Hyperprogression as a distinct outcome after immunotherapy.

Cancer research is living a time of unparalleled expectations around immunotherapy, a therapeutic strategy that materializes the elegant idea of weaponizing our immune system to eradicate tumor cells. In an everchanging standard of care, a growing number of studies have shown that immunotherapy may accelerate tumor progression in a significant subset of patients ranging from 4% to 29% across multiple histologies. The identification of hyperprogression poses a challenge for RECIST criteria, which fail to capture pre- and post-treatment tumor growth kinetics at early times of disease. To this end, parameters such as the TGR (Tumor Growth Rate), TGK (Tumor Growth Kinetics), and TTF (Time to Treatment Failure) have been proposed. Although the definition of hyperprogression is not consistent among research groups, it may be depicted as a RECIST progression at the first on-treatment scan with at least a doubling in growth pace when comparing pre- and post-treatment periods. Unlike pseudoprogression, patients displaying hyperprogression present worse survival outcomes. This phenomenon has been independently associated to older age, higher metastatic load, and previous irradiation, but remarkably failed to show association to tumor burden or aggressive pre-treatment growth. Despite the pivotal interest of recognizing subjects at increased risk of hyperprogression, only MDM2 amplification and EGFR aberrations have been described as potential biomarkers and require further validation. In addition, tumor mutation burden and circulating DNA may be valuable to this purpose. This work provides an update on epidemiology, clinical predictors, biomarkers, and a plausible molecular rationale of hyperprogressive disease after immunotherapy.

Updating experimental models of diabetic cardiomyopathy.

Diabetic cardiomyopathy entails a serious cardiac dysfunction induced by alterations in structure and contractility of the myocardium. This pathology is initiated by changes in energy substrates and occurs in the absence of atherothrombosis, hypertension, or other cardiomyopathies. Inflammation, hypertrophy, fibrosis, steatosis, and apoptosis in the myocardium have been studied in numerous diabetic experimental models in animals, mostly rodents. Type I and type II diabetes were induced by genetic manipulation, pancreatic toxins, and fat and sweet diets, and animals recapitulate the main features of human diabetes and related cardiomyopathy. In this review we update and discuss the main experimental models of diabetic cardiomyopathy, analysing the associated metabolic, structural, and functional abnormalities, and including current tools for detection of these responses. Also, novel experimental models based on genetic modifications of specific related genes have been discussed. The study of specific pathways or factors responsible for cardiac failures may be useful to design new pharmacological strategies for diabetic patients.

Activation of toll-like receptors and inflammasome complexes in the diabetic cardiomyopathy-associated inflammation.

Diabetic cardiomyopathy is defined as a ventricular dysfunction initiated by alterations in cardiac energy substrates in the absence of coronary artery disease and hypertension. Hyperglycemia, hyperlipidemia, and insulin resistance are major inducers of the chronic low-grade inflammatory state that characterizes the diabetic heart. Cardiac Toll-like receptors and inflammasome complexes may be key inducers for inflammation probably through NF-κB activation and ROS overproduction. However, metabolic dysregulated factors such as peroxisome proliferator-activated receptors and sirtuins may serve as therapeutic targets to control this response by mitigating both Toll-like receptors and inflammasome signaling.

Methylmercury decreases cellular excitability by a direct blockade of sodium and calcium channels in bovine chromaffin cells: an integrative study.

Methylmercury, a potent environmental pollutant responsible for fatal food poisoning, blocked calcium channels of bovine chromaffin cells in a time- and concentration-dependent manner with an IC50 of 0.93 µM. This blockade was not reversed upon wash-out and was greater at more depolarising holding potentials (i.e. 21 % at -110 mV and 60 % at -50 mV, after 3 min perfusion with methylmercury). In ω-toxins-sensitive calcium channels, methylmercury caused a higher blockade of I Ba than in ω-toxins-resistant ones, in which a lower blockade was detected. The sodium current was also blocked by acute application of methylmercury in a time- and concentration-dependent manner with an IC50 of 1.05 µM. The blockade was not reversed upon wash-out of the drug. The drug inhibited sodium current at all test potentials and shows a shift of the I-V curve to the left of about 10 mV. Intracellular dialysis with methylmercury caused no blockade of calcium or sodium channels. Voltage-dependent potassium current was not affected by methylmercury. Calcium- and voltage-dependent potassium current was also drastically depressed. This blockade was related to the prevention of Ca(2+) influx through voltage-dependent calcium channels coupled to BK channels. Under current-clamp conditions, the blockade of ionic current present during the generation and termination of action potentials led to a drastic alteration of cellular excitability. The application of methylmercury greatly reduced the shape and the number of electrically evoked action potentials. Taken together, these results point out that the neurotoxic action evoked by methylmercury may be associated to alteration of cellular excitability by blocking ionic currents responsible for the generation and termination of action potentials.

Adaptation of the medullary thick ascending limb to dietary protein intake.

Since the renal growth response to a high-protein diet is characterized by prominent hypertrophy of the medullary thick ascending limb of Henle's loop (MTAL), the functional and metabolic adaptations of this nephron segment to dietary protein were investigated. MTAL suspensions were obtained from rats fed equal amounts of isocaloric food containing either 10% (LP) or 32% (HP) casein for 4-6 weeks. The results show that intact MTAL of HP rats exhibit a blunted respiration rate, sodium pump activity, hormone-sensitive cAMP production and leucine oxidation rate in comparison with those of LP rats. On the other hand, adenylate cyclase and leucine transaminase activities, measured on permeabilized or homogenized MTAL, are enhanced by a HP diet. We conclude that the MTAL adapts to high dietary protein by increasing its maximal enzyme activities, but certain factors, present in intact cells, limit transport and metabolism in HP- more than in LP-fed rats. This reduced function per unit MTAL protein in HP rats is more than compensated for by hypertrophy of the MTAL tissue mass.

Beta-adrenergic-cyclic AMP signalling pathway modulates cell function at the transcriptional level in 3T3-F442A adipocytes.

We studied the role of cAMP in the regulation of the expression of the adipsin gene and of some other adipose-specific genes including lipoprotein lipase (LPL), glycerophosphate dehydrogenase (G3PDH), and adipocyte P2 (aP2) in 3T3-F442A adipocytes. Northern blot analysis of isoproterenol (10(-6) M)-, forskolin (10(-5) M)- or 8-bromo-cAMP (10(-3) M)-treated adipocytes showed that the steady-state levels of adipsin mRNA were strongly reduced in a time-dependent and reversible manner. The concentration of isoproterenol giving a half-maximal effect in the down-regulation of the adipsin message was approximately 5 x 10(-8) M. Similarly, cell treatment by forskolin elicited a down-regulation of LPL and G3PDH mRNA levels but did not alter aP2 mRNA level. As determined by nuclear run-on assays, the rate of transcription of adipsin, LPL and G3PDH in isoproterenol-treated adipocytes was respectively 3, 3, and 2 times lower than in control adipocytes. These results indicate (1) that cAMP plays a dominant antilipogenic role in the fat cell through the transcriptional down-regulation of the expression of two major genes involved in triglyceride biosynthesis; (2) that cAMP does not reverse the adipocyte character; (3) hence, that cAMP suppresses adipsin expression at the transcriptional level, providing additional support for the role of adipsin protein in adipocyte metabolism.

Adipsin gene expression in 3T3-F442A adipocytes is posttranscriptionally down-regulated by retinoic acid.

Retinoic acid (RA) has been shown to inhibit the differentiation of 3T3 adipogenic cell lines. In this report, the steady-state levels of several adipose-specific mRNAs were studied in mature adipocytes treated with RA. Northern blot analysis showed that, following a 24-h exposure of 3T3-F442A adipocytic cultures to RA (10 microM), there was a 4-5-fold decrease of adipsin mRNA level. In contrast, actin, adipocyte P2, lipoprotein lipase, and glycerophosphate dehydrogenase mRNA levels were unchanged during the same interval. The rate of adipsin and actin gene transcription, assessed by nuclear run-on assays, remained unchanged in adipocytes exposed to RA. The half-life (t1/2) of adipsin mRNA, determined by pulse-chase with [3H] uridine, was greatly shortened in RA-treated adipocytes (t1/2 approximately 7.3 h) as compared with untreated cells (t1/2 approximately 37.6 h). Conversely, actin mRNA stability was not altered by the drug. These results suggest that RA can specifically down-regulate adipsin expression in adipocytes at a posttranscriptional level without inducing the reversal of adipocyte differentiation.

The antiglucocorticoid RU38486 is a potent accelerator of adipose conversion of 3T3-F442A cells.

We examined the effects of RU38486, a potent glucocorticoid and progestin antagonist, upon several aspects of 3T3-F442A adipocyte differentiation. RU38486 accelerated the onset of differentiation, as monitored by cell morphological changes, accumulation of lipid droplets and widespread increases in the rate of expression of several enzyme adipose markers and specific mRNAs. RU38486, at a maximal concentration of 1 microM, dramatically hastened the emergence of both fatty-acid synthetase (FAS) and glycerol-3-phosphate dehydrogenase (G3PDH) enzyme activities (550% and 450% above control values 4 days after confluence, respectively). RU38486 induction of G3PDH-specific activity ran parallel to an increase in G3PDH mRNA content (2.4-fold the control content 4 days after confluence). Moreover, RU38486-treated cells exhibited enhancement of adenylate cyclase sensitivity to both isoproterenol and ACTH (160% and 350% above control activities 8 days after confluence, respectively). While the level of expression of lipogenic markers reached similar values at the mature stage, RU38486 enabled cells to acquire hypersensitivity in terms of ACTH-stimulated adenylate cyclase activity. Similarly, adipsin gene expression was highly potentiated by the drug at day 15 post-confluence (5-fold the control value). RU38486 responsiveness observed in differentiating 3T3-F442A cells is dependent upon their prior developmental activation; none of the studied markers could be induced by the drug in the undifferentiating 3T3-C2 cell subclone. Finally, this antiglucocorticoid appears to be a useful tool for studies on adipose conversion in vitro; it could permit a re-evaluation of the role of glucocorticoids in the understanding of adipocyte development.

Decreased biosynthesis of actin and cellular fibronectin during adipose conversion of 3T3-F442A cells. Reorganization of the cytoarchitecture and extracellular matrix fibronectin.

Differentiation of 3T3-F442A cells was accompanied by changes in cell morphology, decreased synthesis and assembly of actin and fibronectin. The network of microfilament stress fibers detected with NBD-phallacidin was altered during adipose conversion of 3T3-F442A cells. Parallel to this, the disappearance of fibrillar bundles of extracellular matrix fibronectin was observed by immunofluorescence staining. The pericellular fibronectin content, detected by immunoblotting, strongly diminished during the differentiation process. An altered rate of biosynthesis of both proteins was also measured by [35S]-methionine pulse-labeling and immunoprecipitation. A 4-5-fold decrease in cellular fibronectin synthesis was observed in adipocytes compared to control preadipocytes. Conversely, non-differentiating 3T3-C2 control cells did not reorganize either the cytoskeletal architecture or the extracellular matrix fibronectin in the resting state. These results suggest that the decreased rate of biosynthesis of cell-associated fibronectin is correlated with that of actin. Moreover, both events can essentially be ascribed to differentiation.