Magdalena Białas

Nicotinamide N-methyltransferase (NNMT) and 1-methylnicotinamide (MNA) in experimental hepatitis induced by concanavalin A in the mouse

Nicotinamide N-methyltransferase (NNMT), which converts nicotinamide (NA) to 1-methylnicotinamide (MNA), is up-regulated in the cirrhotic liver. Because MNA displays PGI(2)-dependent anti-inflammatory effects, the up-regulation of NNMT may play a regulatory role in liver inflammation. In the present work, we analyzed changes in NNMT activity in the liver and concomitant changes in the concentration of endogenous MNA in plasma in T-cell dependent hepatitis induced by concanavalin A (ConA) in BALB/c mice. Furthermore, we tested whether exogenous MNA possessed a protective effect against ConA-induced hepatitis. Development of liver injury induced by ConA (10 mg/kg, iv) was characterized by measurements of plasma concentration of alanine aminotransaminase (ALT), inflammatory cytokines (INF gamma and TNFalpha) and by histopathological examination. ConA-induced hepatitis was characterized by an early activation of inflammatory cytokines (IFN gamma; from below 0.05 ng/ml to 23.72 +/- 8.80 ng/ml; TNFalpha;from 0.07 +/- 0.01 ng/ml to 0.71 +/- 0.12 ng/ml, 2 h after ConA), an elevation of ALT (from 40.65 +/- 3.2 U/l to 5,092.20 +/- 1,129.05 U/l, 8 h after ConA) and by morphological signs of severe liver inflammation and injury (24 h after ConA). In mice injected with ConA, NNMT activity in the liver was up-regulated approximately 2-fold to 3-fold, 8-24 h after ConA injection. The concentration of MNA and its metabolites (Met-2PY and Met-4PY) in plasma were elevated approximately 2-fold 8 h after ConA injection. Exogenous MNA (100 mg/kg, iv) diminished ConA-induced liver injury, and this effect was reversed by an antagonist of the prostacyclin receptor, RO 3244794 (10 mg/kg, po). In conclusion, the present study demonstrated that hepatic NNMT activity and MNA concentration in plasma significantly increased during the progression of ConA-induced hepatitis in mice. This response may play a hepatoprotective role compatible with the PGI(2)-releasing properties of MNA.

CDH1 gene promoter hypermethylation in gastric cancer - Relationship to Goseki grading, microsatellite instability status, and EBV invasion

Hypermethylation of the CDH1 promoter region seems to be the most common epigenetic mechanism in this gene silencing in gastric cancer. In this study, CDH1 promoter hypermethylation was observed in 54.8% (46/84) of the analyzed sporadic gastric carcinomas. We introduce a new relation: clustering of Goseki grading into 3 grade was determined by CDH1 promoter hypermethylation. The percentage of methylation in Goseki III cancers was significantly higher (83%) when compared with other grades; the lowest proportion was detected in IV (36%) and II (38%) groups, whereas grade I demonstrated typical percentage of promoter hypermethylation. A novel polymorphism R732R in exon 14 of the CDH1 gene was detected by mutational analysis. Additionally, all cases with the MSI-high phenotype revealed CDH1 promoter hypermethylation. In MSI-low and MSS gastric cancers, this percentage was lower, reaching 71% and 41%, respectively. Moreover, the methylation status was correlated with the LOH phenotype. We detected CDH1 promoter hypermethylation in all EBV-positive gastric cancers (5/5), whereas methylation in the EBV-negative group occurred in 58% of cases. We also report that “methylated” tumors were slightly larger than “nonmethylated,” whereas the second group revealed a higher probability of longer patient survival, though these relationships were not statistically significant. These results suggest that downregulation of E-cadherin, caused by promoter hypermethylation, in sporadic gastric carcinomas may be associated with a worse prognosis and specific tumor phenotype.

Mitochondrial Aldehyde Dehydrogenase Activation by Alda‐1 Inhibits Atherosclerosis and Attenuates Hepatic Steatosis in Apolipoprotein E‐Knockout Mice

Background Mitochondrial dysfunction has been shown to play an important role in the development of atherosclerosis and nonalcoholic fatty liver disease (NAFLD). Mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme responsible for the detoxification of reactive aldehydes, is considered to exert protective function in mitochondria. We investigated the influence of Alda‐1, an activator of ALDH2, on atherogenesis and on the liver steatosis in apolipoprotein E knockout (apoE−/−) mice. Methods and Results Alda‐1 caused decrease of atherosclerotic lesions approximately 25% as estimated by “en face” and “cross‐section” methods without influence on plasma lipid profile, atherosclerosis‐related markers of inflammation, and macrophage and smooth muscle content in the plaques. Plaque nitrotyrosine was not changed upon Alda‐1 treatment, and there were no changes in aortic mRNA levels of factors involved in antioxidative defense, regulation of apoptosis, mitogenesis, and autophagy. Hematoxylin/eosin staining showed decrease of steatotic changes in liver of Alda‐1‐treated apoE−/− mice. Alda‐1 attenuated formation of 4‐hydroxy‐2‐nonenal (4‐HNE) protein adducts and decreased triglyceride content in liver tissue. Two‐dimensional electrophoresis coupled with mass spectrometry identified 20 differentially expressed mitochondrial proteins upon Alda‐1 treatment in liver of apoE−/− mice, mostly proteins related to metabolism and oxidative stress. The most up‐regulated were the proteins that participated in beta oxidation of fatty acids. Conclusions Collectively, Alda‐1 inhibited atherosclerosis and attenuated NAFLD in apoE−/− mice. The pattern of changes suggests a beneficial effect of Alda‐1 in NAFLD; however, the exact liver functional consequences of the revealed alterations as well as the mechanism(s) of antiatherosclerotic Alda‐1 action require further investigation.

Single center outcomes of laparoscopic transperitoneal lateral adrenalectomy – Lessons learned after 500 cases: A retrospective cohort study

INTRODUCTION Although laparoscopic adrenalectomy is considered relatively safe, the results of treatment vary depending on the profile of the hospital. We would like to present our experience with laparoscopic surgery of the adrenals. METHODS We conducted a retrospective cohort study of consecutive patients operated for adrenal tumours in the years 2003-2014. The study group included 175 (35%) men and 325 (65%) women. The entire group was divided into 4 cohorts of 125 consecutively operated patients. Primary outcomes were operative measures (operative time, its correlation with tumour size, blood loss, conversion rate, use of peritoneal drainage). Secondary outcomes were the intra- and postoperative complications (using the Clavien-Dindo classification), histological type of the tumours and length of hospital stay. RESULTS There were no differences between groups in terms of the size and location of the tumour. The mean operative time in each group was 85.7; 83.7; 89.6; 104.6 min (p < 0.001). The operative time correlated to the size of the tumour. There were no differences in the conversion rates as well as in the blood loss. However, it was observed that the complication rate was declining in subsequent subgroups (14.4%, 11.2%, 8% and 5.6%, respectively, p = 0.013). Length of hospital stay was 4.9 days, 3.9 days, 2.9 days, 2.4 days, respectively (p < 0.001). CONCLUSION The results of laparoscopic adrenalectomy depend not only on the experience of the single surgeon, but on the whole team involved in perioperative care. In high volume centers with extensive experience in surgery of adrenals, this technique may provide an alternative to open surgery, also in selected cases of malignant tumours.

The relationship between gastric cancer cells circulating in the blood and microsatellite instability positive gastric carcinomas.

Cancers characterized by microsatellite instability may be biologically different from their counterparts with stable microsatellite sequences. Circulating cancers cell present in blood prior to surgery may constitute an adverse prognostic finding.

Inactivation of Heparin by Cationically Modified Chitosan

This study was performed to evaluate the ability of N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), the cationically modified chitosan, to form biologically inactive complexes with unfractionated heparin and thereby blocking its anticoagulant activity. Experiments were carried out in rats in vivo and in vitro using the activated partial thromboplastin time (APTT) and prothrombin time (PT) tests for evaluation of heparin anticoagulant activity. For the first time we have found that HTCC effectively neutralizes anticoagulant action of heparin in rat blood in vitro as well as in rats in vivo. The effect of HTCC on suppression of heparin activity is dose-dependent and its efficacy can be comparable to that of protamine-the only agent used in clinic for heparin neutralization. HTCC administered i.v. alone had no direct effect on any of the coagulation tests used. The potential adverse effects of HTCC were further explored using rat experimental model of acute toxicity. When administered i.p. at high doses (250 and 500 mg/kg body weight), HTCC induced some significant dose-dependent structural abnormalities in the liver. However, when HTCC was administered at low doses, comparable to those used for neutralization of anticoagulant effect of heparin, no histopathological abnormalities in liver were observed.

Laparoscopic adrenalectomy for pheochromocytoma is more difficult compared to other adrenal tumors

Introduction Laparoscopic adrenalectomy is the gold standard for the treatment of benign adrenal tumors. However, some authors raise the problem of differences in surgery for pheochromocytoma in comparison to other lesions. Aim To compare laparoscopic adrenalectomy for pheochromocytoma and for other tumors. Material and methods Four hundred and thirty-seven patients with adrenal tumors were included in the retrospective analysis. Patients were divided into two groups: 1 (124 patients treated for pheochromocytoma) and 2 (313 patients with other types of tumor). The two groups were compared with respect to mean operative time, intraoperative blood loss, conversion rate, complication rate and the relationship of tumor size with operative time. Results The mean operative time in group 1 was 91 min, and in group 2 it was 82 min (p = 0.016). In both groups 1 and 2, tumor size correlated with operative time (p < 0.0001 and p = 0.0003, respectively). The mean blood loss in groups 1 and 2 was 117 ml and 54 ml, respectively (p = 0.0011). The complication rate in groups 1 and 2 was 4% and 4.2%, respectively (p = 0.9542). In groups 1 and 2, conversion was necessary in 2 (1.6%) and 5 (1.6%) cases, respectively (p = 0.9925). Conclusions Longer operative time and higher blood loss after laparoscopic adrenalectomy for pheochromocytoma indicate its greater difficulty. However, despite these drawbacks, minimally invasive surgery still seems to be an effective and safe method.

Study of microvessel density and the expression of vascular endothelial growth factors in adrenal gland pheochromocytomas.

Angiogenesis (neoangiogenesis), a process of neovascularization, is an essential step for local tumor growth and distant metastasis formation. We have analysed angiogenesis status: vascular architecture, microvessel density, and vascular endothelial growth factors expression in 62 adrenal pheochromocytomas: 57 benign and 5 malignant. Immunohistochemical evaluation revealed that vascular architecture and vessel density are different in the central and subcapsular areas of the tumor. Furthermore, we have observed a strong correlation between number of macrophages and microvessel density in the central and subcapsular areas of the tumor and between the expression of VEGF-A in tumor cells and microvessel density in central and subcapsular areas of the tumor. Secondary changes in these tumors influence the results and both vascular architecture and microvessel density are markedly disturbed by hemorrhagic and cystic changes in pheochromocytomas. These changes are partially caused by laparoscopic operation technique. However, no differences in vascular parameters were found between pheochromocytomas with benign and malignant clinical behavior. Our observation showed that analysis of angiogenesis, as a single feature, does not help in differentiating malignant and benign pheochromocytomas and has no independent prognostic significance. On the other hand, high microvessel density in pheochromocytoma is a promising factor for antiangiogenic therapy in malignant cases.

The influence of AICAR - direct activator of AMP-activated protein kinase (AMPK) - on liver proteome in apoE-knockout mice

Abstract There is a growing body of evidence that altered functioning of apoE may aggravate cellular energy homeostasis and stress response, leading to oxidative stress, mitochondrial dysfunction, endoplasmic reticulum (ER) stress and inflammation, leading to hypercholesterolemia, dyslipidemia, liver steatosis and neurodegeneration. One of the key cellular responses to mitochondria and ER‐stress related processes and cellular energy imbalance is AMP‐activated protein kinase (AMPK), considered as a cellular master energy sensor and critical regulator of mitochondrial homeostasis. The aim of our study was to use differential proteomics and transcriptomics approach to elucidate the effect of direct AMPK activator AICAR on liver proteome in apoE−/− mice – experimental model of atherosclerosis and moderate nonalcoholic steatosis. We applied Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) labeling and two‐dimensional chromatography coupled with mass spectrometry (2DLC‐MS/MS) MudPIT strategy, as well as RT‐PCR to investigate the changes in mitochondrial and cytosolic proteins and transcripts expression in 6‐month old AICAR‐treated apoE−/−. AICAR elicited induction of proteins related to mitochondrial &bgr;‐oxidation, protein degradation and energy producing pathways (i.a. tricarboxylic acid cycle members and mitochondrial adenylate kinase 2). On the other hand, AICAR repressed inflammatory and pro‐apoptotic markers in the apoE−/− mice liver, alongside reduction in several peroxisomal proteins, possibly suggesting induction of anti‐oxidative pexophagy. Graphical abstract Figure. No Caption available.

Mast cells influence neoangiogenesis in prostatic cancer independently of ERG status

A significant proportion of prostatic adenocarcinomas show recurrent translocation leading to ERG expression. Previously we found that ERG+ cases have higher microvessel density than negative ones. One factor influencing angiogenesis in cancer is mast cells. The aim of the present study was to evaluate the relationship between microvessels, mast cells and ERG status. Tissue microarrays prepared from 113 radical prostatectomy specimens were analyzed with immunohistochemistry for CD31, tryptase and chymase. Vascular profiles and tryptase-positive and chymase-positive cells were counted. The average number of tryptase-positive cells was 28.93/mm2 and chymase-positive cells 9.91/mm2. The average number of CD31+ vascular profiles was 352.66/mm2. The average number of tryptase-positive cells was 26.35/mm2 for ERG- cases and 32.12/mm2 for ERG+ cases. The average number of chymase-positive cells was 8.14/mm2 for ERG- cases and 12.06/mm2 for ERG+ cases. The average number of CD31+ vascular profiles was 321.34/mm2 for ERG- cases and 390.74/mm2 for ERG+ cases. The number of CD31+ vascular profiles was positively correlated with the number of tryptase-positive and chymase-positive cells (R = 0.26 and R = 0.20). In summary, we demonstrated an interrelationship between mast cells, microvascular density and ERG status in prostatic carcinoma.