Multimodal decoding of human liver regeneration.

The liver has a unique ability to regenerate1,2; however, in the setting of acute liver failure (ALF), this regenerative capacity is often overwhelmed, leaving emergency liver transplantation as the only curative option3-5. Here, to advance understanding of human liver regeneration, we use paired single-nucleus RNA sequencing combined with spatial profiling of healthy and ALF explant human livers to generate a single-cell, pan-lineage atlas of human liver regeneration. We uncover a novel ANXA2+ migratory hepatocyte subpopulation, which emerges during human liver regeneration, and a corollary subpopulation in a mouse model of acetaminophen (APAP)-induced liver regeneration. Interrogation of necrotic wound closure and hepatocyte proliferation across multiple timepoints following APAP-induced liver injury in mice demonstrates that wound closure precedes hepatocyte proliferation. Four-dimensional intravital imaging of APAP-induced mouse liver injury identifies motile hepatocytes at the edge of the necrotic area, enabling collective migration of the hepatocyte sheet to effect wound closure. Depletion of hepatocyte ANXA2 reduces hepatocyte growth factor-induced human and mouse hepatocyte migration in vitro, and abrogates necrotic wound closure following APAP-induced mouse liver injury. Together, our work dissects unanticipated aspects of liver regeneration, demonstrating an uncoupling of wound closure and hepatocyte proliferation and uncovering a novel migratory hepatocyte subpopulation that mediates wound closure following liver injury. Therapies designed to promote rapid reconstitution of normal hepatic microarchitecture and reparation of the gut-liver barrier may advance new areas of therapeutic discovery in regenerative medicine.

Serum levels of ADAM10, ADAM12, ADAM17 AND ADAM28 in colorectal cancer patients.

Colorectal cancer is the third most common cancer in the world. Our study analyzed the potential significance of serum levels of selected adamalysines (ADAM10, ADAM12, ADAM17, ADAM28) in colorectal cancer patients. The study was performed on a group of 85 colorectal cancer patients (48 men, 37 women). Serum protein concentrations were measured by ELISA. The ADAMs serum level changes were analyzed according to selected clinical parameters (BMI, sex, age, clinical stage of disease). The following ranges of concentration of analyzed proteins were obtained: ADAM10 min=1.7, max=321.8 [ng/ml]; ADAM12 min=0.6, max=26.7 [ng/ml]; ADAM17 min=0.4, max=9.8 [ng/ml]; ADAM28 min=17.1, max=1545.8 [ng/ml]. In addition, it was stated that there is a relationship between the serum level of ADAM28 and the degree of the clinical stage (p less than 0.04). The obtained results could be the starting point for further research into the role of adamalysines in the development of colorectal cancer, as well as the potential predictive and prognostic value of these proteins.

Assessment of magnesium influence on fatty acid content in isolated rat hepatocytes subjected to incubation.

Magnesium salts are components of many dietary supplements used in treatment or prevention of magnesium deficiency. Hypomagnesemia usually results from an improper lifestyle, including unbalanced diet. Isolated hepatocytes of animals or humans are the preferred model used to study the in vitro effects of exogenous factors on cellular metabolic changes. The aim of this study was to evaluate the content of saturated, monounsaturated and polyunsaturated fatty acids and their esters in isolated rat hepatocytes influenced by different magnesium concentrations. The isolated rat hepatocytes were used as the test material. Hepatocytes were prepared in culture medium (Hepatocyte Medium) + MgCl(2) solution to concentrations of 2 mM/dm(3) MgCl(2), 4 mM/dm(3) MgCl(2). After incubation with different concentrations of magnesium ions, changes in the content of fatty acids and their esters were found for the whole hepatocytes and hepatocyte membranes. Despite changes in the fatty acid content in the whole hepatocytes and their membranes, there were no changes in the coefficient of degree of saturation of fatty acids when different concentrations of MgCl2 were used.

A novel quantitative method of pten expression assessment in tumor tissue.

Phosphatase and Tensin Homolog deleted on chromosome 10 (PTEN) gene is one of the most important tumor suppressor genes which is involved in the regulation of many signaling cascades (AKT/PKB and MAPK). Subtle changes in its activity lead to cancer susceptibility or aggressive tumor behaviour. Despite the diversity of mechanisms leading to PTEN inactivation, it is frequently associated with a decreased or complete loss of protein expression. About 20% decrease in PTEN expression could lead to the development of cancer. There have been no objective, quantitative methods of PTEN expression assessment that allow to measure the subtle variations of the protein concentration in a tissue-contextual manner. A new quantitative algorithm of immunostaining evaluation based on combination of color deconvolution and relative chromogen signal intensity was used in the study. The proposed algorithm was implemented in the popular ImageJ image analysis software and positively verified in cancer cell lines and tissue models as well as in the tissue samples of colorectal cancer (CRC) patients. The proposed quantitative method of PTEN expression assessment creates an alternative to currently available subjective methods and forms the basis for inter-case and inter-tissue comparisons. Using the algorithm it would be possible to identify three groups of patients with advanced colorectal cancer which could significantly differ in the overall survival. The research should be continued.

Epigenetic silencing of endothelin-3 in colorectal cancer.

Endothelins are expressed in a variety of human tissue and are involved in the processes as proliferation, migration and differentiation. The signal transduction pathway is a result of the endothelin-1-3 (ET1-3) binding to their receptors (ETAR, ETBR). ET-3 is a new candidate tumour suppressor gene, which is often downregulated or silenced in human cancer.The aim of the study was to examine DNA methylation of ET-3 genes in colorectal cancer (CRC) tissue samples in relation to the clinical stage (CS) of cancer. The paper is a continuation of our previously published results, which showed a four-fold transcriptional silencing of the ET-3 gene in the samples of colorectal cancer in comparison to normal tissues.A total of 66 paired CRC and normal (surgical margin) tissue samples were used in the study. The tumour tissues were collected from CRC patients in CS I-IV according the 7th edition of UICC TNM Classification of Malignant Tumours (CS I, n = 8; CS II, n = 20; CS III, n = 27; CS IV, n = 11). Assessment of epigenetic silencing of the ET-3 encoding gene was performed in three steps. The silencing of the ET-3 encoding gene was a result from methylation of the promoter sequence using methylation-specific PCR (MS-PCR). Analyses were performed using primers complementary for a CpG island in the first exon of the gene encoding ET-3. An epigenetic silence through methylation of 7.5% (5/66) in comparison to control was observed, including 10% of CS II (2/20), 7% of CS III (2/27) and 9% of CS IV (1/11). The controls and the samples of tumour in CS I showed no epigenetic silencing via methylation. In conclusion, epigenetic silencing of ET-3 in CRC could play a role in the progression than in the induction process. EDN3 would be a future target for epigenetic therapy in colorectal cancer, but further clinical studies are needed.

Expression of ADAM28 and IGFBP-3 genes in patients with colorectal cancer - a preliminary report.

Adamalisynes (ADAMs) play an important role in inter-membrane interactions, cell adhesion and fusion processes and protein shedding from the cell surface. Many reports indicate that members of the ADAMs family are overexpressed in human cancer. The aim of the present study was to evaluate ADAM28 and Insulin Like Growth Factor Binding Protein-3 (IGFBP-3)) gene expression in colorectal carcinoma tissues with regard to the overweight or obese status of the patients using an oligonucleotide microarray technique. Fresh tissue specimens were obtained from colorectal cancer patients during surgical treatment. Eighteen specimens from tumour and 18 normal tissue specimens from colorectal cancer patients at clinical stages III and IV were analysed. The examined patients were divided into two groups; those with BMI greater than or equal to 25 and those with normal BMI. The control group consisted of 18 specimens of non-neoplastic colon tissues, which were divided between overweight/obese and normal body weight patients. The gene transcriptional activity from the specimens was analysed using an oligonucleotide microarray technique. Microarrays and rinsing and marking solutions were prepared according to the procedure in the Gene Expression Analysis Technical Manual. The following conclusions were made: i) change of ADAM28 and IGFBP-3 genes expression are present in the normal tissue in overweight/obese patients with colorectal cancer only; ii) the observed molecular variability of ADAM28 and IGFBP-3 expression may be an initial process of cancer proliferation; iii) the histopathologically normal surgical margin in this group of patients was not equal to the molecular margin.

Changes in the content of short, medium and long-chain fatty acids in isolated hepatocytes incubated in the presence of magnesium ions and/or ethanol.

Magnesium is one of the commonly used dietary supplements. Therefore, this study was to evaluate the content of short, medium and long-chain fatty acids and their esters in isolated rat hepatocytes induced by magnesium and/or ethanol. Isolation of hepatocytes was carried out by the Seglen's enzymatic method using collagenase. To thus prepared samples ethanol and/or MgCl2 solution were added, respectively, so that their concentrations were as follows: 150 mM/dm3 ethanol and/or 2 mM/dm3 MgCl2, 4 mM/dm3 MgCl2. The contents of short, medium and long-chain fatty acids and those of ester-bound acids were determined. The statistical evaluation of the experiment was made by comparing the area normalized for the analysed fatty acids in hepatocytes incubated for 5 h in the presence of the test substances. The effect of magnesium ions on the content of fatty acids and their esters in isolated hepatocytes incubated for 5 h depended on their concentration in the medium. A normalizing effect of magnesium ions on ethanol-induced changes in the content of C14-C17, C18-C20 and C21-C24 fatty acids was demonstrated. A normalizing effect of magnesium on ethanol-induced changes in the content of ester-bound fatty acids in hepatocytes was not confirmed.

Does human papilloma virus participate in colorectal carcinogenesis?

Colorectal cancer is one of the most commonly diagnosed neoplasms still associated with relatively high mortality. Viral infections are often mentioned among the neoplasm transformation risk factors. Incidence of human papilloma virus (HPV), associated with high oncogenic risk, in the large intestine and the meaning of its presence in the colorectal carcinogenesis are still not clear. The aim of the study was to show a presence of HPV in specimens of adenomatous polyps and colorectal cancer using the Q-PCR method. Fifty patients (32 M/18W, mean age 62.8 years) were enrolled in the study, for whom tissue samples were obtained. Study material involved paraffin blocks derived from samples collected by flexible sigmoidoscopy from 10 polyps and 10 large intestine adenocarcinomas and 30 paraffin blocks with specimens of surgically removed large intestine adenocarcinomas. Presence of HPV genome was confirmed by quantitative PCR method using commercially available Abbott RealTime High Risk HPV test. The test is able to detect 14 most prevalent high oncogenic risk subtypes of human papilloma virus. Status of HPV DNA was successfully assessed in all 50 samples. No HPV DNA was discovered in any of the tested samples. Presence of high oncogenic risk HPV subtypes in large intestine adenoma and adenocarcinoma seems to be very rare, and its dominating role in the pathogenesis of colorectal cancer, even if possible, is unlikely.

Effect of magnesium ions on ethanol-induced changes in the pool of saturated, mono- and polyunsaturated fatty acids in isolated rat hepatocytes.

The aim of the paper was the assessment of the effect of magnesium ions on ethanol-induced changes in the content of ester-bound fatty acids in isolated rat hepatocytes and their cell membranes. Hepatocytes were isolated by means of the enzymatic method of Selgen using collagenase. The number and viability of isolated rat hepatocytes, incubated for 5 hours in culture media (Hepatocyte Medium) with ethanol and MgCl2 solutions with concentration amounting respectively to: 150 mM/dm3 of ethanol and/or 2 mM/dm3 of MgCl2, 4 mM/dm3 of MgCl2, were determined. Biochemical tests of hepatocytes were performed, consisting in the determination of the total content of ester-bound fatty acids in whole hepatocytes and their cell membranes after incubation. Confirmed normalising action of magnesium ions with respect to the effects induced in hepatocytes and their membranes by the presence of ethanol should be attributed to the important role of magnesium which it performs during reactions taking place with participation of ATP.

Expression of genes associated with H factor in fibroblasts infected with Borrelia spirochaetes.

Infection with Borrelia spirochaetes leads to the launch of specific and non-specific immunological response in humans. Activation of the complement system is one of the first defence mechanisms against penetrating pathogens. The aim of this study was to select genes related to the alternative pathway of the complement system [including complement factor H (CFH)], differentiating the type of infection in the system model, that is, a culture of normal human diploid fibroblasts infected with three different spirochaete genospecies: Borrelia afzelii, Borrelia garinii and Borrelia burgdorferii sensu stricto, by comparing the infected fibroblast culture with the control fibroblast one. With the use of oligonucleotide microarrays HGU-133A, the differences in the expression of genes selected on the basis of a scientific database Affymetrix were studied by comparing transcriptomes from the four cultures of fibroblasts. In the result of infection of fibroblast cultivation with a specific Borrelia genospecies, a variable expression of certain CFH and complement system-associated genes, specific for one genospecies only, B. afzelii- C1QBP, CD59, C2, CD46 and FHL1; B. garinii - C1S and CLU; Borrelia burgeforii- CFB, A2M and VSIG4, was observed. CFH differentiates infections induced by B. afzelii and B. garinii from infections induced by B. burgdorferii sensu stricto.

Analysis of expression profile of gene encoding proteins of signal cascades activated by insulin-like growth factors in colorectal cancer.

The aim of the study is to analyse gene typing with the use of the microarray technique (HG-U133A, Affymetrix), differentiating colorectal cancer tissues from tissues assessed histopathologically as healthy ones among a panel of 93 mRNA of gene encoding proteins involved in the activation of cellular signal transduction pathways by insulin-like growth factors. The study was conducted on a group of 8 colorectal cancer patients. Frozen tumor and healthy specimens from the patients were used in molecular tests. Transcript IGF2 differentiated cancer from healthy tissue. Among the genes participating in the cascade of signal transfer in cells activated by IGF, GRB10, PIK3R3, PIK3R1, and IRS1 were qualified as differentiating transcripts. IRS1 indicated over-expression in tumour. Transcript SMAD2 showed a significant changed in tumour samples (increased expression).

Statistical analysis of differential gene expression in colorectal cancer using CLEAR-test.

CLEAR test provides a novel method of analysis by combining inference for differential expression and variability. Frozen tumor specimens from 14 (3 coded Stage I, 5 Stage II, 2 Stage III and 4 Stage IV) colon cancer patients were obtained. Archived primary tumor samples were collected at the time of surgery and normal colon mucosae (controls specimens) were also collected. The studied transcriptomes were clustered using hierarchical agglomeration with Ward's method and Tchebychev distance. The separable groups of transcriptomes were classified as high clinical stage of adenocarcinoma (HCS; stages II-IV), low clinical stage of adenocarcinoma (LCS; stages I and 3 controls), and two normal colon mucosae (controls N1 and N2). The results of the CLEAR-test algorithm in normal colon specimens and adenocarcinoma specimens with low and high clinical stage showed 50 most and 50 least significant genes. The list of differential genes (p<0.01) in normal colon specimens and adenocarcinoma specimens with low and high clinical stage presented 58 genes.

Magnesium content in daily food portions and the influence of supplementation.

Magnesium is one of the most important cations for an organism. The aim of our study is to evaluate whether the use of a magnesium formulation as a diet supplement or medical treatment is necessary. The 24-hour recall method was used to obtain information regarding the daily magnesium consumption of 949 people. The results were compared with the Estimated Average Requirement (EAR) and Recommended Daily Allowance (RDA) values. The average daily requirement for magnesium was exceeded by 292 (183 women and 109 men) of the 949 respondents. This research confirmed excessive magnesium intake by both men and women that exceeded both the EAR and the RDA. Uncontrolled, excessive dietary supplementation or medical treatment with magnesium by this group may constitute a health threat.

Leptin and its receptors in obese patients with colorectal cancer.

The purpose of this study is to examine serum concentration of leptin and that of the soluble form, the Ob-Re receptor, in patients with colorectal cancer, as well as to examine the level of leptin mRNA and that of its receptors, Ob-Ra and Ob-Rb, in large intestine specimens collected from patients with colorectal cancer, depending on cancer clinical and pathological progression and BMI. A total of 146 patients with colorectal cancer in a I-IV stage scale according to the TNM Classification were enrolled. The patients were divided into two groups according to BMI calculations based on body weight and height: a Study group (BMI greater than or equal to 25 kg/m2) of 75 patients aged 57 plus or minus 4.5 years and a Control group (20 less than BMI less than 25 kg/m2) of 71 patients aged 60 plus or minus 5 years. The experimental part of the work was performed in two stages: Stage I regarding the assay of leptin concentration and that of its soluble receptor, Ob-Re, in the serum of patients with the use of the ELISA method; and Stage II to determine the number of leptin mRNA copies and two isoforms of leptin receptors, Ob-Ra and Ob-Rb, using the QRT-PCR method in tissue specimens collected from 146 patients. In our results the concentration of serum leptin and Ob-Re was not dependent on the stage of clinical and pathological progression of the cancer. There was a statistically significant higher serum leptin level in colon cancer patients who were overweight or obese compared to patients with normal weight. No presence of mRNA of the gene encoding leptin was found in tissues collected from colorectal cancer patients. The number of mRNA copies of Ob-Rb was statistically significantly higher in all the study groups compared to the reference tissues.

Influence of magnesium on fatty acids and their esters in isolated rat hepatocytes.

The aim of the study is to analyse the changes in the profile of fatty acids and their esters in rat hepatocytes that were incubated for 5 hours with different concentrations of MgCl2 (2 and 4mM) in hepatocyte culture medium. The methyl esters of fatty acids were identified with a GC-MS system included in the Hewlett?Packard quadrupolar mass spectrometer, coupled with a Hewlett-Pacard 5890 gas chromatograph with an ionisation potential of 70 eV and recorded on a Vectra 386 computer. We observed differences in the amount of saturated, monounsaturated and polyunsaturated fatty acids among the examined samples. In the control sample, the largest component consisted of the pool of saturated and monounsaturated fatty acids. Analysing the changes in the profile of ester-bound fatty acids, we found statistically significant differences when 4 mM MgCl2 was presented. The amount of C18:2, C18:1b and C20:4a decreased in comparison with the control sample.

Ethanol-induced changes in profile of fatty acids in isolated rat hepatocytes and the influence of magnesium ions.

The influence of magnesium and ethanol on the fatty acid content in isolated rat hepatocytes was examined in this study. The isolated liver cells were obtained according to the Selgen method and then subjected to ethanol alone or both ethanol and magnesium activity. MgCl2 was used in two concentrations: 2 and 4 mM. The profile of fatty acids in the hepatocytes was evaluated after 5 hours of incubation. Our results revealed that magnesium ions presented together with ethanol in hepatocyte medium changed the hepatocyte fatty acid profile. The total amounts depended on the concentration of magnesium ions.

Genetic disregulation of gene coding tumor necrosis factor alpha receptors (TNFalpha Rs) in colorectal cancer cells.

The expression of TNF ligand by malignant cells might be a mechanism for tumour immune escape. Genetic disregulation of gene coding TNF receptors was observed in neoplastic disease by an increased number of receptors on tumour cells and ligand-receptor activity. It might cause tumour proliferation and metastatic potential. Structure of TNF receptors influences TNF activity in vivo and structure of TNF R2 gene may suggest post-transcription modification based on alternative splicing. The aim of the study was to analyse the expression of gene coding TNF receptors R2 and R2/R7 (without exon 7) by estimation of mRNA expression of colorectal cancer cells in comparison with surrounding tissue free from neoplastic infiltration and searched for differently spliced TNFalphaR2/R7 isoforms. The study included fifty four patients with histopathologically confirmed adenocarcinoma (Stage III according to the AJC TNM Classification). Tissue samples removed from the tumour region were obtained from colorectal cancer patients undergoing surgical treatment. The samples were divided into two parts. The first one--was routinely examined histopathologically, the second one--was used for RNA extraction and the number of TNF and its receptors mRNA copies were subsequently quantified. The TNF and TNFRII genes expression were estimated based on the number of mRNA copies on 1 microg total RNA. The presence of TNFR2 and TNFR2/R7 isoforms in tumour, normal and metastatic cells was observed. The highest number of mRNA TNF copies and over expressed TNF genes were investigated and significantly noticed in metastatic cells (lymph nodes). The decreased number of TNFR2/R7 mRNA copies in metastatic lymph nodes secondarily influenced the decreased TNF soluble receptors' concentration. In conclusion, the genetic disregulation observed in neoplastic disease usually concerns dysfunction of cytokines receptor genes.

On the holistic approach in cancer biology: tumor necrosis factor, colon cancer cells, chaos theory and complexity.

TNFalpha plays a role in the pathogenesis of septic shock, inflammatory diseases, autoimmune diseases, graft rejection reaction, acute, and chronic respiratory inefficiency among others. Its activity depends on the type of target cells and different regulating factors, but the effect of biological activity is conditioned by specific receptors such as p55 (type I, TNF R55) and p75 (type II, TNF R75). The aim of the study was to answer the following questions: 1) Is it possible to apply elements of non-linear dynamics to assess the level of expression of TNF, TNFRI, TNFRII genes in tumor cells, pathologically unchanged tissue and metastatically changed lymph nodes? 2) Is theoretically anticipated variability of cytokine and its receptors in colorectal carcinoma cells and the immediate vicinity justified in the developed mathematical model? The research material--specimens taken from tumor, unchanged tissue and metastatic lymph nodes--were histopathologically and molecularly analysed. Results of the molecular research were used to develop a mathematical model using the basic studies on the theory of chaos and biological system modelling.

Circadian fluctuations of melatonin, tumor necrosis factor-alpha and its soluble receptors in the circulation of patients with advanced gastrointestinal cancer.

The objective of the study was to investigate the dynamic changes of melatonin (MLT), tumor necrosis factor alpha (TNFalpha), soluble TNFalpha receptors ( type I and type II) in serum of advanced cancer patients during 24 hours. The examined group consisted of 42 patients suffering from advanced gastrointestinal neoplasms (colorectal, gastric and pancreatic cancer). Blood samples were collected 6 times a day (8 a.m., 2 p.m., 6 p.m., 10 p.m., 2 a.m., and again 8 a.m.) as well as in healthy controls. Serum levels of TNFalpha and both its receptors were measured using ELISA type and the radioimmunoassay method was used to assess MLT levels. The circadian rhythm of MLT was altered because MLT reached its peak level at 8.50 a.m. with 5 hours delay in respect to average peak time in healthy humans. The presence of circadian rhythm of TNFalpha was proved (acrophase-1.40 a.m.), and no diurnal rhythm of soluble TNF receptors was observed. The concentration of soluble type I (p-55) receptor was distinctly lower than soluble type II (p-75). The peak of soluble type I receptor value appeared at 10.00 p.m. while the type II receptor reached its minimum level at the same time. Although there was no statistical correlation between the receptor concentrations, the shapes of both curves remained inversely proportional. The present results may suggest the presence of complex self-regulation mechanisms between the neuroendocrine system and the cytokine network in advanced gastrointestinal cancer patients.