudomallei, the causative organism of melioidosis, for which there is currently no approved vaccine. We propose that the approach we have taken could be used to screen for the human cellular response to candidate vaccines and formulations, in order to enhance the cell-mediated immunity required to protect against this intracellular pathogen and potentially more broadly against other, difficult-to-treat intracellular pathogens. To date, the polysaccharide capsule of B. pseudomallei, fused to a standard carrier protein, e.g., Crm, looks a likely vaccine candidate. Dendritic cells (DCs), providing, as they do, the first line of defence to infection, process and present microbial products to the immune system to direct downstream immune responses. Here, we have sought to use DCs ex vivo to identify immunogenic products from heat-killed B. pseudomallei. Using practical volumes of fresh human donor blood, we show that heat-killed B. pseudomallei activated and stimulated the expression of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 from both myeloid and plasmacytoid DCs. Furthermore, B. pseudomallei-pulsed DCs cultured with naïve syngeneic T cells ex vivo, induced the activation and proliferation of the CD4+ T-cell population, which was identified by cell surface marker staining using flow cytometry. Thus, both DC subsets are important for driving primary T helper cell responses to B. pseudomallei in healthy individuals and have the potential to be used to identify immunogenic components of B. pseudomallei for future therapies and vaccines., Ministerio de Defensa de Reino Unido - Defence Science and Technology Laboratory (Dstl) - (grant DSTL/AGR/00254/01)

Background Previous studies have examined changes in plasma markers of inflammation and oxidative stress up to 24 months following bariatric surgery, but there is limited evidence on the long-term effects of bariatric surgery. Objectives To examine the effects of bariatric surgery on adipokines (adiponectin, leptin), inflammatory cytokines [C-reactive protein (CRP), interleukin-6 (IL-6), interleukin-10(IL-10)] and global plasma measures of oxidative stress [thiobarbituric acid reactive substances (TBARS) and total antioxidant status (TAOS) 1 and 6 months, and 4 years post-surgery in subjects with obesity and impaired glucose regulation. Methods A prospective study comprising of 19 participants (13 females, mean age 50.4 ± 6.2 years, mean body mass index (BMI) 54 ± 14 kg/m2, 17 type 2 diabetes) undergoing bariatric surgery (10 sleeve gastrectomy, 6 biliopancreatic diversion, 2 Roux-en-Y gastric bypass and 1 laparoscopic adjustable gastric banding). Serial measurements of the above markers were made pre-operatively, 1 and 6 months and 4 years post-operatively. Results Compared to pre-operative levels, significant decreases were seen 4 years post-operatively in CRP (11.4 vs 2.8 ng/mL, p p p = 0.001). At 4 years, both fasting and 120 min TAOS significantly increased by 35% and 19% respectively. However, fasting and 120 min TBARS did not show any significant changes. Conclusion To our knowledge, no other studies have described changes in inflammation and oxidative stress at 4 years following bariatric surgery. This study contributes to the current literature supporting the longer-term beneficial effect of bariatric surgery on chronic inflammation and oxidative stress.

Background Historical data suggesting poor survival in patients with aortic stenosis (AS) who do not undergo treatment are largely confined to patients with severe AS. Objectives This study sought to determine the prognostic impact of all levels of native valvular AS. Methods Severity of AS was characterized by convention and by statistical distribution in 122,809 male patients (mean age 61 ± 17 years) and 118,494 female patients (mean age 62 ± 19 years), with measured aortic valve (AV) mean gradient, peak velocity, and/or area. The relationship between AS severity and survival was then examined during median 1,208 days (interquartile range: 598 to 2,177 days) of follow-up. Patients with previous aortic valve intervention were excluded. Results Overall, 16,129 (6.7%), 3,315 (1.4%), and 6,383 (2.6%) patients had mild, moderate, and severe AS, respectively. On an adjusted basis (vs. no AS; 5-year mortality 19%), patients with mild to severe AS had an increasing risk of long-term mortality (adjusted hazard ratio: 1.44 to 2.09; p 50%) and cardiovascular disease was evident from a mean AV gradient >20.0 mm Hg (moderate AS) after adjusting for age, sex, left ventricular systolic or diastolic dysfunction, and aortic regurgitation. Conclusions These data confirm that when left untreated, severe AS is associated with poor long-term survival. Moreover, they also suggest poor survival rates in patients with moderate AS. (National Echocardiographic Database of Australia [NEDA]; ACTRN12617001387314)