Single-Cell RNA Sequencing Reveals Metabolic Stress-Dependent Activation of Cardiac Macrophages in a Model of Dyslipidemia-Induced Diastolic Dysfunction.

BACKGROUND: Metabolic distress is often associated with heart failure with preserved ejection fraction (HFpEF) and represents a therapeutic challenge. Metabolism-induced systemic inflammation links comorbidities with HFpEF. How metabolic changes affect myocardial inflammation in the context of HFpEF is not known. METHODS: We found that ApoE knockout mice fed a Western diet recapitulate many features of HFpEF. Single-cell RNA sequencing was used for expression analysis of CD45+ cardiac cells to evaluate the involvement of inflammation in diastolic dysfunction. We focused bioinformatics analysis on macrophages, obtaining high-resolution identification of subsets of these cells in the heart, enabling us to study the outcomes of metabolic distress on the cardiac macrophage infiltrate and to identify a macrophage-to-cardiomyocyte regulatory axis. To test whether a clinically relevant sodium glucose cotransporter-2 inhibitor could ameliorate the cardiac immune infiltrate profile in our model, mice were randomized to receive the sodium glucose cotransporter-2 inhibitor dapagliflozin or vehicle for 8 weeks. RESULTS: ApoE knockout mice fed a Western diet presented with reduced diastolic function, reduced exercise tolerance, and increased pulmonary congestion associated with cardiac lipid overload and reduced polyunsaturated fatty acids. The main immune cell types infiltrating the heart included 4 subpopulations of resident and monocyte-derived macrophages, determining a proinflammatory profile exclusively in ApoE knockout-Western diet mice. Lipid overload had a direct effect on inflammatory gene activation in macrophages, mediated through endoplasmic reticulum stress pathways. Investigation of the macrophage-to-cardiomyocyte regulatory axis revealed the potential effects on cardiomyocytes of multiple inflammatory cytokines secreted by macrophages, affecting pathways such as hypertrophy, fibrosis, and autophagy. Finally, we describe an anti-inflammatory effect of sodium glucose cotransporter-2 inhibition in this model. CONCLUSIONS: Using single-cell RNA sequencing in a model of diastolic dysfunction driven by hyperlipidemia, we have determined the effects of metabolic distress on cardiac inflammatory cells, in particular on macrophages, and suggest sodium glucose cotransporter-2 inhibitors as potential therapeutic agents for the targeting of a specific phenotype of HFpEF.

IL10 Secretion Endows Intestinal Human iNKT Cells with Regulatory Functions Towards Pathogenic T Lymphocytes.

BACKGROUND AND AIMS: Invariant natural killer T [iNKT] cells perform pleiotropic functions in different tissues by secreting a vast array of pro-inflammatory and cytotoxic molecules. However, the presence and function of human intestinal iNKT cells capable of secreting immunomodulatory molecules such as IL-10 has never been reported so far. Here we describe for the first time the presence of IL10-producing iNKT cells [NKT10 cells] in the intestinal lamina propria of healthy individuals and of Crohn's disease [CD] patients. METHODS: Frequency and phenotype of NKT10 cells were analysed ex vivo from intestinal specimens of Crohn's disease [n = 17] and controls [n = 7]. Stable CD-derived intestinal NKT10 cell lines were used to perform in vitro suppression assays and co-cultures with patient-derived mucosa-associated microbiota. Experimental colitis models were performed by adoptive cell transfer of splenic naïve CD4+ T cells in the presence or absence of IL10-sufficient or -deficient iNKT cells. In vivo induction of NKT10 cells was performed by administration of short chain fatty acids [SCFA] by oral gavage. RESULTS: Patient-derived intestinal NKT10 cells demonstrated suppressive capabilities towards pathogenic CD4+ T cells. The presence of increased proportions of mucosal NKT10 cells associated with better clinical outcomes in CD patients. Moreover, an intestinal microbial community enriched in SCFA-producing bacteria sustained the production of IL10 by iNKT cells. Finally, IL10-deficient iNKT cells failed to control the pathogenic activity of adoptively transferred CD4+ T cells in an experimental colitis model. CONCLUSIONS: These results describe an unprecedentd IL10-mediated immunoregulatory role of intestinal iNKT cells in controlling the pathogenic functions of mucosal T helper subsets and in maintaining the intestinal immune homeostasis.

Biomarkers and Lung Cancer Early Detection: State of the Art.

Lung cancer burden is increasing, with 2 million deaths/year worldwide. Current limitations in early detection impede lung cancer diagnosis when the disease is still localized and thus more curable by surgery or multimodality treatment. Liquid biopsy is emerging as an important tool for lung cancer early detection and for monitoring therapy response. Here, we reviewed recent advances in liquid biopsy for early diagnosis of lung cancer. We summarized DNA- or RNA-based biomarkers, proteins, autoantibodies circulating in the blood, as well as circulating tumor cells (CTCs), and compared the most promising studies in terms of biomarkers prediction performance. While we observed an overall good performance for the proposed biomarkers, we noticed some critical aspects which may complicate the successful translation of these biomarkers into the clinical setting. We, therefore, proposed a roadmap for successful development of lung cancer biomarkers during the discovery, prioritization, and clinical validation phase. The integration of innovative minimally invasive biomarkers in screening programs is highly demanded to augment lung cancer early detection.

Adoptive transfer of CX3CR1 transduced-T regulatory cells improves homing to the atherosclerotic plaques and dampens atherosclerosis progression.

AIM: Loss of immunosuppressive response supports inflammation during atherosclerosis. We tested whether adoptive cell therapy (ACT) with Tregulatory cells (Tregs), engineered to selectively migrate in the atherosclerotic plaque, would dampen the immune-inflammatory response in the arterial wall in animal models of familial hypercholesterolaemia (FH). METHODS AND RESULTS: FH patients presented a decreased Treg suppressive function associated to an increased inflammatory burden. A similar phenotype was observed in Ldlr -/- mice accompanied by a selective increased expression of the chemokine CX3CL1 in the aorta but not in other districts (lymph nodes, spleen, and liver). Treg overexpressing CX3CR1 were thus generated (CX3CR1+-Tregs) to drive Tregs selectively to the plaque. CX3CR1+-Tregs were injected (i.v.) in Ldlr -/- fed high-cholesterol diet (western type diet, WTD) for 8 weeks. CX3CR1+-Tregs were detected in the aorta, but not in other tissues, of Ldlr -/- mice 24 h after ACT, corroborating the efficacy of this approach. After 4 additional weeks of WTD, ACT with CX3CR1+-Tregs resulted in reduced plaque progression and lipid deposition, ameliorated plaque stability by increasing collagen and smooth muscle cells content, while decreasing the number of pro-inflammatory macrophages. Shotgun proteomics of the aorta showed a metabolic rewiring in CX3CR1+-Tregs treated Ldlr -/- mice compared to controls that was associated with the improvement of inflammation-resolving pathways and disease progression. CONCLUSION: ACT with vasculotropic Tregs appears as a promising strategy to selectively target immune activation in the atherosclerotic plaque.

Single-Cell Sequencing of Mouse Heart Immune Infiltrate in Pressure Overload-Driven Heart Failure Reveals Extent of Immune Activation.

BACKGROUND: Inflammation is a key component of cardiac disease, with macrophages and T lymphocytes mediating essential roles in the progression to heart failure. Nonetheless, little insight exists on other immune subsets involved in the cardiotoxic response. METHODS: Here, we used single-cell RNA sequencing to map the cardiac immune composition in the standard murine nonischemic, pressure-overload heart failure model. By focusing our analysis on CD45+ cells, we obtained a higher resolution identification of the immune cell subsets in the heart, at early and late stages of disease and in controls. We then integrated our findings using multiparameter flow cytometry, immunohistochemistry, and tissue clarification immunofluorescence in mouse and human. RESULTS: We found that most major immune cell subpopulations, including macrophages, B cells, T cells and regulatory T cells, dendritic cells, Natural Killer cells, neutrophils, and mast cells are present in both healthy and diseased hearts. Most cell subsets are found within the myocardium, whereas mast cells are found also in the epicardium. Upon induction of pressure overload, immune activation occurs across the entire range of immune cell types. Activation led to upregulation of key subset-specific molecules, such as oncostatin M in proinflammatory macrophages and PD-1 in regulatory T cells, that may help explain clinical findings such as the refractivity of patients with heart failure to anti-tumor necrosis factor therapy and cardiac toxicity during anti-PD-1 cancer immunotherapy, respectively. CONCLUSIONS: Despite the absence of infectious agents or an autoimmune trigger, induction of disease leads to immune activation that involves far more cell types than previously thought, including neutrophils, B cells, Natural Killer cells, and mast cells. This opens up the field of cardioimmunology to further investigation by using toolkits that have already been developed to study the aforementioned immune subsets. The subset-specific molecules that mediate their activation may thus become useful targets for the diagnostics or therapy of heart failure.

Ceruloplasmin replacement therapy ameliorates neurological symptoms in a preclinical model of aceruloplasminemia.

Aceruloplasminemia is a monogenic disease caused by mutations in the ceruloplasmin gene that result in loss of protein ferroxidase activity. Ceruloplasmin plays a role in iron homeostasis, and its activity impairment leads to iron accumulation in liver, pancreas, and brain. Iron deposition promotes diabetes, retinal degeneration, and progressive neurodegeneration. Current therapies mainly based on iron chelation, partially control systemic iron deposition but are ineffective on neurodegeneration. We investigated the potential of ceruloplasmin replacement therapy in reducing the neurological pathology in the ceruloplasmin-knockout (CpKO) mouse model of aceruloplasminemia. CpKO mice were intraperitoneal administered for 2 months with human ceruloplasmin that was able to enter the brain inducing replacement of the protein levels and rescue of ferroxidase activity. Ceruloplasmin-treated mice showed amelioration of motor incoordination that was associated with diminished loss of Purkinje neurons and reduced brain iron deposition, in particular in the choroid plexus. Computational analysis showed that ceruloplasmin-treated CpKO mice share a similar pattern with wild-type animals, highlighting the efficacy of the therapy. These data suggest that enzyme replacement therapy may be a promising strategy for the treatment of aceruloplasminemia.

Is there a role for maintenance therapy in non-small-cell lung cancer? An emerging issue.

Standard treatment of advanced non-small-cell lung cancer consists of platinum-based (doublet) polychemotherapy. The treatment should last for a maximum of four to six cycles. Several randomized clinical trials and some meta-analyses showed that a longer duration of treatment does not lead to an increase in survival. Recent data with new-generation agents (chemo- or targeted therapies) suggest that a maintenance treatment until progression of disease in patients with response or stable disease after the first cycles of induction (platinum-based) chemotherapy confers an advantage in time-to-progression or even overall survival (with pemetrexed) compared with no maintenance treatment. The advent of new agents and the better selection of patients according to histology or mutation of specific tyrosine kinase receptors (e.g., EGF receptor) seems to increase the therapeutic options and improve the prognosis in the advanced disease setting. The paradigm of the duration of therapy in advanced lung cancer appears to change from the use of second-line treatment only at progression of disease to an early introduction of an alternative (second-line) therapy as consolidation and chronic phase of maintenance.

Targeted therapies for the treatment of breast cancer in the post-trastuzumab era.

Targeted therapies for breast cancer are evolving rapidly. Trastuzumab has revolutionized breast cancer treatment and outcome, reducing the risk for recurrence and significantly increasing survival, at least for a subgroup of patients. Other targeted therapies, such as bevacizumab, a monoclonal antibody targeting angiogenesis, lapatinib, a dual human epidermal growth factor receptor (HER)-1 and HER-2 inhibitor, other small-molecule tyrosine kinase inhibitors, and mammalian target of rapamycin inhibitors, have been developed in phase II and III clinical trials. Although there has been rapid approval of these new drugs by health authorities, some questions have emerged about their application in clinical practice. What is the appropriate drug or sequence of drugs? What is the ideal target? How should tumor response be evaluated? Are financial resources sufficient to treat patients? How do we design trials with these molecules? These are emerging as current dilemmas for clinical oncologists.

Optimizing use of opiates in the management of cancer pain.

Cancer pain is often suboptimally managed. The underestimation and undertreatment continues to be a problem despite the availability of consensus-based guidelines. Most patients with cancer develop pain. The prevalence and severity of pain among cancer patients varies according to primary and metastatic sites and stage of disease. Opioid therapy is the cornerstone of management of severe chronic pain in the field of cancer patients and in general in palliative care medicine. Since this class of drugs is the cornerstone of the treatment, optimizing its use may be useful in clinical practice. For this purpose we focused on 4 distinct issues: 1) How to implement the use the opioids in cancer patients; 2) How to optimise the use of morphine in cancer patients; 3) The management of side effects and opioid switching; 4) What is the role of other potent opioids. A holistic approach including an appropriate use of opioids may improve pain control in most cancer patients, particularly for those with advanced disease.

Primary chemotherapy with epirubicin and vinorelbine in women with locally advanced breast cancer.

BACKGROUND: To assess the activity and toxicity of primary chemotherapy with epirubicin (60 mg/m2 every other week) and vinorelbine (25 mg/m2, weekly) plus granulocyte colony-stimulating factor (G-CSF) for 12 weeks, in patients with locally advanced breast cancer in a multicenter setting. PATIENTS AND METHODS: Patients with stage IIIA or IIIB breast cancer, not older than 70, were eligible. A two-stage phase II design was applied. Response was assessed clinically, instrumentally and pathologically. RESULTS: Out of 48 enrolled patients, 87.5% received all planned cycles, with a median dose-intensity of 30 mg/m2/week for epirubicin and 23.8 mg/m2/week for vinorelbine. A clinical or instrumental objective response was reached in 42 patients (87.5%, exact 95% CI: 74.7-95.3); significant downstaging was reached in all but one patient; 6 cases had a pathological complete response in the breast, and 2 cases in the lymph nodes too (pathological complete response rate 4.2%, exact 95% CI: 0.5-14.2); a further 2 patients had only microscopic cancer foci at pathological examination of the breast. Radiological tests underestimated the treatment effect on the breast. Toxicity was mild, neutropenia being the most frequent (grade 3-4 in 47% of patients), but never complicated with fever or sepsis. Mild constipation (< or =grade 2) occurred in 35% of patients. Moderate to severe asthenia occurred in 12% of 6 patients. No cardiac toxicity was reported. At 3 years, disease-free survival was 68% and overall survival 81%. CONCLUSION: Primary chemotherapy with epirubicin every other week, weekly vinorelbine and G-CSF support is highly active and well tolerated in patients with locally advanced breast cancer.

Venous thromboembolism and cancer: new issues for an old topic.

Thromboembolic complications represent the second leading cause of death for cancer patients. Even though the correlation between cancer and a hypercoagulable state has been widely recognised, the pathogenesis of thromboembolism during malignancy is not yet entirely understood. The direct or indirect activation of the coagulation cascade favours neoplastic dissemination and metastasis. Disordered coagulation is encountered in up to 90% of cancer patients, although only 15% of them develop a localised acute or chronic deep venous thrombosis or a disseminated intravascular coagulation. This risk is significantly increased by chemotherapy, hormone therapy, surgery and central venous catheters. Therefore, much effort is needed to develop efficient prophylaxis and treatment, to reduce recurrence and bleeding and finally, to improve quality of life. Better knowledge about the biochemical bases of the coagulation process represents a pivotal step in cancer biology comprehension and global therapeutic management.