Immune checkpoints in aggressive breast cancer subtypes.

Immune checkpoints are molecules referred to inhibitory pathways in the immune system that play a pivotal role in prevention of autoimmunity and oncogenesis. The aim of the study was to evaluate expression levels of selected immune checkpoints- PD-1 (programmed cell death protein 1), and PD-L1 (programmed cell death 1 ligand 1) in breast cancer patients, suitable for breast conservation and sentinel node biopsy and determine their associations with clinicopathological factors.Expression of the genes coding for PD-1 and PD-L1 was analyzed in formalin-fixed paraffin-embedded specimens using real-time PCR. mRNA expression levels were determined using beta actin (ACTB) as an endogenous control. There was a trend towards significance between higher PD-1 and PD-L1 levels in triple negative breast cancers (p=0.1). Higher PD-L1 expression was also found in aggressive breast cancer subtypes e.g. triple negative and HER2 (human epidermal growth factor receptor 2) -positive as compared with subtypes with better prognosis such as luminal A and luminal BHER2-negative (p=0.05). There was a trend towards significance in higher PD-1 levels in triple negative and HER-2 positive breast cancers (p=0.1). A statistically significant difference was found between PD-L1 expression and tumor grade (p=0.01). Elevated PD-L1 levels were noted in G3 tumors. Immunogenicity appears to be gaining importance in triple negative and HER2-positive molecular subtypes of breast cancer, and the results in this study provide a basis for further investigation into the role of immune checkpoints in breast cancer.

Apoptosis-, proliferation, immune function-, and drug resistance- related genes in ER positive, HER2 positive and triple negative breast cancer.

The aim of our study was to examine an association between gene expression assessed using a 23-gene microarray and receptor status of breast cancer samples categorized as ER positive, HER2 positive and triple negative subtypes. The ER positive cohort was subsequently divided into Luminal A, Luminal B HER2 negative and Luminal B HER2 positive subtypes. Core- needle biopsies were collected from 78 female patients with inoperable locally advanced breast cancer or resectable tumors suitable for downstaging, before any treatment. Expressions of 23 genes were determined by means of TagMan Low Density Arrays. Analysis of variance was used to select genes with discriminatory potential between receptor subtypes. We introduced a correction for false discovery rates (presented as q values) due to testing multiple hypothesis. Pairwise post-hoc comparisons of receptor subtypes were performed using Tukey 's HSD test. Five genes out of a 23-gene microarray differed significantly in relation to breast cancer receptor-based subtypes. Among these five genes, we identified: BCL2 (p=0.0002, q=0.0009), MKI67 (p=0.0037, q=0.0064), IGF1R (p=0.0040, q=0.0064), FOXC1 (p=0.0113, q=0.0135) and IRF1 (p=0.0435, q=0.0416) as ones showing ER positive, HER2 positive and triple negative -subtype specific expression profiles. When incorporating Luminal A, Luminal B HER2 negative, Luminal B HER2 positive subtypes into analysis, four genes: BCL2 (p=0.0006, q=0.0034), MKI67 (p=0.0078, q=0.0198), FOXC1 (p=0.0102, q=0.0198) and IGF1R (p=0.0174, q=0.0254) were selected. Elevated levels of IGF1R and BCL2 were significantly linked with Luminal A subtype. Triple negative breast cancer subtype was associated with higher expression of IRF1, FOXC1 and MKI67. In HER2 positive cohort lower expression of all five analyzed genes was noted.

Intracellular signaling pathways involved in inhibition of PAI-1 expression by CNP in endothelial cells.

PAI-1 is a multifunctional protein stimulated by infectious agents and its activation is mediated by inflammatory cytokines such as TNFalpha. Recent studies demonstrate that natriuretic peptides, particularly C-type (CNP), can affect PAI-1 expression in bovine aortic smooth muscle cells and rat aortic endothelial cells. We have previously shown that CNP inhibits both basal and TNFalpha induced expression of PAI-1 in human endothelial cells. Herein, we describe mechanism by which CNP modulates signaling engaged in controlling PAI-1 expression in human endothelial cells. To examine which pathway initiated by TNFalpha is influenced, we tested kinase activity of MAP, PI3K/AKT and involvement of cGMP in endothelial cells exposed to CNP. CNP significantly increased cGMP level in endothelial cells. Its analogue, 8-Br-cGMP alone had no effect but significantly inhibited TNFalpha induced expression of PAI-1. Similarly, CNP and the inhibitors of ERK1/2 (PD098059) and PI3K (LY294002) attenuated PAI-1 expression induced by TNFalpha. CNP almost abolished TNFalpha induced phosphorylation of ERK1/2 but did not affect JNK phosphorylation, indicating that its effect on ERK1/2 was specific. These data suggest that CNP might function as the natural defense of vascular wall against cytokine induced PAI-1 release through its ability to inactivate PI3K/AKT and MEK/ERK pathways.

Regulation of PAI-1 concentration in platelets by systemic administration of antisense oligonucleotides to rats.

In this report we tested the effect of oligodeoxyribonucleotides antisense to PAI-1 mRNA administered into rats on PAI-1 concentration in platelets. Low doses of the antisense oligonucleotide (MPO-16R) reduced PAI-1 activity, both in rat blood plasma and platelet lysates by 20.5% and 28.7%, respectively. There was no change in platelet count after treatment with MPO-16R but treated platelets showed lower aggregability as compared with controls (37 +/- 13% and 54 +/- 12%, respectively). In an experimental model of rat arterial thrombosis, low doses of MPO-16R caused a significant delay in the occlusion time (31.8%). These data further support for the role of PAI-1 as a major determinant of arterial thrombolysis resistance and for the first time demonstrate the possibility of reduction of platelet PAI-1 concentration by antisense approach.

Impact of economic incentives on costs and benefits of occupational health and safety.

The most common type of economic incentive used in the field of health and safety is experience rating of insurance premiums. The impact of this incentive on occupational health and safety (OHS) costs in the company was analysed by comparing insurance costs with other OHS costs associated with inadequate working conditions, such as accident costs borne by a company. Accident costs were estimated on the basis of research carried out in 10 companies. Insurance costs and their adjustments according to the health and safety level in a company were calculated according to an experience rating model developed in the Central Institute for Labour Protection.

Dual regulatory effects of nitric oxide on plasminogen activator inhibitor type 1 expression in endothelial cells.

In this report we compared the mechanism by which nitric oxide (NO), generated exogenously and endogenously, affects the plasminogen activator inhibitor type 1 (PAI-1) expression in endothelial cells. For this purpose, we stimulated the endothelial cell line EA.hy 926 with tumour necrosis factor alpha (TNFalpha) in the presence of the exogenously NO-releasing donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine, or regulators of nitric oxide synthase (NOS) inhibitor N-nitro-L-arginine-methyl ester hydrochloride and substrate L-Arg. Expression of PAI-1 in EA.hy 926 cells was determined by measuring the level of mRNA, using relative quantitative reverse transcriptase PCR, and protein, using ELISA. In addition, we estimated the level of activation of two mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK1/2), in the cells before and after treatment with TNFalpha, in the presence or absence of NO donors and inhibitors. In contrast to exogenously released NO that significantly reduced mostly basal PAI-1 expression, endogenously generated NO by NOS potentiated TNFalpha-induced upregulation of PAI-1 expression. Exogenously and endogenously generated NO causes different effects on activation of the MAPKs ERK1/2 and JNK1/2. Specifically, the SNP-released NO activates only ERK1/2, while endogenously generated NO in a pathway induced by TNFalpha activates both MAPKs. Thus our data indicate that due to different cellular locations and mechanisms of generation, NO may participate in various signalling pathways leading to opposite effects on PAI-1 expression in endothelial cells.

The cytotoxicity of anti-PAI-1 oligonucleotides and their conjugates.

The cytotoxicity of anti-PAI-5 hexadecanucleotides (phosphodiesters and phosphorothioates) and their conjugates with lipophilic alcohols was tested in EA.hy 926 hybrid endothelial cells. Some cytotoxicity was found for cholesteryl and bornyl conjugates at concentrations higher than those used for antisense inhibition experiments.

Inhibition of plasminogen activator inhibitor release in endothelial cell cultures by antisense oligodeoxyribonucleotides with a 5'-end lipophilic modification.

A series of conjugates containing residues of lipophilic alcohols covalently bound to 5' end of oligodeoxyribonucleotides targeted against human plasminogen activator inhibitor (PAI-1) mRNA was synthesized via the oxathiaphospholane approach. The highest anti-PAI-1 activity in EA.hy 926 endothelial cell cultures was found for conjugates containing menthyl or heptadecanyl groups linked with an oligonucleotide complementary to a segment of human PAI-1 mRNA. The phosphodiester antisense oligonucleotides, which otherwise exhibit only limited anti-PAI-1 activity, were found to be more active than phosphorothioate oligonucleotides when conjugated to lipophilic alcohol residues. For menthyl conjugates an evidence of antisense mechanism of inhibition was found.

Interferon gamma bound to endothelial cells is phosphorylated by ecto-protein kinases.

The presence of protein kinase activity and its phosphorylated products has been demonstrated on the outer surface of the plasma membrane of endothelial cells. Extracellular phosphorylation was detected by incubation of primary endothelial cells (HUVEC's) and endothelial cell line EA.hy 926 with [gamma-32P]ATP. The reaction products were subjected to SDS/PAGE, autoradiography and scanning densitometry. Under the experimental conditions, five proteins with apparent molecular masses of 19, 23, 55, 88, and 110 kDa were prominently phosphorylated in both types of cells. Phosphorylation of the 19 kDa protein was the most rapid reaching maximum after 60 s and then the protein became dephosphorylated. Ecto-protein kinases responsible for the surface labeling of membrane proteins were characterized by using (a) protein kinase C inhibitors: K-252b, chelerythrine chloride, and [Ala113] myelin basic protein (104-118), (b) protein kinase A inhibitor Kemptide 8334, and (c) casein kinase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB). Stimulation of endothelial cells with tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) is associated with 20-80% reduction of extracellular phosphorylation of all membrane proteins. IFN gamma bound to membrane receptors becomes rapidly phosphorylated. Only in the case of IFN gamma it was associated with the appearance of a strongly phosphorylated band of 17 kDa corresponding to IFN gamma itself. Phosphorylation of this 17 kDa exogenous substrate was prevented by an ecto-kinase inhibitor K-252b. The existence of ecto-phosphoprotein phosphatase activity in endothelial cells was evidenced by testing the effect of microcystin LR--a membrane impermeable reagent that inhibits both PP-1 and PP-2a phosphoprotein phosphatases. The extent of phosphorylation of 19 kDa and 110 kDa phosphoproteins significantly increased in the presence of microcystin. Our results suggest the presence of at least two ecto-kinase activities on endothelial cells that may play a significant role(s) in the regulation of cytokines function.

Synthesis, biochemical and biological studies on oligonucleotides bearing a lipophilic dimethoxytrityl group.

Dimethoxytritylphosphono-oligonucleotide conjugates have been prepared. They are totally resistant to nucleases present in human serum and do not affect cleavage of a complementary oligoribonucleotide by RNase H. Conjugates possessing a phosphate backbone gave better antisense inhibition of expression of plasminogen activator inhibitor type-1 within endothelial cells as compared with unconjugated oligonucleotides.

Phosphorothioate oligodeoxyribonucleotides antisense to PAI-1 mRNA increase fibrinolysis and modify experimental thrombosis in rats.

The effect of systemic inhibition of PAI-1 expression in rats by PS-16R, a phosphorothioate analogue of hexadecadeoxyribonucleotide complementary to a signal peptide coding sequence of rat PAI-1 mRNA, on PAI-1 activity in blood plasma and thrombus formation was studied in rat models for experimental thrombosis. In previous in vitro studies, oligonucleotides of PS-16R family have been shown to inhibit efficiently PAI-1 synthesis in endothelial cells by antisense mechanism. When PS-16R was administered intravenously as a single bolus injection (1 to 5 mg per rat), it produced a significant reduction in PAI-1 activity of blood plasma. This effect was both time- and concentration-dependent. Under the same conditions, three groups of rats were treated with control oligodeoxynucleotides such as PS-16R with double mismatches, with scrambled sequence, and an oligodeoxynucleotide with sense sequence (complementary to PS-16R), respectively. Based on these preliminary experiments, a low dose of 1.5 mg per rat was selected to produce approximately 20-30% reduction of PAI-1 activity in blood plasma and the effect of such a decrease in PAI-1 expression was tested on thrombus formation in two rat models for experimentally induced thrombosis. Such a limited decrease in PAI-1 activity produced a significant antithrombotic effect in the arterial thrombosis model. There was a profound delay in the occlusion time in rats treated with PS-16R when compared to control animals (80 +/- 3 and 55 +/- 3 h, respectively), although blood plasma activity of PAI-1 in the same groups of rats differed only by 20%. There was also a tendency to reduce both an incidence of venous thrombosis (58.33 and 68.11%, respectively) and thrombus weight (2.1 +/- 0.4 and 2.9 +/- 0.9 mg, respectively) in the animals treated with PS-16R. However, this effect was not significant. Thus, low dose of PS-16R through inhibition of PAI-1 synthesis in targeted cells in rats reduced PAI-1 activity in blood plasma and protected against arterial thrombus formation in the rat.

Stereodependent inhibition of plasminogen activator inhibitor type 1 by phosphorothioate oligonucleotides: proof of sequence specificity in cell culture and in vivo rat experiments.

Hexadecadeoxyribonucleotides complementary to a fragment of human PAI-1 mRNA located upstream of the start codon and their phosphorothioate analogs were studied in cultured HUVECs as sequence-dependent inhibitors of PAI-1 expression. The activity of the random mixture of diastereomers of phosphorothioate hexadecanucleotide PS-16H has been compared with that of isosequential, stereoregular [All-Sp] and [All-Rp] isomers. The highest inhibitory effect on PAI-1 synthesis was observed with the [All-Sp] diastereomer. Stereorandom phosphorothioate oligonucleotide PS-16R complementary to the same region of rat PAI-1 mRNA, when injected into tail vein of rats, substantially decreased the level of PAI-1 in blood plasma.

Modulation of plasminogen activator inhibitor type-1 biosynthesis in vitro and in vivo with oligo(nucleoside phosphorothioate)s and related constructs.

Oligonucleotides with a nucleotide sequence complementary to various regions of human plasminogen activator inhibitor type-1 (PAI-1) mRNA have been studied as antisense inhibitors of expression of PAI-1 protein in cultured cells [human umbilical vein endothelial cells (HUVEC), human aortic smooth muscle cells, human hybrid endothelial cells]. Hexadeca(deoxyribonucleoside phosphorothioate) 13 complementary to a fragment of a signal peptide PAI-1 mRNA was found to be most active, giving ca. 70% inhibition of PAI-1 release in a time- and dose-dependent way. The stereo-regular All-S(P) and All-R(P) diastereomers of 13 were studied and found to inhibit PAI-1 synthesis in HUVEC in a stereo-dependent manner, with the All-S(P) diastereomer considerably more active than the stereo-random construct and All-R(P) isomer. The observed stereo-dependent activity of oligonucleotide phosphorothioate constructs is presumably governed by their resistance to nucleases. The corresponding phosphodiester analogue of 13 was not active unless covalently bound at its 5'-end to a lipophilic alcohol residue (menthol, heptadecanol). The observed antisense activity of phosphodiester oligonucleotide bioconjugates in cultured human hybrid endothelial cells was paralleled by their increased stability in human plasma with respect to unconjugated oligonucleotide. The oligo(deoxyribonucleoside phosphorothioate) complementary to the same signal peptide region of rat PAI-1 mRNA was found to reduce the PAI-1 level in blood plasma of rats after intravenous administration into the tail vein. The effect was both time- and dose-dependent. The same oligonucleotide was found to protect against arterial thrombus formation in the rat (lower incidence of venous thrombosis, lower thrombus weight, and increased occlusion time in experimentally induced thrombosis). An anti-PAI-1 inhibitory activity has been independently reported for a 20-mer oligo(2'-O-methyl-ribonucleoside phosphorothioate) complementary to a 3'-untranslated region of human PAI-1 mRNA in cultured HUVEC and human aortic smooth muscle cells.

Changes in platelet response to adenosine 5'-diphosphate caused by verapamil.

Verapamil is widely used in the treatment of patients with coronary artery disease. The effect of verapamil on vascular smooth muscle cells is well documented. This effect is mediated by the inhibition of calcium fluxes across plasma membranes. Some data suggest that verapamil may affect platelet functions in thrombosis, but those observations were made for much higher verapamil concentration than could be achieved in vivo. Our current investigations are focused on an influence of low doses of verapamil (0.1-1.0 microM) on platelet response to ADP. We have found that verapamil at concentration of 0.1 microM can inhibit platelet aggregation (by 10%) evoked in PRP by 1.0-1.5 microM ADP. Moreover, the inhibitory effect is potentiated by prolonged time of platelet preincubation with verapamil. On the other hand, we have found a significant reduction in the number of fibrinogen receptors exposed on the platelet surface of patients (n = 21) treated with therapeutic doses (240 mg/day) of verapamil during two weeks of drug administration. The mean number of exposed receptors was reduced from 75,000 to 40,000 per platelet, with significance p < 0.0001. In vitro platelet preincubation with verapamil, even in much higher concentrations, did not affect fibrinogen binding to ADP activated platelets. It suggests, that in vitro exposure of platelets to verapamil for a short time has no effect on the expression of fibrinogen receptors on platelets, but prolonged in vivo interaction of this drug with platelets results in reduction of the fibrinogen receptor exposition. Thus, observed inhibition of platelet aggregation does not relay on a simple reduction of the number of exposed receptors, but intraplatelet signalling has to be affected. In fact, we have observed, in platelets pretreated with low doses of verapamil, significantly reduced release of calcium ions upon activation by ADP, whereas the calcium influx under such conditions does not seem to be affected.

Internalization of oligodeoxynucleotide antisense to type-1 plasminogen activator inhibitor mRNA in endothelial cells: a three-dimensional reconstruction by confocal microscopy.

A three-dimensional reconstruction analysis of localization of phosphodiester and phosphorothioate oligonucleotide antisense to type-1 plasminogen activator inhibitor (PAI-1) mRNA within endothelial cells is described. When EA.hy 926 cells were incubated with fluorescently labelled phosphodiester (PO-16) or phosphorothioate (PS-16) oligonucleotides at low, not cytotoxical concentrations, the relative brightness composition of the images of the particular samples was much higher for PS-16 than PO-16 and dependent upon the extracellular concentration and the incubation time. The 3-D reconstructions based on the series of optical sections of the samples, spaced every 1.5 microns, showed the punctuate accumulation of the oligonucleotides and a striking difference in a spatial distribution between PO-16 and PS-16 within the cytoplasm. Even after 24 h incubation of endothelial cells with 2.5 microM of PO-16 and PS-16 oligonucleotides, there was a predominant oligonucleotide localization within the cytoplasm and only traces of oligonucleotides could be seen in the cell nucleus and/or perinuclear organelles.

Surface phosphorylation by ecto-protein kinase C in brain neurons: a target for Alzheimer's beta-amyloid peptides.

The powerful regulatory machinery of protein phosphorylation operates in the extracellular environment of the brain. Enzymatic activity with the catalytic specificity of protein kinase C (PKC) was detected on the surface of brain neurons, where it can serve as a direct target for neurotrophic and neurotoxic substances that control neuronal development and cause neurodegeneration. This activity fulfilled all the criteria required of an ecto-protein kinase (ecto-PK). Detailed analysis of surface protein phosphorylation in cultured brain neurons using specific exogenous substrates (casein, histones, and myelin basic protein), inhibitors (PKC-pseudosubstrate 19-36; K252b) and antibodies (anti-PKC catalytic region M.Ab.1.9, antibodies to the carboxy-terminus of eight PKC isozymes) revealed several types of ecto-PK activity, among them ecto-PKs with catalytic specificity of the PKC isozymes zeta and delta. The activity of the neuronal ecto-PKC is constitutive and not stimulated by phorbol esters. the phosphorylation of a 12K/13K surface protein duplex by ecto-PKC-delta was found to be developmentally regulated, with peak activity occurring during the onset of neuritogenesis. Alzheimer's amyloid peptides beta 1-40 and beta 25-35 applied at neurotrophic concentrations stimulated the phosphorylation of endogenous substrates of ecto-PKC activity in brain neurons but inhibited specifically this surface phosphorylation activity with the same dose-response relationships that cause neurodegeneration. As may be expected from a relevant pathophysiological activity, beta-amyloid peptide 1-28 did not inhibit this surface phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of fibrinogen receptors on platelets of uremic patients is correlated with the content of GPIIb and plasma level of creatinine.

Platelets of uremic patients, activated with ADP, exposed less fibrinogen receptors than control platelets, i.e. 24612 +/- 5541 and 33400 +/- 4302 receptors per platelet, respectively. However, this difference was not statistically significant. When compared with the total number of GPIIb/IIIa complexes, quantified from platelet glycoprotein IIb (GPIIb) contents, active receptors on the platelet surface represented 13.6% and 35.1% of total pool of fibrinogen receptors in uremic and control platelets, respectively. The number of exposed fibrinogen receptors was positively correlated with the amount of GPIIb copies in both uremic and normal platelets. In uremic platelets, both the number of exposed receptors and the number of GPIIb copies were correlated with the plasma creatinine concentration suggesting, that binding of fibrinogen to uremic platelets depends upon the degree of renal failure. Uremic platelets contain similar amounts of fibrinogen as control ones i.e. 13.2 +/- 2.3 micrograms and 17.6 +/- 2.2 micrograms per 1 x 10(8) platelets, respectively. Whereas for beta-thromboglobulin (beta-TG) there was a significant difference of 392 +/- 102 ng and 803 +/- 202 ng per 1 x 10(8) platelets, respectively. Reduced beta-TG content in uremic platelets suggests limited platelet activation in vivo. These results support the concept that uremic platelets have impaired functions and indicate that there is a relationship between the progression in renal failure and disability of platelets in thrombosis.