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Background: The immunotherapy has revolutionized the world of oncology in the last decades with considerable advantages in terms of overall survival in cancer patients. The association of Pembrolizumab and Trastuzumab was recently proposed in clinical trials for the treatment of Trastuzumab-resistant advanced HER2-positive breast cancer. Although immunotherapies are frequently associated with a wide spectrum of immune-related adverse events, the cardiac toxicity has not been properly studied., Purpose: We studied, for the first time, the putative cardiotoxic and pro-inflammatory effects of Pembrolizumab associated to Trastuzumab., Methods: Cell viability, intracellular calcium quantification and pro-inflammatory studies (analyses of the production of Interleukin 1β, 6 and 8, the expression of NF-kB and Leukotriene B4) were performed in human fetal cardiomyocytes. Preclinical studies were also performed in C57BL6 mice by analyzing fibrosis and inflammation in heart tissues., Results: The combination of Pembrolizumab and Trastuzumab leads to an increase of the intracellular calcium overload (of 3 times compared to untreated cells) and to a reduction of the cardiomyocytes viability (of 65 and 20-25%, compared to untreated and Pembrolizumab or Trastuzumab treated cells, respectively) indicating cardiotoxic effects. Notably, combination therapy increases the inflammation of cardiomyocytes by enhancing the expression of NF-kB and Interleukins. Moreover, in preclinical models, the association of Pembrolizumab and Trastuzumab increases the Interleukins expression of 40-50% compared to the single treatments; the expression of NF-kB and Leukotriene B4 was also increased., Conclusion: Pembrolizumab associated to Trastuzumab leads to strong cardiac pro-inflammatory effects mediated by overexpression of NF-kB and Leukotriene B4 related pathways., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The subcommissural organ (SCO) is a circumventricular organ of glial origin typical of all vertebrates. The SCO releases its secretion into the third ventricle to constitute Reissner's fibre (RF). Reportedly, in reptiles, SCO has cyclic secretory activity related to the reproductive cycle. In this immunocytochemical study we show that, in females of oviparous reptiles (Lacertidae: Podarcis sicula) and in a viviparous species (Scincidae: Chalcides chalcides), SCO secretion consists of hormones, in part of the oxytocin-like (OXY-like) type. The amount of OXY-like material in the cells and in the third ventricle varies according to the different stages of the reproductive cycle. In the oviparous species, OXY-like hormone secretion can be induced by FSH administration at 28 degrees C, in the period of winter reproductive stasis as well. In the viviparous skink, showing an annual single ovulatory cycle, OXY-like secretion is present in the basal region of the cells, and is released into the third ventricle only at delivery. The role of an OXY-like hormone in the SCO is here discussed in relation to the different stages of the reproductive cycle. Its influence on the hypothalamus-hypophysis-gonad axis and its role in the transport of eggs into the ducts in the oviparous species, and at delivery in the viviparous one, are also suggested.

We have previously reported that polyamines interfere with protein kinase C-membrane interactions. With the aim of clarifying the influence of the relationship between calcium and polyamines on this process we have investigated the effect of spermine on the formation of active protein kinase C-membrane complexes as a function of Ca++ concentrations. Protein kinase C, purified from rat brain, was allowed to interact with phospholipid vesicles of defined composition. The active complex protein kinase C-liposomes was determined by its ability to bind radioactive phorbol ester with an exact 1:1 stoichiometry. The results show that, at Ca++ levels below 0.1 microM, spermine inhibits the formation of complexes between protein kinase C and membranes. At higher Ca++ concentrations, spermine does not prevent the association process but does influence the ratio between the enzyme molecules irreversibly inserted into the membrane and those reversibly associated with it. We have also demonstrated that spermine, by reducing the density of acidic component of liposomes, influences the calcium requirement for protein kinase C-membrane binding. This study indicates that spermine may regulate the activation of protein kinase C and affects the calcium requirement for the association of this enzyme with the phospholipid bilayer. The results suggest a possible role for polyamines in signal transduction when protein kinase C is involved.

Protein kinase C is reported to exist in two membrane-bound states: a reversible one which can be dissociated by calcium chelators (membrane-associated form) and an irreversible one which is chelator stable (membrane-inserted form). In the present work the effects of a naturally occurring polyamine (spermine) on the membrane-associated and membrane-inserted forms of protein kinase C were investigated using a reconstituted system consisting of partially purified protein kinase C from rat brain and phospholipid vesicles of defined composition. The active membrane-bound complex was conveniently determined by its ability to bind radioactive phorbol ester with an exact 1:1 stoichiometry. Our experimental data show that, in the absence of calcium ions, the amount of enzyme bound to phospholipids vesicles was dramatically reduced by the presence of spermine whereas the PDBu binding affinity was not significantly affected. The addition of the divalent cation increased the affinity of phorbol ester for the active complex but had no effect on Nmax; spermine added in this experimental conditions was no longer able to decrease the total number of enzyme molecules bound to liposomes. Moreover gel filtration experiments of the protein kinase C-phospholipids complex formed in the presence of calcium, indicated that polyamine added during the association process was able to reduce the extent of enzyme insertion into liposomes. Since the increase in phospholipid concentration resulted in a higher level of non-dissociable protein kinase C-liposomes complex we propose that spermine, complexing to membrane binding sites both in the absence and in the presence of Ca++, could promote binding conditions that oppose to the formation of the inserted form of the enzyme. As a consequence the distribution between the reversible and the irreversible membrane-bound forms of protein kinase C is affected.

The in vitro mechanism by which polyamines affect protein kinase C (PK C) activation process was investigated in a reconstituted system consisting of purified enzyme and phospholipid vesicles of various phosphatidylserine content. It was found that the addition of spermine greatly interferes with the association of PK C to liposomes. This tetramine, at micromolar concentrations, was most potently effective while other polyamines such as spermidine and putrescine were almost ineffective; therefore the modulatory action appeared to be structure specific. The spermine effect is dramatically influenced by the density of the phosphatidylserine present on the liposome, suggesting the complex formation with the acidic component on phospholipid vesicles to be the mechanism by which this polyamine exerts its modulatory action.

The spermine-binding activity of a cytosolic protein from chick intestine increases during embryogenesis and in the first week of life. Ornithine and S-adenosylmethionine decarboxylase activities assayed under the same experimental conditions increase showing a maximum at day 18 and 20 respectively. The behaviour of either enzyme activity is reflected in the pattern of duodenal polyamine concentration measured during the same period. The possibility that duodenal spermine-binding protein may be correlated with spermine accumulation in the tissue is discussed.

A protein fraction extracted from the liver of rats accustomed to eating during the first 8 h of a daily 12 h dark period is able to increase homologous form-B RNA polymerase activity. When the protein factor is added to RNA polymerase extracted at the end of the daily fasted period (0.9:OOh) the sensitivity of enzyme to ionic strength is modified, and resembles that of the enzyme extracted during the eating period (15:00 h) either using liver deproteinized DNA or chromatin as template. The chromatin-bound RNA polymerase activity is also modified in the presence of the factor, showing a modification of enzyme sensitivity to ionic strength similar to the one induced by food intake.

Chick duodenal mucosa contains an endogenous factor which is capable to inhibit selectively a homologous polyamine-sensitive protein kinase. The inhibitor was partially purified and characterized, and it was found to contain typical mucopolysaccharidic components. Glycosidases digestion studies, selective degradation analysis and spectrophotometric titrations with metachromatic dyes indicated that the inhibitor preparation contained two major moieties identified as heparin-like and heparan sulfate-like structures. In chick intestine the inhibitor was specific for polyamine-sensitive protein kinase since selectively interacted with it and was inert towards other cAMP-independent and cAMP-dependent protein kinases. The inhibitory effect of the endogenous factor was counteracted by naturally occurring polyamines such as spermine. The order of potency of various polyamines was: spermine greater than thermine much greater than spermidine much greater than diamines. The release of inhibition by addition of physiological concentrations of spermine was also apparent when using cytosolic proteins as endogenous phosphate acceptors. These results suggest that a possible role of polyamine in the regulation of polyamine-sensitive protein kinase in the intestine is to protect the enzyme from the inhibitory action of endogenous heparinoids.

Human histiocytic lymphoma cells (U-937) contain high affinity binding protein for 1 alpha, 25-dihydroxycholecalciferol. The Kd (0.2 nM) and sedimentation coefficient on sucrose gradients (3.7 S) are similar to those reported for 1 alpha, 25-dihydroxycholecalciferol receptor in other tissues. This secosteroid added to the culture was able to inhibit cell proliferation in a dose dependent manner. Differentiation-associated properties such as phagocytic ability, C3 rosette formation, positive reaction for alpha- naphtyl acetate esterase as well as electron microscopy examination indicate that 1 alpha, 25-dihydroxycholecalciferol induces in vitro monocyte-macrophage differentiation in this monoblast-like cell line.

The effect of polyamines on the catalytic domain of protein kinase C from rat brain was investigated. It was found that the addition of spermine strongly inhibited phosphorylation activity toward histone H1 as substrate. This tetramine, at millimolar concentrations, was most potently effective while triamines and diamines were almost uneffective, therefore the inhibitory action appeared to be structural specific. Data shown here suggest that polyamine by interacting with the catalytic domain of the enzyme may contribute to its regulation.

Human histiocytic lymphoma cells (U-937) undergo similar differentiation when incubated with the phorbol ester 12-0-tetradecanoyl phorbol-13-acetate (TPA) and 1,25-dihydroxycholecalciferol. In this action, TPA somehow implicates calcium-sensitive and phospholipid-dependent protein kinase (protein kinase C), which is rapidly and significantly affected by this inducer. On the contrary, 1,25-dihydroxycholecalciferol in its differentiating action does not involve protein kinase C thus suggesting that the secosteroid induces monocytic differentiation possible through a different mechanism of that of phorbol ester.