Cardioprotective effect of nicorandil on isoproterenol induced cardiomyopathy in the Mdx mouse model.

BACKGROUND: Duchenne muscular dystrophy (DMD) associated cardiomyopathy is a major cause of morbidity and mortality. In an in vitro DMD cardiomyocyte model, nicorandil reversed stress-induced cell injury through multiple pathways implicated in DMD. We aimed to test the efficacy of nicorandil on the progression of cardiomyopathy in mdx mice following a 10-day treatment protocol. METHODS: A subset of mdx mice was subjected to low-dose isoproterenol injections over 5 days to induce a cardiac phenotype and treated with vehicle or nicorandil for 10 days. Baseline and day 10 echocardiograms were obtained to assess cardiac function. At 10 days, cardiac tissue was harvested for further analysis, which included histologic analysis and assessment of oxidative stress. Paired student's t test was used for in group comparison, and ANOVA was used for multiple group comparisons. RESULTS: Compared to vehicle treated mice, isoproterenol decreased ejection fraction and fractional shortening on echocardiogram. Nicorandil prevented isoproterenol induced cardiac dysfunction. Isoproterenol increased cardiac fibrosis, which nicorandil prevented. Isoproterenol increased gene expression of NADPH oxidase, which decreased to baseline with nicorandil treatment. Superoxide dismutase 2 protein expression increased in those treated with nicorandil, and xanthine oxidase activity decreased in mice treated with nicorandil during isoproterenol stress compared to all other groups. CONCLUSIONS: In conclusion, nicorandil is cardioprotective in mdx mice and warrants continued investigation as a therapy for DMD associated cardiomyopathy.

Epitope Mapping of SERCA2a Identifies an Antigenic Determinant That Induces Mainly Atrial Myocarditis in A/J Mice.

Sarcoplasmic/endoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA)2a, a critical regulator of calcium homeostasis, is known to be decreased in heart failure. Patients with myocarditis or dilated cardiomyopathy develop autoantibodies to SERCA2a suggesting that they may have pathogenetic significance. In this report, we describe epitope mapping analysis of SERCA2a in A/J mice that leads us to make five observations: 1) SERCA2a contains multiple T cell epitopes that induce varying degrees of myocarditis. One epitope, SERCA2a 971-990, induces widespread atrial inflammation without affecting noncardiac tissues; the cardiac abnormalities could be noninvasively captured by echocardiography, electrocardiography, and magnetic resonance microscopy imaging. 2) SERCA2a 971-990-induced disease was associated with the induction of CD4 T cell responses and the epitope preferentially binds MHC class II/IAk rather than IEk By creating IAk/and IEk/SERCA2a 971-990 dextramers, the T cell responses were determined by flow cytometry to be Ag specific. 3) SERCA2a 971-990-sensitized T cells produce both Th1 and Th17 cytokines. 4) Animals immunized with SERCA2a 971-990 showed Ag-specific Abs with enhanced production of IgG2a and IgG2b isotypes, suggesting that SERCA2a 971-990 can potentially act as a common epitope for both T cells and B cells. 5) Finally, SERCA2a 971-990-sensitized T cells were able to transfer disease to naive recipients. Together, these data indicate that SERCA2a is a critical autoantigen in the mediation of atrial inflammation in mice and that our model may be helpful to study the inflammatory events that underlie the development of conditions such as atrial fibrillation in humans.

Immunotherapy in Anal Cancer.

The incidence and mortality of squamous cell carcinoma of the anus has been gradually increasing globally over the last few decades. The evolution of different modalities, including immunotherapies, has changed the treatment paradigm of metastatic anal cancers. Chemotherapy, radiation therapy, and immune-modulating therapies form the backbone of treatment of anal cancer in various stages. Most anal cancers are linked to high-risk human papilloma virus (HPV) infections. HPV oncoproteins E6 and E7 are responsible for an anti-tumor immune response triggering the recruitment of tumor-infiltrating lymphocytes. This has led to the development and utilization of immunotherapy in anal cancers. Current research in anal cancer is moving forward to discover ways to incorporate immunotherapy in the treatment sequencing in various stages of anal cancers. Immune checkpoint inhibitors alone or in combination, adoptive cell therapy, and vaccines are the areas of active investigations in anal cancer in both locally advanced and metastatic settings. Immunomodulating properties of non-immunotherapies are incorporated to enhance immune checkpoint inhibitors' effectiveness in some of the clinical trials. The aim of this review is to summarize the potential role of immunotherapy in anal squamous cell cancers and future directions.

Oligometastatic Melanoma Treated by Metastasectomy in Combination with Immune Checkpoint and BRAF Inhibitors: A Case Series.

BACKGROUND Early therapies for metastatic melanoma improved patient quality of life; however, median survival remained unaffected. Studies are showing that surgical excision with the combination of immune checkpoint inhibitor (ICI) therapy has better outcomes than systemic therapy alone. This single-center case series describes 7 patients with oligometastatic melanoma treated by metastasectomy in combination with ICI and BRAF inhibitors. CASE REPORT One female and 6 male patients are included in our study, with ages ranging from 34 to 82 years. Oligometastatic melanoma is defined was having no more than 5 metastatic regions. Each patient had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Patients received either ICI therapy with ipilimumab, nivolumab, and/or pembrolizumab, or targeted therapy with encorafenib and binimetinib, or a combination. Patients underwent metastasectomies with curative intent. The main outcome and measurements obtained were the duration of disease-free survival, based on radiographic evidence. The range of disease-free survival in our population was 13 to 67 months, with the lower end limited by patient death and the upper limit being the present day. CONCLUSIONS This case series reiterates survival benefit for patients who received metastasectomy after exhibiting good response to ICI therapy. ICI and/or BRAF inhibitor therapy combined with metastasectomy provides a possible curative option for patients who may have previously been relegated to palliative-focused care. By using a multimodal approach with oncologists and surgeons, we can challenge our understanding of what constitutes a resectable cancer.

Clinical presentation of Warburg effect in aggressive lymphoma: a case report.

BACKGROUND: The Warburg effect is a rare condition in tumor biology, illustrated by significant lactate production in the presence of oxygen. The Warburg effect is associated with very poor prognosis in patients with malignancy. CASE PRESENTATION: We report a 76-year-old Caucasian woman with double-expressor diffuse large B cell lymphoma who presented with severe lactic acidosis and extreme hypoglycemia with normal mentation. Her lactic acidosis was initially controlled with a bicarbonate infusion, and the patient was started promptly on steroids, followed by chemotherapy, but her clinical course was complicated by tumor lysis syndrome, acute renal failure requiring hemodialysis, and progressive liver failure. She manifested a temporary clinical response to chemotherapy but eventually died of complications. CONCLUSIONS: This case demonstrates the importance of prompt recognition of the Warburg effect, aggressive supportive measures, and early initiation of chemotherapy. Future studies are needed to characterize the role of hemodialysis in this setting.

Response to the Rechallenge With Talimogene Laherparepvec (T-VEC) After Ipilimumab/Nivolumab Treatment in Patient With Cutaneous Malignant Melanoma Who Initially Had a Progression on T-VEC With Pembrolizumab.

Talimogene laherparepvec (T-VEC) is approved for unresected stage III-IV malignant melanoma. T-VEC has a direct cytotoxic effect and enhances the antitumor immunity of host cells. Immune checkpoints inhibitors also enhance the immunity of host cells by increasing the recruitment of antigen-presenting cells or activation and restoration of T-cell functions. Both type of therapies can potentiate the effect of the other therapy. We are reporting a case of T-VEC rechallenge who initially progressed on T-VEC with pembrolizumab but then responded to T-VEC rechallenge after intervening ipilimumab/nivolumab. An 83-year-old man developed a subungual lesion of the left thumb and found to have AJCC V. 7 stage IIIb melanoma. Few months later, he developed axillary lymphadenopathy and multiple subcutaneous nodules (AJCC V. 7 stage IIIc). The patient was started on intralesional rose Bengal and pembrolizumab. After 4 cycles of pembrolizumab with rose Bengal, a positron-emission tomography/computerized tomography scan showed the progression of disease. He was started on T-VEC intralesional injections with concurrent pembrolizumab, however, after 3 T-VEC injections and 2 more cycles of pembrolizumab, there was the progression of disease. Subsequently, ipilimumab/nivolumab was started and patient responded partially. Ipilimumab/nivolumab was held due to toxicity. Eight weeks from the last dose of ipilimumab/nivolumab, he experienced locoregional progression and was rechallenged with T-VEC monotherapy. The patient showed a significant response after second T-VEC injection and continued to show response 6 months since rechallenge. After, initial progression on T-VEC with pembrolizumab, intervening immune checkpoints inhibitors may favorably modify the antitumor immunity and potentiate antitumor effect of T-VEC rechallenge.

Prognostic Significance of Hematological Indices in Malignant Melanoma Treated With Immune Checkpoint Inhibitors.

Local and systemic inflammation significantly effects tumor progression and its response to therapy. We aim to evaluate the prognostic significance of inflammatory cells, their ratios, and a change in these indices while patients are receiving immune checkpoint inhibitors (ICIs). We retrospectively reviewed 120 malignant melanoma patients who had received any ICIs from 2011 until December 2017 and evaluated the effect of hematological indices on survival and radiographic responses. We followed the trends of these indices at 0, 6, and 12 weeks while on ICIs. Univariate and multivariate survival analyses were performed. The Student t tests and logistic regression were performed as well. Patients with neutrophil to lymphocyte ratio (NLR) <5 and derived neutrophil to lymphocyte ratio (dNLR) <3 had better overall survival and progression-free survival. The objective response rate was significantly higher in patients with absolute neutrophil count (ANC) <5 and dNLR<3 at baseline. Responder to ICIs had downtrending median ANC, NLR, dNLR, and an uptrending median lymphocyte to monocyte ratio compared with those of nonresponders. Moreover, in responders, the decrease in mean ANC, NLR, and dNLR were statistically significant compared with that of nonresponders at 6 and 12 weeks while on ICIs. Hematological indices can predict the response to ICIs and prognosis in malignant melanoma. Besides, the changes in these indices from their baseline values could be monitored in real-time to predict an earlier response even before a radiographic evaluation. However, the prospective and validation studies are needed before these models can be used in routine clinical practices.

Clinical outcomes in non-small-cell lung cancer patients receiving concurrent metformin and immune checkpoint inhibitors.

AIM: To study the clinical benefits of concurrent metformin and immune checkpoint inhibitors (ICIs) in non-small-cell lung cancer patients. MATERIALS & METHODS: This is a retrospective review of 50 non-small-cell lung cancer patients receiving ICIs with metformin (cohort A) or without metformin (cohort B). Patients were also stratified by ICIs as second-/third-line therapy. RESULTS: Overall response rate and disease control rate were higher in cohort A (41.1 vs 30.7%, p = 0.4 and 70.5 vs 61.6%, p = 0.5, respectively). Median overall survival and progression-free survival were also higher in cohort A (11.5 vs 7.6 months, p = 0.5 and 4.0 vs 3.0 months, p = 0.6, respectively). On subset analysis (second-/third-line ICIs), overall response rate, disease control rate, median overall survival, progression-free survival were also higher in cohort A. CONCLUSION: Despite the small-sample size, we observed improved clinical outcomes in patients who received ICIs in combination with metformin.

Sleeve gastrectomy in obese Wistar rats improves diastolic function and promotes cardiac recovery independent of weight loss.

BACKGROUND: Heart failure with preserved ejection fraction is the most common cause of heart failure and is characterized by impaired diastolic relaxation. Bariatric surgery significantly improves diastolic relaxation, but a mechanism beyond weight loss remains unknown. OBJECTIVES: We tested the hypothesis that a sleeve gastrectomy (SG) will improve diastolic dysfunction independent of weight loss due to postoperative alterations in the enterocardiac axis. SETTING: University research laboratory. METHODS: Male Wistar rats were fed a high-fat diet (HFD) or low-fat diet (LFD) for 10 weeks and then divided into SG-HFD, pair-fed sham HFD, ad-lib sham HFD, or ad-lib sham LFD groups (n = 9-14 per group). At least 2 months postoperatively, cardiac function, meal tolerance, glucose tolerance, and cardiac gene expression were compared between groups. RESULTS: Only the SG cohort showed significant improvements in postoperative diastolic relaxation (isovolumetric relaxation time pre-SG: 14.7 ± 2.3 msec, post-SG: 11.2 ± 1.8 msec, P < .001). SG significantly increased active glucagon-like peptide-1 (P = .03). Compared to pair-fed sham HFD rats, SG-HFD rats had significantly altered mRNA cardiac gene expression, including sarco/endoplasmic reticulum Ca2+-ATPase 2 a (SERCA2 a) (P < .001). CONCLUSIONS: SG improves diastolic function independent of weight loss in a rat model of obesity with beneficial alterations in cardiac gene expression of multiple known targets related to cardiac failure, including SERCA2 a. These data support that a greater curve gastrectomy induces beneficial intracellular cardiac signaling for diastolic function mediated by the enterocardiac axis that is independent of weight loss. These findings could translate to offering metabolic surgery to patients with heart failure with preserved ejection fraction.

Immune checkpoint inhibitor (anti-CTLA-4, anti-PD-1) therapy alone versus immune checkpoint inhibitor (anti-CTLA-4, anti-PD-1) therapy in combination with anti-RANKL denosumuab in malignant melanoma: a retrospective analysis at a tertiary care center.

Denosumab is a monoclonal antibody against RANK ligand with a role in the prevention of skeletal-related events and is also known to possess antitumor properties. In this retrospective review, we aim to evaluate the synergist effect of a combination therapy with immune checkpoint inhibitors and denosumab in malignant melanoma patients. Patients of 18 years of age or older with a diagnosis of malignant melanoma who have received immune checkpoint inhibitors and denosumab between June 2015 and May 2017 were divided into two cohorts: cohort A (immune checkpoint inhibitors only) and cohort B (immune checkpoint inhibitors and denosumab). Overall survival, progression-free survival, objective response rate, and safety analysis were performed. Stratified analysis based on metastatic (M) status was performed as well. Eleven (29.72%) out of 37 patients received immune checkpoint inhibitors and denosumab combination. Median overall survival in cohort B was 57 months compared with 22.8 months in cohort A and 22 months in M1c patients from cohort A. Median progression-free survival was 4.15 months in cohort B compared with 11.6 months in cohort A and 5.12 months in M1c patients from cohort A. The mean number of distant sites involved in metastasis were significantly higher in cohort B (3.54 vs. 2.23, P=0.0015). Cohort B also had more patients with more than two distant metastatic sites (90.9 vs. 30.8%, P=0.001). A combination therapy with denosumab and immune checkpoint inhibitors may have a beneficial effect on survival and progression as in our study; the patients receiving combination therapy did not behave poorly despite having poor prognostic features.

Molecular Approaches in HFpEF: MicroRNAs and iPSC-Derived Cardiomyocytes.

Heart failure with preserved left ventricular ejection fraction (HFpEF) has emerged as one of the largest unmet needs in cardiovascular medicine. HFpEF is increasing in prevalence and causes significant morbidity, mortality, and health care resource utilization. Patients have multiple co-morbidities which contribute to the disease complexity. To date, no effective treatment for HFpEF has been identified. The paucity of cardiac biopsies from this patient population and the absence of well-accepted animal models limit our understanding of the underlying molecular mechanisms of HFpEF. In this review, we discuss combining state-of-the-art technologies of microRNA profiling and human induced pluripotent cell-derived cardiomyocytes (iPSC-CMs) in order to uncover novel molecular pathways that may contribute to the development of HFpEF. Here, we focus the advantages and limitations of microRNA profiling and iPSC-CMs as a disease model system to discover molecular mechanisms in HFpEF.

ß1-Adrenergic Receptor Contains Multiple IAk and IEk Binding Epitopes That Induce T Cell Responses with Varying Degrees of Autoimmune Myocarditis in A/J Mice.

Myocarditis/dilated cardiomyopathy (DCM) patients can develop autoantibodies to various cardiac antigens and one major antigen is ß1-adrenergic receptor (ß1AR). Previous reports indicate that animals immunized with a ß1AR fragment encompassing, 197-222 amino acids for a prolonged period can develop DCM by producing autoantibodies, but existence of T cell epitopes, if any, were unknown. Using A/J mice that are highly susceptible to lymphocytic myocarditis, we have identified ß1AR 171-190, ß1AR 181-200, and ß1AR 211-230 as the major T cell epitopes that bind major histocompatibility complex class II/IAk or IEk alleles, and by creating IAk and IEk dextramers, we demonstrate that the CD4 T cell responses to be antigen-specific. Of note, all the three epitopes were found also to stimulate CD8 T cells suggesting that they can act as common epitopes for both CD4 and CD8 T cells. While, all epitopes induced only mild myocarditis, the disease-incidence was enhanced in animals immunized with all the three peptides together as a cocktail. Although, antigen-sensitized T cells produced mainly interleukin-17A, their transfer into naive animals yielded no disease. But, steering for T helper 1 response led the T cells reacting to one epitope, ß1AR 181-200 to induce severe myocarditis in naive mice. Finally, we demonstrate that all three ß1AR epitopes to be unique for T cells as none of them induced antibody responses. Conversely, animals immunized with a non-T cell activator, ß1AR 201-220, an equivalent of ß1AR 197-222, had antibodies comprising of all IgG isotypes and IgM except, IgA and IgE. Thus, identification of T cell and B cell epitopes of ß1AR may be helpful to determine ß1AR-reactive autoimmune responses in various experimental settings in A/J mice.

Nicorandil, a Nitric Oxide Donor and ATP-Sensitive Potassium Channel Opener, Protects Against Dystrophin-Deficient Cardiomyopathy.

BACKGROUND: Dystrophin-deficient cardiomyopathy is a growing clinical problem without targeted treatments. We investigated whether nicorandil promotes cardioprotection in human dystrophin-deficient induced pluripotent stem cell (iPSC)-derived cardiomyocytes and the muscular dystrophy mdx mouse heart. METHODS AND RESULTS: Dystrophin-deficient iPSC-derived cardiomyocytes had decreased levels of endothelial nitric oxide synthase and neuronal nitric oxide synthase. The dystrophin-deficient cardiomyocytes had increased cell injury and death after 2 hours of stress and recovery. This was associated with increased levels of reactive oxygen species and dissipation of the mitochondrial membrane potential. Nicorandil pretreatment was able to abolish these stress-induced changes through a mechanism that involved the nitric oxide-cyclic guanosine monophosphate pathway and mitochondrial adenosine triphosphate-sensitive potassium channels. The increased reactive oxygen species levels in the dystrophin-deficient cardiomyocytes were associated with diminished expression of select antioxidant genes and increased activity of xanthine oxidase. Furthermore, nicorandil was found to improve the restoration of cardiac function after ischemia and reperfusion in the isolated mdx mouse heart. CONCLUSION: Nicorandil protects against stress-induced cell death in dystrophin-deficient cardiomyocytes and preserves cardiac function in the mdx mouse heart subjected to ischemia and reperfusion injury. This suggests a potential therapeutic role for nicorandil in dystrophin-deficient cardiomyopathy.

Generation of induced pluripotent stem cells from muscular dystrophy patients: efficient integration-free reprogramming of urine derived cells.

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy(1). Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2-3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.