Novel potential biomarkers for the diagnosis and monitoring of patients with ulcerative colitis.

Unambiguously, great progress has been achieved in the unraveling of more pathological pathways implicated in the development and progression of ulcerative colitis during the last decades. Novel effective drugs that have augmented the management armamentarium have been developed alongside this growing comprehension of the disease, rendering mucosal healing not only a feasible but the optimal goal of every therapy. Clinical evaluation, colonoscopy and biomarkers are the tools used by practitioners for the diagnosis and assessment of the status of the disease in order to achieve clinical remission and mucosal healing for their patients. Among these tools, colonoscopy is the gold method for the cause but is still an invasive, high-cost procedure with possible adverse events such as perforation. While clinical evaluation entails much subjectivity, biomarkers are objective, easily reproducible, non-invasive, cheap and potent surrogate tools of mucosal inflammation. Unfortunately, the well-established, currently in use serum biomarkers, such as C-reactive protein, erythrocyte sedimentation rate and others, do not display sufficiently acceptable sensitivity and specificity rates for the diagnosis of ulcerative colitis and, most importantly, do not represent precisely the mucosal inflammation status of the disease. Therefore, the discovery of new serum biomarkers has been the cause of several studies attempting to discover an "optimal" serum biomarker during the recent years. After thorough research, collection and examination of current data, this review focuses on and selectively presents promising, potential, novel serum biomarkers of ulcerative colitis as they are indicated by studies on the patient over the last years.

Eltrombopag, a thrombopoietin mimetic, crosses the blood-brain barrier and impairs iron-dependent hippocampal neuron dendrite development.

Essentials Potential neurodevelopmental side effects of thrombopoietin mimetics need to be considered. The effects of eltrombopag (ELT) on neuronal iron status and dendrite development were assessed. ELT crosses the blood-brain barrier and causes iron deficiency in developing neurons. ELT blunts dendrite maturation, indicating a need for more safety studies before neonatal use. SUMMARY: Background Thrombocytopenia is common in sick neonates. Thrombopoietin mimetics (e.g. eltrombopag [ELT]) might provide an alternative therapy for selected neonates with severe and prolonged thrombocytopenia, and for infants and young children with different varieties of thrombocytopenia. However, ELT chelates intracellular iron, which may adversely affect developing organs with high metabolic requirements. Iron deficiency (ID) is particularly deleterious during brain development, impairing neuronal myelination, dopamine signaling and dendritic maturation and ultimately impairing long-term neurological function (e.g. hippocampal-dependent learning and memory). Objective To determine whether ELT crosses the blood-brain barrier (BBB), causes neuronal ID and impairs hippocampal neuron dendrite maturation. Methods ELT transport across the BBB was assessed using primary bovine brain microvascular endothelial cells. Embryonic mouse primary hippocampal neuron cultures were treated with ELT or deferoxamine (DFO, an iron chelator) from 7 days in vitro (DIV) through 14 DIV and assessed for gene expression and neuronal dendrite complexity. Results ELT crossed the BBB in a time-dependent manner. 2 and 6 µm ELT increased Tfr1 and Slc11a2 (iron-responsive genes involved in neuronal iron uptake) mRNA levels, indicating neuronal ID. 6 µm ELT, but not 2 µm ELT, decreased BdnfVI, Camk2a and Vamp1 mRNA levels, suggesting impaired neuronal development and synaptic function. Dendrite branch number and length were reduced in 6 µm ELT-treated neurons, resulting in blunted dendritic arbor complexity that was similar to DFO-treated neurons. Conclusions Eltrombopag treatment during development may impair neuronal structure as a result of neuronal ID. Preclinical in vivo studies are warranted to assess ELT safety during periods of rapid brain development.

Liver preservation with machine perfusion and a newly developed cell-free oxygen carrier solution under subnormothermic conditions.

We describe a new preservation modality combining machine perfusion (MP) at subnormothermic conditions(21 °C) with a new hemoglobin-based oxygen carrier (HBOC) solution. MP (n=6) was compared to cold static preservation (CSP; n=6) in porcine orthotopic liver transplants after 9 h of cold ischemia and 5-day follow-up. Recipients' peripheral blood, serial liver biopsies, preservation solutions and bile specimens were collected before, during and after liver preservation. Clinical laboratorial and histological analyses were performed in addition to mitochondrial functional assays, transcriptomic, metabolomic and inflammatory inflammatory mediator analyses. Compared with CSP, MP animals had: (1) significantly higher survival (100%vs. 33%; p<0.05); (2) superior graft function (p<0.05);(3) eight times higher hepatic O2 delivery than O2 consumption (0.78 mL O2/g/h vs. 0.096 mL O2/g/h) during MP; and (4) significantly greater bile production (MP=378.5 ± 179.7; CS=151.6 ± 116.85). MP downregulated interferon (IFN)-α and IFN-γ in liver tissue. MP allografts cleared lactate, produced urea, sustained gluconeogenesis and produced hydrophilic bile after reperfusion. Enhanced oxygenation under subnormothermic conditions triggers regenerative and cell protective responses resulting in improved allograft function. MP at 21 °C with the HBOC solution significantly improves liver preservation compared to CSP.

MCM7 amplification and overexpression are associated with prostate cancer progression.

The genomic DNA profiles of prostate cancers with aggressive features were compared to the profiles of matched normal DNA to identify genes that are selectively amplified in the cancer cells. One of the identified genes, MCM7, which is a component of the DNA replication licensing complex, has been studied extensively both at the DNA and protein levels in human prostate tissues. Approximately half of the prostate cancer specimens studied showed MCM7 gene amplification, and 60% of the aggressive prostate cancer specimens had increased MCM7 protein expression. Amplification or overexpression of MCM7 was significantly associated with relapse, local invasion and a worse tumor grade. Constitutive expression of MCM7 in a human prostate cancer cell line, DU145, resulted in markedly increased DNA synthesis and cell proliferation compared to vector-only controls, and an increased cell invasion in vitro. Indeed, MCM7 overexpression produced primary tumors 12 times larger than vector-only controls and resulted in a rapid demise of mice bearing those tumors. These studies implicate MCM7, and the DNA replication licensing gene family, in prostate cancer progression, growth and invasion.

Myopodin, a synaptopodin homologue, is frequently deleted in invasive prostate cancers.

Prostate cancer is one of the leading causes of cancer-related deaths for men in the United States. Like other malignancies, prostate cancer is underscored by a variety of aberrant genetic alterations during its development. Although loss of heterozygosity or allelic loss is frequently identified among prostate cancers, few genes have been identified thus far as critical to the development of invasive prostate cancers. In this report, we used the recently developed technology, the "differential subtraction chain," to perform a genome-wide search for sequences that are deleted in an aggressive prostate cancer. Among the deleted sequences, we found that one sequence was deleted in >50% of prostate cancers we tested. We mapped this sequence to chromosome 4q25 by screening the Genebridge 4 hamster radiation panel with primers specific to this probe, and subsequently identify a 54-kb minimal common deletion region that contains the sequence encoding myopodin. Sequence analysis indicates that myopodin shares significant homology with synaptopodin, a protein closely associated with podocyte and neuron differentiation. Further study shows that frequent complete or partial deletions of the myopodin gene occurred among invasive prostate cancer cases (25 of 31 cases, or 80%). Statistical analysis indicates that deletion of myopodin is highly correlated with the invasiveness of prostate cancers, and thus may hold promise as an important prognostic marker for prostate cancers.

Histological organization in hepatocyte organoid cultures.

Hepatocytes and other cellular elements isolated by collagenase perfusion of the liver and maintained in defined culture conditions undergo a series of complex changes, including apoptosis and cell proliferation, to reconstruct tissue with specific architecture. Cultures in collagen-coated pleated surface roller bottles, with hepatocyte growth medium medium and in the presence of hepatocyte growth factor (HGF) and epidermal growth factor (EGF), form characteristic and reproducible tissue architecture composed of a superficial layer of biliary epithelial cells, an intermediate layer of connective tissue and hepatocytes, and a basal layer of endothelial cells. Dexamethasone, EGF, and HGF are required for the complete histological organization. Analysis of the structures formed demonstrates that the receptor tyrosine kinase ligands HGF and EGF are required for the presence, growth, and phenotypic maturation of the biliary epithelium on the surface of the cultures and for the formation of connective tissue in the cultures. Dexamethasone, in the presence of HGF and EGF, was required for the phenotypic maturation of hepatocytes. The results demonstrate the role of these molecules for the formation and phenotypic maturation of specific histological elements of the liver and suggest roles for these signaling molecules in the formation and structure of the in vivo hepatic architecture.

The processing and utilization of hepatocyte growth factor/scatter factor following partial hepatectomy in the rat.

Hepatocyte growth factor/scatter factor (HGF/SF) is a pluripotent growth factor capable of acting as a motogen, a morphogen, and a mitogen. Originally, HGF/SF was found as a blood-borne mitogen for hepatocytes and has since been determined to be very important in liver repair. Previous studies have established that HGF/SF must be proteolytically cleaved to elicit its effects. After liver injury by toxins such as carbon tetrachloride or after surgical resection, partial hepatectomy (PHX), HGF/SF concentrations increase in the blood. The aims of this study were to examine (1) which form of HGF/SF is present in the normal liver, (2) which form is present in the regenerating liver after PHX, and (3) if the HGF/SF used after PHX is derived from existing liver reservoirs. Both single-chain HGF/SF and active two-chain HGF/SF are present in normal liver, with the former being the dominant form. After PHX, the liver can be described as having two phases with regard to the use of endogenous HGF/SF. The first phase from 0 to 3 hours is the consumptive phase and is characterized by a decrease in both single-chain HGF/SF and active two-chain HGF/SF. The second phase is the productive phase. It is characterized by a pronounced reappearance of both single-chain HGF/SF as well as two-chain HGF/SF. The activation index shows a 5-fold increase over sham operations during the productive phase. The use of radiolabeled HGF/SF showed that during the first 3 hours, HGF/SF is used in part from hepatic stores. Furthermore, during the first 3 hours after PHX, only active two-chain HGF/SF is seen in the plasma.

Hepatocellular carcinomas in native livers from patients treated with orthotopic liver transplantation: biologic and therapeutic implications.

The gross and histopathologic characteristics of 212 nonfibrolamellar hepatocellular carcinomas (HCCs) discovered in native livers removed at the time of liver transplantation were correlated with features of invasive growth and tumor-free survival. The results show that most HCCs begin as small well-differentiated tumors that have an increased proliferation rate and induce neovascularization, compared with the surrounding liver. But at this stage, they maintain a near-normal apoptosis/mitosis ratio and uncommonly show vascular invasion. As tumors enlarge, foci of dedifferentiation appear within the neoplastic nodules, which have a higher proliferation rate and show more pleomorphism than surrounding better-differentiated areas. Vascular invasion, which is the strongest predictor of disease recurrence, correlates significantly with tumor number and size, tumor giant cells and necrosis, the predominant and worst degree of differentiation, and the apoptosis/mitosis ratio. In the absence of macroscopic or large vessel invasion, largest tumor size (P <.006), apoptosis/mitosis ratio (P <.03), and number of tumors (P <.04) were independent predictors of tumor-free survival and none of 24 patients with tumors having an apoptosis/mitosis ratio greater than 7.2 had recurrence. A minority of HCCs (<15%) quickly develop aggressive features (moderate or poor differentiation, low apoptosis/mitosis ratio, and vascular invasion) while still small, similar to flat carcinomas of the bladder and colon. In conclusion, hepatic carcinogenesis in humans is a multistep and multifocal process. As in experimental animal studies, aggressive biologic behavior (vascular invasion and recurrence) correlates significantly with profound alterations in the apoptosis/mitosis ratio and with architectural and cytologic alterations that suggest a progressive accumulation of multiple genetic abnormalities.

Changes in WNT/beta-catenin pathway during regulated growth in rat liver regeneration.

The wnt/beta-catenin pathway is important during embryogenesis and carcinogenesis. beta-Catenin interaction with E-cadherin has been shown to be crucial in cell-cell adhesion. We report novel findings in the wnt pathway during rat liver regeneration after 70% partial hepatectomy using Western blot analyses, immunoprecipitation studies, and immunofluorescence. We found wnt-1 and beta-catenin proteins to be predominantly localized in hepatocytes. Immediately following partial hepatectomy, we observed an initial increase in beta-catenin protein during the first 5 minutes with its translocation to the nucleus. We show this increase to be the result of decreased degradation of beta-catenin (decrease in serine phosphorylated beta-catenin) as seen by immunoprecipitation studies. We observed activation of beta-catenin degradation complex comprising of adenomatous polyposis coli gene product (APC) and serine-phosphorylated axin protein, beginning at 5 minutes after hepatectomy, leading to its decreased levels after this time. Quantitative changes observed in E-cadherin protein during liver regeneration are, in general, reverse to those seen in beta-catenin. In addition, using immunoprecipitation, we observe elevated levels of tyrosine-phosphorylated beta-catenin at 6 hours onward. Thus, changes in the wnt pathway during regulated growth seem to tightly regulate cytosolic beta-catenin levels and may be contributing to induce cell proliferation and target gene expression. Furthermore, these changes might also be intended to negatively regulate cell-cell adhesion for structural reorganization during the process of liver regeneration.

Sustained expression of naked plasmid DNA encoding hepatocyte growth factor in mice promotes liver and overall body growth.

To understand the physiological functions of exogenous hepatocyte growth factor (HGF) on normal adult animals, we delivered human HGF gene into mice by a hydrodynamics-based in vivo gene transfection approach using a naked plasmid vector. Systemic administration of naked plasmid containing HGF cDNA driven under cytomegalovirus promoter (pCMV-HGF) by rapid injection via the tail vein produced a remarkable level of human HGF protein in the circulation, beginning to appear at 4 hours and peaking at 12 hours following injection. Tissue distribution studies identified the liver as the organ with the highest level of transgene expression. Through weekly repeated injections of plasmid vector, we achieved sustained, long-term, high levels of exogenous HGF expression in mice for 8 weeks. Increases of more than 31% and 16% in liver and body weights were found, respectively, in the mice that received pCMV-HGF plasmid compared with that given the control vector for 8 weeks. Expression of exogenous HGF in vivo activated mitogen-activated protein kinases and induced proliferating cell nuclear antigen expression in normal adult liver and kidneys. These data suggest that systemic administration of naked plasmid vector is a convenient, safe, and highly efficient approach to introduce and maintain exogenous HGF gene expression in vivo in a controllable fashion. Our results also indicate that long-term expression of human HGF in mice markedly activates growth-related signal transduction events, promotes cell proliferation, and leads to liver and overall body growth in whole adult animals.

Comparative genomic hybridization of hepatocellular carcinoma: correlation with fluorescence in situ hybridization in paraffin-embedded tissue.

BACKGROUND: Proto-oncogene MYC, mapped to chromosomal band 8q24 and the genes for hepatocyte growth factor (HGF at 7q21) and its receptor, MET, at chromosomal band 7q31, have an important role in the biology and growth of normal and neoplastic liver. Comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) studies have reported frequent abnormalities of chromosomes 1 and 8 in hepatocellular carcinomas (HCCs) of various clinical and pathological stages. Chromosome 7 involvement is reported to be less frequent. MATERIALS AND METHODS: Frozen tissue from 17 HCCs was used for CGH analysis and sections of corresponding formalin-fixed, paraffin-embedded HCC tissue were used for dual-color FISH with locus-specific (LSI-cMYC for chromosome 8q24 and LSI D7S486 for chromosome 7q31) and centromeric probes, CEP8 (8p11.1-q11.2) and CEP7 (7p11.1-q11.2) (Vysis, Inc, Downers Grove, IL). This study intended to determine the pattern of chromosomal aberrations in early-stage (incidental) HCC and large surgically resected HCC, and also compared the efficiency and usefulness of the two cytogenetic methods. RESULTS: CGH showed abnormalities on chromosomes 1q, 5q, 7q, 8q, 9, 10, 13q, 15, 16, 17p, 18q, 19, 20, 21, 22, and X. Gains of 8q were noted in 50% of the HCCs, including five cases of incidental HCCs by CGH. Increase in copy numbers of MYC detected by FISH was noted in 25% of tumors that had shown 8q gains by CGH and in five cases with no chromosome abnormalities noted by CGH. Three cases with 7q31 copy number abnormalities were found by FISH in addition to those detected by CGH. CONCLUSION: Combined use of CGH and FISH may provide important information about early and/or primary genetic changes in the development of HCC.

Induction of cytochrome P450 (CYP)1A1, CYP1A2, and CYP3A4 but not of CYP2C9, CYP2C19, multidrug resistance (MDR-1) and multidrug resistance associated protein (MRP-1) by prototypical inducers in human hepatocytes.

Human hepatocytes cultured serum-free for up to 6 weeks were used to study expression and induction of enzymes and membrane transport proteins involved in drug metabolism. Phase I drug metabolizing enzymes cytochrome P450 (CYP)1A1, CYP1A2, CYP2C9, CYP2C19, CYP2E1, and CYP3A4 were detected by Western blot analyses and, when appropriate, by enzymatic assays for ethoxyresorufin-O-deethylase(EROD)-activity and testosterone-6beta-hydroxylase(T6H)-activity. Expression of the membrane transporter multi-drug resistance protein (P-glycoprotein, MDR-1), multidrug resistance-associated protein (MRP-1), and lung-resistance protein (LRP) was maintained during the culture as detected by RT-PCR and Western blot analyses. Model inducers like rifampicin, phenobarbital, or 3-methylcholanthrene and beta-naphtoflavone were able to induce CYP1A or CYP3A4 as well as EROD or T6H activities for up to 30 days. CYP2C9, CYP2C19 and CYP2E1 expression was maintained but not inducible for 48 days. Also, rifampicin and phenobarbital were unable to increase MDR-1 and MRP-1 protein levels significantly.

Serum-free, long-term cultures of human hepatocytes: maintenance of cell morphology, transcription factors, and liver-specific functions.

Since human hepatocytes are available only in limited number, the development of a serum-free culture system for long-term cultivation of differentiated and functional hepatocytes is of great importance. Here we describe the culture of human hepatocytes in a chemically defined serum-free medium for up to 5 weeks. Cell morphology was assayed by light and electron microscopy and revealed a well-preserved cellular morphology. Marker proteins for epithelial and bile duct cells, cytokeratin (CK) 18 and 19, and liver-specific proteins, like phosphoenolpyruvate carboxykinase-2 (PCK2) and serum proteins, were expressed. Liver-enriched transcription factors CCAAT/enhancer binding protein alpha (C/EBPalpha) and hepatocyte nuclear factor-4 (HNF-4), cytokine and mitogen activated factors (nuclear factor kappa B) NFkappaB, and activator protein-1 (AP-1) were maintained and active for several weeks in our cultures. In summary, our serum-free culture system allows the culture of differentiated human hepatocytes for several weeks. It may serve as a model system for metabolic, pharmacologic-toxicologic studies, and studies on human pathogens under defined chemical conditions.

Cross-talk between epidermal growth factor receptor and c-Met signal pathways in transformed cells.

In rat liver epithelial cells constitutively expressing transforming growth factor alpha (TGFalpha), c-Met is constitutively phosphorylated in the absence of its ligand, hepatocyte growth factor. We proposed that TGFalpha and the autocrine activation of its receptor, epidermal growth factor receptor (EGFR), leads to phosphorylation and activation of c-Met. We found that there is constitutive c-Met phosphorylation in human hepatoma cell lines and the human epidermoid carcinoma cell line, A431 which express TGFalpha, but not in normal human hepatocytes. Constitutive c-Met phosphorylation in A431, HepG2, AKN-1, and HuH6 cells was inhibited by neutralizing antibodies against TGFalpha and/or EGFR. Exposure to exogenous TGFalpha or EGF increased the phosphorylation of c-Met in the human epidermoid carcinoma cell line, A431. The increase of c-Met phosphorylation by TGFalpha in A431 cells was inhibited by neutralizing antibodies against TGFalpha and/or EGFR and by the EGFR-specific inhibitor tyrphostin AG1478. These results indicate that constitutive c-Met phosphorylation, and the increase of c-Met phosphorylation by TGFalpha or EGF, in tumor cell lines is the result of the activation via EGFR. We found that c-Met in tumor cells co-immunoprecipitates with EGFR regardless of the existence of their ligands in tumor cells, but not in normal human hepatocytes. We conclude that c-Met associates with EGFR in tumor cells, and this association facilitates the phosphorylation of c-Met in the absence of hepatocyte growth factor. This cross-talk between c-Met and EGFR may have significant implications for altered growth control in tumorigenesis.

Expression and activation of pro-MMP-2 and pro-MMP-9 during rat liver regeneration.

Partial hepatectomy triggers a variety of biological phenomena, which culminate in regeneration of the liver mass. Hepatocyte proliferation is a major feature of the regenerating liver after partial hepatectomy. Previous studies in our laboratory suggested that hepatic matrix remodeling might be a prerequisite process for hepatocyte proliferation in the regenerating liver. In the present study we use immunohistochemical staining, Western blot analysis, and gelatin zymography to show that the inactive matrix metalloproteinases (MMPs), pro-MMP-2 and pro-MMP-9, are elevated at 30 minutes and activated at 6 to 12 hours and at 3 to 6 hours, respectively, after hepatectomy. Sham-operated livers did not show an increase in inactive pro-MMP-2 and pro-MMP-9 and did not contain active MMP-2 or MMP-9. To examine whether tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) is changed to regulate the activities of MMPs after partial hepatectomy, the level of TIMP-1 protein was analyzed by both immunohistochemistry and Western blot analysis. The level of TIMP-1 protein appears to increase by 6 to 18 hours, implying that an increase in TIMP-1 regulates activities of active MMP-2 and MMP-9. Taken together, these results suggest that hepatic matrix remodeling is mediated by activated MMPs, which contribute to modulation of the environment surrounding hepatocytes during rat liver regeneration.

Cationic colloidal silica membrane perturbation as a means of examining changes at the sinusoidal surface during liver regeneration.

By employing the cationic colloidal silica membrane density perturbation technique, we examined growth factor receptor and extracellular matrix (ECM) changes at the sinusoidal surface during rat liver regeneration 72 hours after 70% partial hepatectomy (PHx). At this time after PHx, hepatocyte division has mostly subsided, while sinusoidal endothelial cell (SEC) proliferation is initiating, resulting in avascular hepatocyte islands. Because of the discontinuous nature of the surface of liver SEC, ECM proteins underlying the SEC, as well as SEC luminal membrane proteins, are available to absorption to the charged silica beads when the liver is perfused with the colloid. Subsequent liver homogenization and density centrifugation yield two separate fractions, enriched in SECs as well as hepatocyte basolateral membrane-specific proteins up to 50-fold over whole liver lysates. This technique facilitates examination of changes in protein composition that influence or occur as a result of SEC mitogenesis and migration during regeneration of the liver. When ECM and receptor proteins from SEC-enriched fractions were examined by Western immunoblotting, urokinase plasminogen activator receptor, fibronectin, and plasmin increased at the SEC surface 72 hours after PHx. Epidermal growth factor receptor, plasminogen, SPARC (secreted protein, acidic and rich in cysteine, also called osteonectin or BM40), and collagen IV decreased, and fibrinogen subunits and c-Met expression remained constant 72 hours after PHx when compared to control liver. These results display the usefulness of the cationic colloidal silica membrane isolation protocol. They also show considerable modulation of surface components that may regulate angiogenic processes at the end stage of liver regeneration during the reformation of sinusoids.