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The extracellular signal-regulated kinase (ERK) has been shown to mediate cisplatin (CP)-induced toxicity to renal proximal tubule cells. Here, we demonstrate that ERK serves as the kinase that phosphorylates the pro-apoptotic p66shc protein at its Serine36 residue in CP-treated renal proximal tubule cells. Pharmacologic or dominant-negative inhibition of ERK mitigates cisplatin-induced Ser36 phosphorylation of p66shc. Overexpression of p66shc exacerbates while its knockdown or mutation of the Serine36 site to alanine ameliorates CP-induced nephrotoxicity in vitro. Since p66shc is Serine36 phosphorylated in the kidneys of mice after treatment with CP, a similar mechanism might exist in vivo. [ABSTRACT FROM AUTHOR]

Cisplatin is a widely used antineoplastic agent that has nephrotoxicity as a major side effect. The underlying mechanism of this nephrotoxicity is still not well known. Iron has been implicated to play an important role in several models of tissue injury, presumably through the generation of hydroxyl radicals via the Haber-Weiss reaction or other highly toxic free radicals. In the present study we examined the catalytic iron content and the effect of iron chelators in an in vitro model of cisplatin-induced cytotoxicity in LLC-PK1 cells (renal tubular epithelial cells) and in an in vivo model of cisplatin-induced acute renal failure in rats. Exposure of LLC-PK1 cells to cisplatin resulted in a significant increase in bleomycin-detectable iron (iron capable of catalyzing free radical reactions) released into the medium. Concurrent incubation of LLC-PK1 cells with iron chelators including deferoxamine and 1,10-phenanthroline significantly attenuated cisplatin-induced cytotoxicity as measured by lactate dehydrogenase (LDH) release. Bleomycin-detectable iron content was also markedly increased in the kidney of rats treated with cisplatin. Similarly, administration of deferoxamine in rats provided marked functional (as measured by blood urea nitrogen and creatinine) and histological protection against cisplatin-induced acute renal failure. In a separate study, we examined the role of hydroxyl radical in cisplatin-induced nephrotoxicity. Incubation of LLC-PK1 cells with cisplatin caused an increase in hydroxyl radical formation. Hydroxyl radical scavengers, dimethyl sulfoxide, mannitol and benzoic acid, significantly reduced cisplatin-induced cytotoxicity and, treatment with dimethyl sulfoxide or dimethylthiourea provided significant protection against cisplatin-induced acute renal failure. Taken together, our data strongly support a critical role for iron in mediating tissue injury via hydroxyl radical (or a similar oxidant) in this model of nephrotoxicity. [ABSTRACT FROM AUTHOR]

Most kidney transplant patients who undergo biopsies are classified as having no rejection based on consensus thresholds. However, we hypothesized that because these patients have normal adaptive immune systems, T cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR) may exist as subthreshold activity in some transplants currently classified as no rejection. To examine this question, we studied genome-wide microarray results from 5086 kidney transplant biopsies (from 4170 patients). An updated molecular archetypal analysis designated 56% of biopsies as no rejection. Subthreshold molecular TCMR and/or ABMR activity molecular activity was detectable as elevated classifier scores in many biopsies classified as no rejection, with ABMR activity in many TCMR biopsies and TCMR activity in many ABMR biopsies. In biopsies classified as no rejection histologically and molecularly, molecular TCMR classifier scores correlated with increases in histologic TCMR features and molecular injury, lower estimated glomerular filtration rate, and higher risk of graft loss, and molecular ABMR activity correlated with increased glomerulitis and donor-specific antibody. No rejection biopsies with high subthreshold TCMR or ABMR activity had a higher probability of having TCMR or ABMR, respectively, diagnosed in a future biopsy. We conclude that many kidney transplant recipients have unrecognized subthreshold TCMR or ABMR activity, with significant implications for future problems., Competing Interests: Declaration of competing interest The authors of this manuscript have conflicts of interest to disclose as described by the American Journal of Transplantation. P.F. Halloran holds shares in Transcriptome Sciences Inc., a University of Alberta research company dedicated to developing molecular diagnostics, supported in part by a licensing agreement between Transcriptome Sciences Inc. and Thermo Fisher Scientific, and by a research grant from Natera, Inc. P.F. Halloran is a consultant to Natera, Inc. and Argenx BV. The other authors have declared no conflict of interest exists., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aldosterone-producing adenoma is a rare cause of hypertension in children. Only a limited number of cases of aldosterone-producing adenomas with somatic KCNJ5 gene mutations have been described in children. Blacks are particularly more susceptible to developing long-standing cardiovascular effects of aldosterone-induced severe hypertension. Somatic CACNA1D gene mutations are particularly more prevalent in black males whereas KCNJ5 gene mutations are most frequently present in black females. We present here a novel somatic KCNJ5 p.I157S mutation in an aldosterone-producing adenoma from a 16-year-old black female whose severe drug-resistant hypertension significantly improved following unilateral adrenalectomy. Prompt diagnosis of aldosterone-producing adenoma and early identification of gene mutation would enable appropriate therapy and significantly reduce cardiovascular sequelae., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gomez-Sanchez, van Rooyen, Rainey, Nanba, Blinder and Baliga.)

Background: IgA-dominant infection-associated glomerulonephritis is well-documented in adults but has not been studied in depth in children. We assessed the incidence of pediatric IgA-dominant infection-associated glomerulonephritis and clinical and kidney biopsy findings., Methods: Pediatric native kidney biopsies over a 10-year period with IgA dominance, strong C3, and findings indicative of infection-associated etiology were identified., Results: We identified 9 cases of IgA-dominant infection-associated glomerulonephritis, 0.8% of pediatric native kidney biopsies. Seven patients presented with elevated creatinine. All had hematuria and proteinuria. Eight patients had clinical evidence of infection: one each with central port infection by methicillin-sensitive Staphylococcus aureus, recurrent streptococcal pharyngitis and recent otitis media, streptococcal pharyngitis demonstrated 8 months after biopsy, suspected streptococcal scalded skin syndrome, and viral gastroenteritis, and three with serologic evidence of Streptococcal infection but no identified site of infection. All but one patient experienced short-term normalization of creatinine and resolution of proteinuria, though two eventually progressed to kidney failure: one 3 years later due to progressive disease and one 11 years later due to focal segmental glomerulosclerosis without concurrent immune deposits., Conclusions: Pediatric IgA-dominant infection-associated glomerulonephritis is rare, and generally has a favorable prognosis, contrasting that seen in adults with severe comorbidities. A higher resolution version of the Graphical abstract is available as Supplementary., (© 2021. IPNA.)

Background: Gilteritinib is a novel FMS-like tyrosine kinase 3 inhibitor recently approved by the United States Food and Drug Administration in 2018 for relapsed or refractory acute myeloid leukemia. However, gilteritinib may be associated with underrecognized cardiotoxicities., Case Presentation: This case describes a patient with a history significant for hyperlipidemia who was diagnosed with relapsed acute myeloid leukemia. After four doses of gilteritinib monotherapy, she abruptly developed acute systolic heart failure with global hypokinesis and septal wall motion abnormalities. Two days after discontinuation, cardiac magnetic resonance imaging showed partial recovery of her left ventricular ejection fraction as well as myocardial edema and non-ischemic fibrosis suggestive of inflammatory cardiomyopathy. She underwent intravenous diuresis and eventually started guideline-directed heart failure therapy. Follow-up cardiac magnetic resonance imaging five months later showed improved ejection fraction with mild non-ischemic fibrosis and resolution of myocardial edema and inflammation. She later received an allogeneic stem cell transplant from a matched unrelated donor., Conclusions: Gilteritinib may be associated with early cardiotoxicities, including non-ischemic cardiomyopathy and myocarditis. Cardiac magnetic resonance imaging can be an important modality to help differentiate or diagnose early cardiotoxicities associated with novel targeted therapies., (© 2021. The Author(s).)

Background: Chimeric antigen receptor T-cell (CAR-T) infusion is associated with early toxicity. Yet, whether early toxicity development holds ramifications for long-term outcomes is unknown., Methods: From a large cohort of consecutive adult patients treated with CAR-T therapies for relapsed or refractory lymphomas from 2016 to 2019, we assessed progression-free survival (PFS), by toxicity development (cytokine release syndrome (CRS), neurotoxicity, or cardiotoxicity]. We also assessed the relationship of toxicity development to objective disease response, and overall survival (OS). Multivariable regression was utilized to evaluate relationships between standard clinical and laboratory measures and disease outcomes. Differences in outcomes, by toxicity status, were also assessed via 30-day landmark analysis. Furthermore, we assessed the effects of early anti-CRS toxicity therapy use (at ≤grade 2 toxicity) on maximum toxicity grade observed, and long-term disease outcomes (PFS and OS)., Results: Overall, from 102 CAR-T-treated patients, 90 were identified as treated with single-agent therapy, of which 88.9% developed toxicity (80 CRS, 41 neurotoxicity, and 17 cardiotoxicity), including 28.9% with high-grade (≥3) events. The most common manifestations were hypotension at 96.6% and fever at 94.8%. Among patients with cardiac events, there was a non-significant trend toward a higher prevalence of concurrent or preceding high-grade (≥3) CRS. 50.0% required tocilizumab or corticosteroids. The median time to toxicity was 3 days; high grade CRS development was associated with cardiac and neurotoxicity. In multivariable regression, accounting for disease severity and traditional predictors of disease response, moderate (maximum grade 2) CRS development was associated with higher complete response at 1 year (HR: 2.34; p=0.07), and longer PFS (HR: 0.41; p=0.02, in landmark analysis), and OS (HR: 0.43; p=0.03). Among those with CRS, relative blood pressure (HR: 2.25; p=0.004), respectively, also associated with improved PFS. There was no difference in disease outcomes, or maximum toxicity grade (CRS, neurotoxicity, or cardiotoxicity) observed, based on the presence or absence of the use of early CRS-directed therapies., Conclusions: Among adult lymphoma patients, moderate toxicity manifest as grade 2 CRS after CAR-T infusion may associate with favorable clinical outcomes. Further studies are needed to confirm these findings., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Opinion Statement: Prostate cancer is the second leading cause of cancer death in men, and cardiovascular disease is the number one cause of death in patients with prostate cancer. Androgen deprivation therapy, the cornerstone of prostate cancer treatment, has been associated with adverse cardiovascular events. Emerging data supports decreased cardiovascular risk of gonadotropin releasing hormone (GnRH) antagonists compared to agonists. Ongoing clinical trials are assessing the relative safety of different modalities of androgen deprivation therapy. Racial disparities in cardiovascular outcomes in prostate cancer patients are starting to be explored. An intriguing inquiry connects androgen deprivation therapy with reduced risk of COVID-19 infection susceptibility and severity. Recognition of the cardiotoxicity of androgen deprivation therapy and aggressive risk factor modification are crucial for optimal patient care.

Background: Vitamin B 6 is a rate-limiting coenzyme that plays an important role in the biosynthesis of heme and the incorporation of iron into protoporphyrin. Its deficiency is often seen in chronic kidney disease (CKD), particularly those requiring dialysis and following administration of erythropoietin-stimulating agent (ESA)., Case- Diagnosis/treatment: A 16-year-old African-American male with stage 5 CKD on chronic hemodialysis experienced a decrease in hemoglobin over a 3-month period from 11 to 6.5 g/dl while receiving ESA, resulting in multiple blood transfusions. His transferrin saturation was 41%, ferritin level 706 [80-388] ng/mL, mean corpuscular volume (MCV) 87 [78-98] μm 3 , corrected reticulocytes count 2.3% [0.2-1.8%], and vitamin B 6 1.2 [5.3-46.7] μg/L. Bone marrow biopsy was normocellular (65%) with erythroid hyperplasia and rare dyserythropoiesis. Prussian blue staining showed increased iron storage. Supplemental vitamin B 6 (100 mg daily) was initiated at hemoglobin 7.3 g/dL with correction of anemia. Eighteen months later, his hemoglobin is 11.7 g/dL, transferrin saturation 45%, with no additional blood transfusions., Conclusions: Vitamin B 6 deficiency anemia should be considered in any pediatric patient on hemodialysis who does not respond to standard ESA and iron therapy.

Background: Trastuzumab-induced cardiotoxicity (TIC) can lead to early discontinuation of adjuvant therapy, however there is limited evidence on long-term survival outcomes in patients with operable human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC) experiencing treatment interruption or discontinuation., Methods: The primary objective of the study was to evaluate disease-free survival (DFS) in non-metastatic, HER2-positive, female BC patients who experienced treatment interruption or early discontinuation of trastuzumab therapy. Clinical and histopathological data were collected on 400 patients at The Ohio State University, an NCI-designated comprehensive cancer center between January 2005 and December 2015. Treatment interruption was defined as any delay of ≥2 weeks during trastuzumab therapy, including permanent cessation prior to completing planned therapy. TIC was defined as LVEF < 50% or > 15 points decline from baseline as evaluated by 2D echocardiogram after initiation of (neo) adjuvant therapy. DFS was defined as the time from diagnosis to first recurrence (loco-regional or distant recurrence) including second primary BC or death. Overall survival (OS) was defined as the time from diagnosis to death or last known follow up. OS/DFS estimates were generated using Kaplan-Meier methods and compared using Log-rank tests. Cox proportional hazard models were used to calculate adjusted hazard ratios (aHR) for OS/DFS., Results: A total of 369 patients received trastuzumab therapy; 106 (29%) patients experienced treatment interruption at least once and 42 (11%) permanently discontinued trastuzumab prior to completing planned therapy. TIC was the most common reason for interruption (66 patients, 62%). The median duration of trastuzumab in patients with treatment interruption was 11.3 months (range: 0.5-16.9) with 24 (23%) patients receiving ≤6 months of therapy. This duration includes the time delay related to treatment interruption. Patients with any treatment interruption had worse DFS (aHR: 4.4, p = 0.001) and OS (aHR: 4.8, p < 0.001) after adjusting for age, stage, grade, ER, node status and TIC., Conclusions: Treatment interruption or early discontinuation of trastuzumab therapy in early HER2-positive BC, most often from TIC, is an independent prognostic marker for worse DFS and OS in operable HER2-positive BC. Future prospective studies should consider targeting at-risk populations and optimizing cardiac function to avoid interruption in trastuzumab therapy.

Natural immunoglobulin M (IgM) antibodies are pentameric or hexameric macro-immunoglobulins and have been highly conserved during evolution. IgMs are initially expressed during B cell ontogeny and are the first antibodies secreted following exposure to foreign antigens. The IgM multimer has either 10 (pentamer) or 12 (hexamer) antigen binding domains consisting of paired µ heavy chains with four constant domains, each with a single variable domain, paired with a corresponding light chain. Although the antigen binding affinities of natural IgM antibodies are typically lower than IgG, their polyvalency allows for high avidity binding and efficient engagement of complement to induce complement-dependent cell lysis. The high avidity of IgM antibodies renders them particularly efficient at binding antigens present at low levels, and non-protein antigens, for example, carbohydrates or lipids present on microbial surfaces. Pentameric IgM antibodies also contain a joining (J) chain that stabilizes the pentameric structure and enables binding to several receptors. One such receptor, the polymeric immunoglobulin receptor (pIgR), is responsible for transcytosis from the vasculature to the mucosal surfaces of the lung and gastrointestinal tract. Several naturally occurring IgM antibodies have been explored as therapeutics in clinical trials, and a new class of molecules, engineered IgM antibodies with enhanced binding and/or additional functional properties are being evaluated in humans. Here, we review the considerable progress that has been made regarding the understanding of biology, structure, function, manufacturing, and therapeutic potential of IgM antibodies since their discovery more than 80 years ago.

Purpose of Review: Radiation-induced cardiovascular disease, including coronary artery disease, is a well-known sequela of radiation therapy and represents a significant source of morbidity and mortality for cancer survivors. This review examines current literature and guidelines to care for this growing population of cancer survivors., Recent Findings: The development of radiation-induced ischemic heart disease following radiation can lead even to early cardiotoxicities, inclusive of coronary artery disease, which limit cancer treatment outcomes. These coronary lesions tend to be diffuse, complex, and proximal. Early detection with multimodality imaging and targeted intervention is required to minimize these risks. Early awareness, detection, and management of radiation-induced cardiovascular disease are paramount as cancer survivorship continues to grow.

The 143 low- and middle-income countries (LMICs) of the world constitute 80% of the world's population or roughly 5.86 billion people with much variation in geography, culture, literacy, financial resources, access to health care, insurance penetration, and healthcare regulation. Unfortunately, their burden of cardiovascular disease in general and acute ST-segment-elevation myocardial infarction (STEMI) in particular is increasing at an unprecedented rate. Compounding the problem, outcomes remain suboptimal because of a lack of awareness and a severe paucity of resources. Guideline-based treatment has dramatically improved the outcomes of STEMI in high-income countries. However, no such focused recommendations exist for LMICs, and the unique challenges in LMICs make directly implementing Western guidelines unfeasible. Thus, structured solutions tailored to their individual, local needs, and resources are a vital need. With this in mind, a multicountry collaboration of investigators interested in LMIC STEMI care have tried to create a consensus document that extracts transferable elements from Western guidelines and couples them with local realities gathered from expert experience. It outlines general operating principles for LMICs focused best practices and is intended to create the broad outlines of implementable, resource-appropriate paradigms for management of STEMI in LMICs. Although this document is focused primarily on governments and organizations involved with improvement in STEMI care in LMICs, it also provides some specific targeted information for the frontline clinicians to allow standardized care pathways and improved outcomes.

Amber codon suppression for the insertion of non-natural amino acids (nnAAs) is limited by competition with release factor 1 (RF1). Here we describe the genome engineering of a RF1 mutant strain that enhances suppression efficiency during cell-free protein synthesis, without significantly impacting cell growth during biomass production. Specifically, an out membrane protease (OmpT) cleavage site was engineered into the switch loop of RF1, which enables its conditional inactivation during cell lysis. This facilitates extract production without additional processing steps, resulting in a scaleable extract production process. The RF1 mutant extract allows nnAA incorporation at previously intractable sites of an IgG1 and at multiple sites in the same polypeptide chain. Conjugation of cytotoxic agents to these nnAAs, yields homogeneous antibody drug conjugates (ADCs) that can be optimized for conjugation site, drug to antibody ratio (DAR) and linker-warheads designed for efficient tumor killing. This platform provides the means to generate therapeutic ADCs inaccessible by other methods that are efficient in their cytotoxin delivery to tumor with reduced dose-limiting toxicities and thus have the potential for better clinical impact.

Thrombosis and thromboembolic events contribute to significant morbidity in cancer patients. Venous thrombosis embolism (which includes deep vein thrombosis and pulmonary embolism) accounts for a large percentage of thromboembolic events. Appropriate identification of cancer patients at high risk for venous thromboembolism and management of thromboembolic event is crucial in improving the quality of care for cancer patients. However, thromboembolism in cancer patients is a complex problem and the management has to be tailored to each individual. The focus of this review is to understand the complex pathology, physiology and risk factors that drive the process of venous thrombosis and embolism in cancer patients and the current guidelines in management.

Rhabdomyolysis accounts for ∼10% of acute kidney injuries. In glycerol-induced myoglobinuric acute kidney injury, we found an increase in the nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear protein, a key redox-sensitive transcription factor, and Nrf2-regulated genes and proteins including upregulation of heme oxygenase-1. In in vitro studies, pretreatment of LLC-PK1 cells with an activator of Nrf2 before myoglobin exposure significantly decreased oxidant generation and cytotoxicity, whereas Nrf2 inhibition and gene silencing exacerbated the injury. Chlormethiazole, a specific CYP2E1 transcription inhibitor, prevented an increase in catalytic iron in the kidneys, decreased oxidative stress, blocked nuclear translocation of the Nrf2 protein, decreased heme oxygenase-1 upregulation, and provided functional and histological protection against acute kidney injury. CYP2E1 inhibitors and gene silencing in renal tubular epithelial cells significantly decreased reactive oxygen species generation and provided marked protection against myoglobin-induced cytotoxicity. Thus, during CYP2E1-induced oxidative stress, the transcription factor Nrf2 has a pivotal role in the early adaptive response. Inhibition of CYP2E1 coupled with the prior induction of Nrf2 may be a valuable tool to reduce CYP2E1-mediated rhabdomyolysis-induced acute kidney injury.

Antibodies are well-established as therapeutics, and the preclinical and clinical pipeline of these important biologics is growing rapidly. Consequently, there is considerable interest in technologies to engineer and manufacture them. Mammalian cell culture is commonly used for production because eukaryotic expression systems have evolved complex and efficient chaperone systems for the folding of antibodies. However, given the ease and manipulability of bacteria, antibody discovery efforts often employ bacterial expression systems despite their limitations in generating high titers of functional antibody. Open-Cell Free Synthesis (OCFS) is a coupled transcription-translation system that has the advantages of prokaryotic systems while achieving high titers of antibody expression. Due to the open nature of OCFS, it is easily modified by chemical or protein additives to improve the folding of select proteins. As such, we undertook a protein additive screen to identify chaperone proteins that improve the folding and assembly of trastuzumab in OCFS. From the screen, we identified the disulfide isomerase DsbC and the prolyl isomerase FkpA as important positive effectors of IgG folding. These periplasmic chaperones function synergistically for the folding and assembly of IgG, and, when present in sufficient quantities, gram per liter IgG titers can be produced. This technological advancement allows the rapid development and manufacturing of immunoglobulin proteins and pushes OCFS to the forefront of production technologies for biologics.

Objective: To delineate the constitutive pulmonary vascular phenotype of the TβRIIΔk-fib mouse model of scleroderma, and to selectively induce pulmonary endothelial cell injury using vascular endothelial growth factor (VEGF) inhibition to develop a model with features characteristic of pulmonary arterial hypertension (PAH)., Methods: The TβRIIΔk-fib mouse strain expresses a kinase-deficient transforming growth factor β (TGFβ) receptor type II driven by a fibroblast-specific promoter, leading to ligand-dependent up-regulation of TGFβ signaling, and replicates key fibrotic features of scleroderma. Structural, biochemical, and functional assessments of pulmonary vessels, including in vivo hemodynamic studies, were performed before and following VEGF inhibition, which induced pulmonary endothelial cell apoptosis. These assessments included biochemical analysis of the TGFβ and VEGF signaling axes in tissue sections and explanted smooth muscle cells., Results: In the TβRIIΔk-fib mouse strain, a constitutive pulmonary vasculopathy with medial thickening, a perivascular proliferating chronic inflammatory cell infiltrate, and mildly elevated pulmonary artery pressure resembled the well-described chronic hypoxia model of pulmonary hypertension. Following administration of SU5416, the pulmonary vascular phenotype was more florid, with pulmonary arteriolar luminal obliteration by apoptosis-resistant proliferating endothelial cells. These changes resulted in right ventricular hypertrophy, confirming hemodynamically significant PAH. Altered expression of TGFβ and VEGF ligand and receptor was consistent with a scleroderma phenotype., Conclusion: In this study, we replicated key features of systemic sclerosis-related PAH in a mouse model. Our results suggest that pulmonary endothelial cell injury in a genetically susceptible mouse strain triggers this complication and support the underlying role of functional interplay between TGFβ and VEGF, which provides insight into the pathogenesis of this disease., (Copyright © 2013 by the American College of Rheumatology.)

In the past decade, in vitro transcription/translation technologies have emerged as discovery tools for screening large protein expression libraries, for the selection of engineered polypeptide libraries, and as alternatives to conventional heterologous expression for protein production. Therapeutic proteins and peptides discovered using ribosome-based display methods that link genetic information to the encoded polypeptide generated by cell-free extracts, or purified translation components, are beginning to move forward into human clinical trials. This review details the significant progress in in vitro translation for novel protein and non-natural amino acid containing peptide discovery platforms, as well as advances in the clinical-scale production of therapeutic proteins using cell-free transcription/translation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Aims: Myocardial tumour necrosis factor-α (TNF-α) expression induces cardiac dysfunction in endotoxemia. The aim of this study was to investigate the role of mitogen-activated protein kinase phosphatase-1 (MKP1) pathway in myocardial TNF-α expression and cardiac function during endotoxemia., Methods and Results: Lipopolysaccharide (LPS) increased MKP1 expression in the myocardium in vivo and in cultured neonatal cardiomyocytes in vitro. LPS-induced extracellular signal-regulated kinase (ERK) 1/2 and p38 phosphorylation in the myocardium was prolonged in MKP1(-/-) mice. Myocardial TNF-α mRNA and protein levels were enhanced in MKP1(-/-) compared with wild-type (WT) mice in endotoxemia, leading to a further decrease in cardiac function. To study if Rac1/p21-activated kinase 1 (PAK1) signalling regulates MKP1 expression, cardiomyocytes were treated with LPS. Inhibition of Rac1 and PAK1 by a dominant negative Rac1 adenovirus (Ad-Rac1N17) and PAK1 siRNA, respectively, blocked LPS-induced MKP1 expression in cardiomyocytes. PAK1 siRNA also decreased p38 and c-Jun N-terminal kinase (JNK) activation, and TNF-α expression induced by LPS. Furthermore, deficiency in either Rac1 or JNK1 decreased myocardial MKP1 expression in endotoxemic mice., Conclusion: LPS activates the Rac1/PAK1 pathway, which increases myocardial MKP1 expression via JNK1. MKP1 attenuates ERK1/2 and p38 activation, inhibits myocardial TNF-α expression, and improves cardiac function in endotoxemia. Thus, MKP1 represents an important negative feedback mechanism limiting pro-inflammatory response in the heart during sepsis.

Breathing is the most vital function for maintenance of life. Slow and deep breathing is an integral part of Pranayama and it reduces dead space ventilation and renews air throughout the lungs. The reported beneficial effects of deep breathing as a part of either long term or short term practice of Pranayama are well documented. However our knowledge about the effects of a few minutes' of deep breathing on human ventilatory parameters is poor. In the present study, we examined the relationship between exposure to short duration of deep breathing and performance on pulmonary function tests before and after the deep breathing. The study was conducted in a homogenous group of 12 volunteers containing 4 females and 8 males who were well trained in pulmonary function testing (PFT) before the start of the study. The volunteers performed deep breathing (DB) exercise for 2, 5 and 10 minutes at the rate of 6 breaths per minute under guidance, and the duration of DB exercise for that day was randomly selected for each group. PFT was done before and after the DB exercise. There was a significant (P < 0.05) increase in vital capacity (VC) after 2 and 5 minutes' DB exercise and a consistent improvement in tidal volume (TV) and minute ventilation (MV) after the DB exercise in all the three groups, though it wasn't statistically significant. There was a significant (P < 0.05) increase in forced vital capacity (FVC) after 2 minutes' of DB exercise and a consistent increase in all the three groups in forced inspiratory vital capacity (FIVC) and peak inspiratory flow rate (PIFR), though this increase was not statistically significant. This shows that deep breathing exercise, even for a few minutes' duration is beneficial for the lung functions.

Decreased cardiac contractility is a central feature of systolic heart failure. Existing drugs increase cardiac contractility indirectly through signaling cascades but are limited by their mechanism-related adverse effects. To avoid these limitations, we previously developed omecamtiv mecarbil, a small-molecule, direct activator of cardiac myosin. Here, we show that it binds to the myosin catalytic domain and operates by an allosteric mechanism to increase the transition rate of myosin into the strongly actin-bound force-generating state. Paradoxically, it inhibits adenosine 5'-triphosphate turnover in the absence of actin, which suggests that it stabilizes an actin-bound conformation of myosin. In animal models, omecamtiv mecarbil increases cardiac function by increasing the duration of ejection without changing the rates of contraction. Cardiac myosin activation may provide a new therapeutic approach for systolic heart failure.

Although the adverse allograft outcomes associated with HLA antibodies are well documented, some controversy exists regarding the importance of low-level donor specific anti-HLA antibodies (DSA). To provide further detail on this controversy, we prospectively looked at low-level DSA in negative T- and B-cell flow cytometric crossmatch (FCXM) or acceptable reactive crossmatch (ARC) patients who each underwent protocol based post-transplant antibody monitoring. HLA Class I and II antibody screening and specificity determination was conducted via a solid phase assay (SPA) and FCXM versus donor and autologous T and B cells. Post-transplant patients were immunosuppressed with quadruple maintained immunosuppressive therapy, rabbit anti-thymocyte globulin induction, and HLA antibody monitoring. Out of 31 ARC patients transplanted, 65% had a PRA > 50% and 26% showed increased DSA at 7-14 days post-transplant. Antibody mediated rejection (AMR) was treated with pharmacological and/or plasmapheresis (PP) therapy. DSA were lowered and remained at low-levels (MFI 1000- 3000) or below FCXM cutoffs. None of the 31 patients transplanted developed de-novo antibodies. Two patients lost their allografts, one to polyoma (BK) virus, and one to antibody mediated rejection (AMR). In conclusion, our experience demonstrates that patients deemed higher risk for an immunological event due to low-level DSA should be transplanted with an ARC and followed post-transplant according to an established alloantibody monitoring protocol. With close monitoring, 5-year outcomes can be expected to approach that of low-immunologic risk transplant patients.

Membranous nephropathy is a disease that affects the filtering units of the kidney, the glomeruli, and results in proteinuria accompanied by loss of kidney function. Passive Heymann nephritis is an experimental model that mimics membranous nephropathy in humans, wherein the glomerular epithelial cell (GEC) injury induced by complement C5b-9 leads to proteinuria. We examined the role of cytochrome P450 2B1 (CYP2B1) in this complement-mediated sublytic injury. Overexpression of CYP2B1 in GECs significantly increased the formation of reactive oxygen species, cytotoxicity, and collapse of the actin cytoskeleton following treatment with anti-tubular brush-border antiserum (anti-Fx1A). In contrast, silencing of CYP2B1 markedly attenuated anti-Fx1A-induced reactive oxygen species generation and cytotoxicity with preservation of the actin cytoskeleton. Gelsolin, which maintains an organized actin cytoskeleton, was significantly decreased by complement C5b-9-mediated injury but was preserved in CYP2B1-silenced cells. In rats injected with anti-Fx1A, the cytochrome P450 inhibitor cimetidine blocked an increase in catalytic iron and ROS generation, reduced the formation of malondialdehyde adducts, maintained a normal distribution of nephrin in the glomeruli, and provided significant protection at the onset of proteinuria. Thus, GEC CYP2B1 contributes to complement C5b-9-mediated injury and plays an important role in the pathogenesis of passive Heymann nephritis.

Background: Orthostatic hypotension (OH) is common among older people and is more prevalent in elderly with various disorders and on medications., Aims: The objective of the study was to know the prevalence of orthostatic hypotension in healthy geriatric subjects., Subjects and Methods: The study group comprised of healthy non hypertensive, non diabetic elderly individuals aged 60 years and above (n=80) and another group, healthy aged 30 to 50 years age (n=80, mean age39.2±5.3). Orthostatic hypotension was defined as a decline in systolic/diastolic blood pressure of ≥20/10 mmHg when an individual changed from a supine to a standing position within 3 minutes of standing. Systolic and Diastolic blood pressure was measured in supine position and within 3 minutes of standing., Results: 1 out of 80 (1.25%) in the elderly subjects was found to have orthostatic hypotension., Conclusion: The study concluded that the orthostatic hypotension is less prevalent in healthy elderly subjects without any illness or without on any medications.

Background and Purpose: Excessive production of nitric oxide (NO) by inducible NO synthase (iNOS) is thought to underlie the vascular dysfunction, systemic hypotension and organ failure that characterize endotoxic shock. Plasma levels of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are raised in animal models and humans with endotoxic shock and correlate with the associated cardiovascular dysfunction. Since both NO and natriuretic peptides play important roles in cardiovascular homeostasis via activation of guanylate cyclase-linked receptors, we used mice lacking natriuretic peptide receptor (NPR)-A (NPR1) to establish if natriuretic peptides contribute to the cardiovascular dysfunction present in endotoxic shock., Experimental Approach: Wild-type (WT) and NPR-A knockout (KO) mice were exposed to lipopolysaccharide (LPS) and vascular dysfunction (in vitro and in vivo), production of pro-inflammatory cytokines, and iNOS expression and activity were evaluated., Key Results: LPS-treated WT animals exhibited a marked fall in mean arterial blood pressure (MABP) whereas NPR-A KO mice maintained MABP throughout. LPS administration caused a greater suppression of vascular responses to the thromboxane-mimetic U46619, ANP, acetylcholine and the NO-donor spermine-NONOate in WT versus NPR-A KO mice. This differential effect on vascular function was paralleled by reduced pro-inflammatory cytokine production, iNOS expression and activity (plasma [NO(x)] and cyclic GMP)., Conclusions and Implications: These observations suggest that NPR-A activation by natriuretic peptides facilitates iNOS expression and contributes to the vascular dysfunction characteristic of endotoxic shock. Pharmacological interventions that target the natriuretic peptide system may represent a novel approach to treat this life-threatening condition.

Previously, we demonstrated that cytochrome P450 2B1 (CYP2B1) can generate reactive oxygen species in puromycin aminonucleoside (PAN)-induced nephrotic syndrome, an animal model of minimal-change disease in humans. In this study we found that overexpression of CYP2B1 in rat glomerular epithelial cells in vitro significantly increased PAN-induced reactive oxygen species generation, cytotoxicity, cell death, and collapse of the actin cytoskeleton. All of these pathological changes were markedly attenuated by siRNA-induced CYP2B1 silencing. The cellular CYP2B1 protein content was significantly decreased whereas its mRNA level was markedly increased, suggesting regulation by protein degradation rather than transcriptional inhibition in the PAN-treated glomerular epithelial cells. This degradation of CYP2B1 was accompanied by the induction of heme oxygenase-1, an important indicator of heme-induced oxidative stress. In PAN-treated CYP2B1-silenced glomerular epithelial cells the induction of heme oxygenase-1 and caspase-3 activity were significantly decreased. Further, cleavage of the stress-induced pro-apoptotic endoplasmic reticulum-specific pro-caspase-12 was prevented in the silenced cells. Our results support a pivotal role of CYP2B1 for reactive oxygen species production in the endoplasmic reticulum in PAN-induced cytotoxicity.

Mitochondrial dysfunction is involved in pathopysiology of ischemia-reperfusion-induced acute kidney injury (AKI). The p66shc adaptor protein is a newly recognized mediator of mitochondrial dysfunction, which might play a role in AKI-induced renal tubular injury. Oxidative stress-mediated Serine36 phosphorylation of p66shc facilitates its transportation to the mitochondria where it oxidizes cytochrome c and generates excessive amount of reactive oxygen species (ROS). The consequence is mitochondrial depolarization and injury. Earlier we determined that p66shc plays an essential role in injury of cultured mouse renal proximal tubule cells during oxidative stress. Here, we studied the role of p66shc in ROS generation and consequent mitochondrial dysfunction during oxidative injury in renal proximal tubule cells. We employed p66shc knockdown renal proximal tubule cells and cells that overexpress wild-type, Serine phosphorylation (S36A), or cytochrome c-binding (W134F) mutants of p66shc. Inhibition of the mitochondrial electron transport chain or the mitochondrial permeability transition revealed that hydrogen peroxide-induced injury is mitochondrial ROS and consequent mitochondrial depolarization dependent. We also found that through Ser36 phosphorylation and mitochondria/cytochrome c binding, p66shc mediates those effects. We propose a similar mechanism in vivo as we demonstrated mitochondrial binding of p66shc as well as its association with cytochrome c in the postischemic kidneys of mice. Thus, manipulating p66shc might offer a new therapeutic modality to ameliorate renal ischemic injury.

Inhibition of mitotic kinesins represents a novel approach for the discovery of a new generation of anti-mitotic cancer chemotherapeutics. We report here the discovery of the first potent and selective inhibitor of centromere-associated protein E (CENP-E) 3-chloro-N-{(1S)-2-[(N,N-dimethylglycyl)amino]-1-[(4-{8-[(1S)-1-hydroxyethyl]imidazo[1,2-a]pyridin-2-yl}phenyl)methyl]ethyl}-4-[(1-methylethyl)oxy]benzamide (GSK923295; 1), starting from a high-throughput screening hit, 3-chloro-4-isopropoxybenzoic acid 2. Compound 1 has demonstrated broad antitumor activity in vivo and is currently in human clinical trials.

Background: Cancer therapies and cancer progression can increase oxidative stress that might account for renal toxicity in cancer patients. Flavin 7 (F7) is a natural polyphenol-containing dietary supplement with potential antioxidant activity. Therefore, it might help to attenuate renal toxicity of chemotherapeutics., Materials and Methods: Cultured mouse renal proximal tubule cells were subjected to H(2)O(2)-mediated oxidative stress. Potential antioxidant effects of F7 were assessed by measuring the production of reactive oxygen species (ROS), mitochondrial depolarization and injury (lactate dehydrogenase release as well as trypan blue exclusion) in cells that were pretreated with F7 prior to treatment with H(2)O(2)., Results: F7 pretreatment significantly attenuated H(2)O(2)-induced ROS production, mitochondrial depolarization and consequent injury in renal proximal tubule cells., Conclusion: F7 supplementation might be beneficial for cancer patients in order to prevent renal toxicity of anticancer drug- or cancer progression-related oxidative stress.

Type 1 diabetes is associated with a unique form of cardiomyopathy that is present without atherosclerosis. Redox imbalance and/or changes in vascular endothelial growth factor (VEGF) expression have been associated with diabetes-related cardiomyopathy. However, the mechanisms of these changes and their interrelationships remain unclear. Using a murine type 1 diabetes model, we tested the hypothesis that alterations in cardiac performance are associated with decreased cardiac microvascular prevalence, as well as downregulation of VEGF isoforms. We also investigated oxidative stress as a contributor to regulate individual VEGF isoforms and microvascular rarefaction. Significant and rapid hyperglycemia was observed at 1 wk post-streptozotocin (STZ) and persisted throughout the 5-wk study. Left ventricular (LV) fractional shortening was reduced at week 1 and 5 post-STZ insult relative to age-matched controls. We also observed the early reduction in E/A ratio at 1 wk. Immunostaining for CD31 and digital image analysis demonstrated a 35% reduction in microvessels/myocardial area, indicative of rarefaction, which was highly correlated with fractional shortening. Furthermore, a significant increase in the prevalence of protein 3-nitrotyrosine was observed in the diabetic cardiac tissue, which was inversely associated with microvascular rarefaction. The expressions of three VEGF isoforms were significantly reduced to different extents. The reduction of VEGF(164) was associated with GSSG accumulation. These data demonstrate that the mouse model of STZ-induced diabetes has hallmark features observed in humans with respect to nonischemic systolic and diastolic performance and microvascular rarefaction, which are associated with changes in VEGF isoform expression and redox imbalance in the myocardium.

Background: Increased production of reactive oxygen species (ROS) by anticancer drugs has been described in patients with various malignancies, which might attribute to their nephrotoxicity., Materials and Methods: The effects of two epigenetic modifiers - trichostatin A (TSA) and 5-aza-deoxycytidine (5AZA) - on ROS production and cell injury alone or in combination with mild oxidative stress were studied in mouse renal proximal tubule cells., Results: Both agents increased mitochondrial ROS production and consequent lactate dehydrogenase (LDH) release either alone or in combination with a low dose of H(2)O(2). The antioxidant N-acetyl-cysteine (NAC) abolished LDH release. It was also found that CREB-mediated transcription, vital for survival of proximal tubule cells, is attenuated by these anticancer agents., Conclusion: The ROS-inducing activity of TSAI and 5AZA might explain the in vivo nephrotoxicity of epigenetic modifiers. The mechanisms that are responsible for this injury could involve attenuation of pro-survival signaling and/or activation of death signaling pathway(s) associated with mitochondrial ROS release.

The pathogenesis of coronary lesion development is a multi-factorial process involving a number of different cell types and covariates, and injury and dysfunction of the vascular endothelium is an important marker and likely participant in the initiation and/or progression of most forms of heart disease. In addition to chronic dysfunction of endothelial responses in patients with established heart disease, there is evidence that 'acute insults' can cause measurable dysfunction in vascular response in humans (drug toxicities, hypoxia, high fat meal). Such repeated acute insults may contribute to disease risk in otherwise healthy individuals or promote disease progression in established patients. Consumption of grape products, especially wine, has been linked to lower cardiovascular risk but the vascular endothelial effects of grape products in healthy normal subjects, in the absence of ethanol, have not been evaluated. We therefore tested the hypotheses that 1) a standardized product derived from fresh grapes (GP, acute and chronic consumption) improves endothelial performance in healthy normal young subjects, and 2) that concomitant grape consumption affects the 'acute endothelial insult' caused by a single standardized high fat meal (HF). Acute consumption of GP equivalent to 1.25 cups of fresh grapes caused significant improvement in brachial artery flow mediated dilation (FMD) within 3 h of consumption, when compared to control consumption of sugar solution (p<0.05). No acute changes in heart rate, hemodynamics, or lipid profiles were observed. When this 'dose' was then consumed twice daily for 3 weeks FMD was further improved and total antioxidant capacity in plasma was slightly increased (p<0.05), with no change in heart rate, hemodynamics, or lipid profiles. A single HF meal (900 cal, 49 g total fat) caused a 50% reduction in FMD response when consumed alone, and this effect coincided with increased blood triglyceride levels within 3 h post-consumption. In contrast the concomitant consumption of GP with the HF meal completely prevented this HF-induced vascular endothelial dysfunction (p<0.05), but had no effect on rising triglycerides. These data demonstrate that a modest intake of fresh grapes can have acute favorable effects on vascular endothelial function in normal healthy subjects, that chronic intake can further improve performance and concomitant intake can blunt the 'acute insult' to endothelium caused by a typical western HF meal. This effect is likely to be related to antioxidant effects at the endothelium, rather than changes in blood lipids. These data support epidemiological data of the health benefits of grapes, and demonstrate that 'favorable' food consumption can apparently reduce some toxicities induced by 'unfavorable' food consumption.