Temporal changes in the expression and distribution of adhesion molecules during liver development and regeneration

We have compared by immunocytochemistry and immunoblotting the expression and distribution of adhesion molecules participating in cell-matrix and cell-cell interactions during embryonic development and regeneration of rat liver. Fibronectin and the fibronectin receptor, integrin alpha 5 beta 1, were distributed pericellularly and expressed at a steady level during development from the 16th day of gestation and in neonate and adult liver. AGp110, a nonintegrin fibronectin receptor was first detected on the 17th day of gestation in a similar, nonpolarized distribution on parenchymal cell surfaces. At that stage of development haemopoiesis is at a peak in rat liver and fibronectin and receptors alpha 5 beta 1 and AGp110 were prominent on the surface of blood cell precursors. During the last 2 d of gestation (20th and 21st day) hepatocytes assembled around lumina. AGp110 was initially depolarized on the surface of these acinar cells but then confined to the lumen and to newly-formed bile canaliculi. At birth, a marked increase occurred in the canalicular expression of AGp110 and in the branching of the canalicular network. Simultaneously, there was enhanced expression of ZO-1, a protein component of tight junctions. On the second day postpartum, presence of AGp110 and of protein constituents of desmosomes and intermediate junctions, DGI and E-cadherin, respectively, was notably enhanced in cellular fractions insoluble in nonionic detergents, presumably signifying linkage of AGp110 with the cytoskeleton and assembly of desmosomal and intermediate junctions. During liver regeneration after partial hepatectomy, AGp110 remained confined to apical surfaces, indicating a preservation of basic polarity in parenchymal cells. A decrease in the extent and continuity of the canalicular network occurred in proliferating parenchyma, starting 24 h after resection in areas close to the terminal afferent blood supply of portal veins and spreading to the rest of the liver within the next 24 h. Distinct acinar structures, similar to the ones in prenatal liver, appeared at 72 h after hepatectomy. Restoration of the normal branching of the biliary tree commenced at 72 h. At 7 d postoperatively acinar formation declined and one-cell-thick hepatic plates, as in normal liver, were observed.

Role of hepatocyte wounding on " Plasmodium " liver-stage development and survival

RESUME La première étape primordiale au cycle de vie du Plasmodium dans un hôte mammifère est l'invasion des hepatocytes par des sporozoites. L'infection finale des hepatocytes est précédée de la traversée de plusieurs cellules hôtes, rompant les membranes plasmiques et ayant comme résultat la sécrétion des facteurs cytotoliques dans le micro-environnement. Ce matériel endogène libéré est fortement stimulant/immunogène et peut servir de signal de danger initiant des réponses distinctes dans diverses cellules. De nos jours, le caractère essentiel et salutaire de la migration des sporozoites comme étape d'infection du Plasmodium est vivement controversée. Ainsi, notre étude a visé à caractériser l'effet de l'interaction du parasite avec ses cellules hôtes d'un point de vue immunologique. En particulier, nous avons voulu évaluer l'effet de la perte de matériel cellulaire pendant l'infection de Plasmodium sur les hepatocytes primaires de souris et sur des cultures cellulaires HepG2. Nous avons observé que les facteurs cytotoxiques dérivés des cellules endommagés activent NF-κB - un important régulateur de réponse inflammatoires -dans des cellules voisines des cellules endommagés, qui sont des cellules hôtes potentielles pour l'infection finale du parasite. Cette activation de NF-κB s'est produite peu de temps après l'infection et a mené in vitro et in vivo à une réduction d'infection de façon dépendante du temps, un effet qui a pu être compensé par l'addition de BAY11-7082, un inhibiteur spécifique de NF-κB. De plus, aucune activation de NF-κB avec des parasites SPECT-/-, incapables de traverser les hepatocytes, n'a été observée. Nous avons montré parla suite que l'activation de NF-κB induit l'expression de l'enzyme iNOS dans les hepatocytes, qui est responsable d'une diminution des hepatocytes infectés. En outre, les hepatocytes primaires des souris MyD88-/- n'ont montré ni activation de NF-κB, ni expression d'iNOS lors de l'infection, ce qui suggère la participation des membres de famille du Toll/IL-1 récepteur dans la reconnaissance des facteurs cytosoxiques. En effet, le manque de MyD88 a augmenté significativement l'infection in vitro et in vivo. D'autre part, un rôle bénéfique pour l'activation de NF-κB a été évalué. Les cellules infectées étaient plus résistantes contre l'apoptose induite par Fas (CD95/Apo-1) que les cellules non infectées ou les cellules infectées dans lesquelles NF-κB a été bloqué par BAY11-7082 in vitro. Paradoxalement, l'expression d'iNOS contribue à la protection des cellules infectées contre l'apoptose pax Fas, puisque le traitement avec l'inhibiteur spécifique SMT (S-methylisothiourea) a rendu les cellules infectées plus susceptibles à l'apoptose. Un effet bénéfique additionnel pour le parasite est que la plupart des cellules hôtes traversées présentent des peptides du parasite aux cellules T cytotoxiques spécifiques et peuvent donc réorienter la réaction immune spécifique sur les cellules non infectées. Nous montrons que les cellules hôtes endommagés par la migration du parasite induit l'inflammation, qui limite l'ampleur de l'infection. D'autre part, nos données soutiennent que la survie du parasite Plasmodium dans le foie est assurée par une augmentation de la résistance des hepatocytes contre l'apoptose. SUMMARY The first obligatory step of the Plasmodium life cycle in the mammalian host is the invasion of hepatocytes by sporozoites. Final hepatocyte infection involves the penetration of several host cells, whose plasma membranes are ruptured in the process, resulting in the release of cytosolic factors into the microenvironment. This released endogenous material is highly stimulatory / immunogenic and can serve as a danger signal initiating distinct responses in various cells. To date, it is highly controversial whether sporozoite migration through hepatocytes is an essential and beneficial step for Plasmodium infection. Thus, our study aimed at characterizing the effect of the interaction of the parasite with its host cells from an immunological point of view In particular, we wanted to evaluate the effect of cell material leakage during Plasmodium infection on cultured mouse primary hepatocytes and HepG2 cells. We observed that wounded cell-derived cytosolic factors activate NF-κB - a main regulator of host inflammatory responses - in cells bordering wounded cells, which are potential host cells for final parasite infection. This activation of NF-κB occurred shortly after infection and led to a reduction of infection load in a time dependent manner in vitro and in viva, an effect that could be reverted by addition of the specific NF-κB inhibitor BAY11-7082. In addition, no NF-κB activation was observed when SPECT-/- parasites, which are devoid of hepatocyte traversing properties, were used. We provide further evidence that NF-κB activation causes the induction of inducible nitric oxide synthase (iNOS) expression in hepatocytes, and this is, in turn, responsible for a decrease in Plasmodium-infected hepatocytes. Furthermore, primary hepatocytes from MyD88-/- mice showed no NF-κB activation and iNOS expression upon infection, suggesting a role of the Toll/IL-1 receptor family members in sensing cytosolic factors. Indeed, lack of MyD88 significantly increased infection in vitro and in vivo. In a further complementary series of experiments, we assessed a possible beneficial role for the activation of NF-κB. Infected cells were more resistant to Fas (CD95/Apo-1)-mediated apoptosis than uninfected cells or infected cells in which NF-κB was blocked by BAYl1-7082 in vitro. Paradoxically, iNOS expression contributes to the protection of infected cells from Fas-induced apoptosis, since treatment with the specific iNOS inhibitor SMT (S-Methylisothiourea Sulfate) rendered the infected cells more susceptible to apoptosis. An additional beneficial effect of host cell traversal for the parasite is the fact that mainly traversed cells present parasite-derived peptides to specific cytotoxic T cells and therefore may redirect the specific immune response to uninfected cells. In summary, we have shown that host cells wounded by parasite migration induce inflammation, which limits the extent of parasite infection. In addition, our data support the notion that survival of Plasmodium parasites in the liver is mediated by increasing the resistance of hepatocytes to Fas-induced apoptosis.

Fsp27/CIDEC is a CREB target gene induced during early fasting in liver and regulated by FA oxidation rate

FSP27 (CIDEC in humans) is a protein associated with lipid droplets that downregulates the fatty acid oxidation (FAO) rate when it is overexpressed. However, little is known about its physiological role in liver. Here, we show that fasting regulates liver expression of Fsp27 in a time-dependent manner. Thus, during the initial stages of fasting a maximal induction of 800-fold was achieved, while during the later phase of fasting, Fsp27 expression decreased. The early response to fasting can be explained by a canonical PKA-CREB-CRTC2 signaling pathway since: i) CIDEC expression was induced by forskolin, ii) Fsp27 promoter activity was increased by CREB, and iii) Fsp27 expression was upregulated in the liver of Sirt1 knockout animals. Interestingly, pharmacological (etomoxir) or genetic (Hmgcs2 interference) inhibition of the FAO rate increases the in vivo expression of Fsp27 during fasting. Similarly, CIDEC expression was upregulated in HepG2 cells by either etomoxir or HMGCS2 interference. Our data indicate that there is a kinetic mechanism of auto-regulation between short- and long-term fasting, by which free fatty acids delivered to the liver during early fasting are accumulated/exported by FSP27/CIDEC, while over longer periods of fasting they are degraded in the mitochondria through the carnitine palmitoyl transferase (CPT) system.

Peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) but not PPARalpha serves as a plasma free fatty acid sensor in liver.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is an important transcription factor in liver that can be activated physiologically by fasting or pharmacologically by using high-affinity synthetic agonists. Here we initially set out to elucidate the similarities in gene induction between Wy14643 and fasting. Numerous genes were commonly regulated in liver between the two treatments, including many classical PPARalpha target genes, such as Aldh3a2 and Cpt2. Remarkably, several genes induced by Wy14643 were upregulated by fasting independently of PPARalpha, including Lpin2 and St3gal5, suggesting involvement of another transcription factor. Using chromatin immunoprecipitation, Lpin2 and St3gal5 were shown to be direct targets of PPARbeta/delta during fasting, whereas Aldh3a2 and Cpt2 were exclusive targets of PPARalpha. Binding of PPARbeta/delta to the Lpin2 and St3gal5 genes followed the plasma free fatty acid (FFA) concentration, consistent with activation of PPARbeta/delta by plasma FFAs. Subsequent experiments using transgenic and knockout mice for Angptl4, a potent stimulant of adipose tissue lipolysis, confirmed the stimulatory effect of plasma FFAs on Lpin2 and St3gal5 expression levels via PPARbeta/delta. In contrast, the data did not support activation of PPARalpha by plasma FFAs. The results identify Lpin2 and St3gal5 as novel PPARbeta/delta target genes and show that upregulation of gene expression by PPARbeta/delta is sensitive to plasma FFA levels. In contrast, this is not the case for PPARalpha, revealing a novel mechanism for functional differentiation between PPARs.

The tumor microenvironment and its contribution to tumor evolution toward metastasis.

Cancer cells acquire cell-autonomous capacities to undergo limitless proliferation and survival through the activation of oncogenes and inactivation of tumor suppressor genes. Nevertheless, the formation of a clinically relevant tumor requires support from the surrounding normal stroma, also referred to as the tumor microenvironment. Carcinoma-associated fibroblasts, leukocytes, bone marrow-derived cells, blood and lymphatic vascular endothelial cells present within the tumor microenvironment contribute to tumor progression. Recent evidence indicates that the microenvironment provides essential cues to the maintenance of cancer stem cells/cancer initiating cells and to promote the seeding of cancer cells at metastatic sites. Furthermore, inflammatory cells and immunomodulatory mediators present in the tumor microenvironment polarize host immune response toward specific phenotypes impacting tumor progression. A growing number of studies demonstrate a positive correlation between angiogenesis, carcinoma-associated fibroblasts, and inflammatory infiltrating cells and poor outcome, thereby emphasizing the clinical relevance of the tumor microenvironment to aggressive tumor progression. Thus, the dynamic and reciprocal interactions between tumor cells and cells of the tumor microenvironment orchestrate events critical to tumor evolution toward metastasis, and many cellular and molecular elements of the microenvironment are emerging as attractive targets for therapeutic strategies.

Fast liver catabolism of C1q in patients with paraproteinaemia and depletion of the classical pathway of complement.

The main clinical features in four patients with IgG1k paraproteinaemia and acquired complement deficiency included xanthomatous skin lesions (in three), panniculitis (in three) and hepatitis (in two). Hypocomplementaemia concerned the early classical pathway components--in particular C1q. Metabolic studies employing 125I-C1q revealed a much faster catabolism of this protein in the four patients than in five normal controls and three patients with cryoglobulinaemia (mean fractional catabolic rates respectively: 23.35%/h; 1.44%/h; 5.84%/h). Various experiments were designed to characterize the mechanism of the hypocomplementaemia: the patients' serum, purified paraprotein, blood cells, bone marrow cells, or xanthomatous skin lesions did not produce significant complement activation or C1q binding. When three of the patients (two with panniculitis and hepatitis) were injected with 123I-C1q, sequential gamma-camera imaging demonstrated rapid accumulation of the radionuclide in the liver, suggesting that complement activation takes place in the liver where it could produce damage.

Care complexity, mood, and quality of life in liver pre-transplant patients.

RATIONALE: This study was intended to document the frequency of care complexity in liver transplant candidates, and its association with mood disturbance and poor health-related quality of life (HRQoL). METHODS: Consecutive patients fulfilling inclusion criteria, recruited in three European hospitals, were assessed with INTERMED, a reliable and valid method for the early assessment of bio-psychosocial health risks and needs. Blind to the results, they were also assessed with the Hospital Anxiety and Depression Scale (HADS). HRQoL was documented with the EuroQol and the SF36. Statistical analysis included multivariate and multilevel techniques. RESULTS: Among patients fulfilling inclusion criteria, 60 patients (75.9%) completed the protocol and 38.3% of them were identified as "complex" by INTERMED, but significant between-center differences were found. In support of the working hypothesis, INTERMED scores were significantly associated with all measures of both the SF36 and the EuroQol, and also with the HADS. A one point increase in the INTERMED score results in a reduction in 0.93 points in EuroQol and a 20% increase in HADS score. CONCLUSIONS: INTERMED-measured case complexity is frequent in liver transplant candidates but varies widely between centers. The use of this method captures in one instrument multiple domains of patient status, including mood disturbances and reduced HRQoL.

Clinical value of immunoscintigraphy in colorectal carcinoma patients: a prospective study.

Fifty-seven patients with suspected CEA-producing tumors were studied prospectively by radioimmunoscintigraphy (RIS) using a 123I-labeled anti-CEA monoclonal antibody (MAb) (essentially the F(ab')2 or Fab fragments) and emission computed tomography (ECT). Results of RIS were compared to those of a comprehensive diagnostic study. Final diagnosis was based on surgery, biopsy and autopsy (n = 39) or follow-up findings (n = 18). Three groups of patients were defined: Group A with suspected primary tumors (n = 11), Group B with probable (n = 19) and Group C with questionable (n = 27) tumor relapse. Eighty-eight per cent, 93% and 71% of the anatomic regions studied were correctly identified as being involved, and 97%, 97%, and 87% as being free from tumor in Groups A, B, and C, respectively. In the 27 patients from Group C with no definite diagnosis of relapse, and in whom diagnosis was most difficult, 38 tumor sites were involved. Of these, 21 were detected by both prospective RIS and repeated comprehensive study, six by RIS only and seven by conventional methods only. Four sites remained undetected by both approaches. Ten of the 21 lesions were detected by RIS more than 1 mo earlier than by any other method. Among the seven tumor sites detected by other diagnostic modalities only, three were identified at the time of RIS and four became positive more than 6 mo later. Overall diagnosis was entirely correct in 30, partially correct in 16 and incorrect in six patients studied. RIS with ECT and 123I-labeled anti-CEA MAb allows early detection of recurrence or metastasis of colorectal cancer. It thus contributes to reduced delay between diagnosis and treatment.

Population-based genome-wide association studies reveal six loci influencing plasma levels of liver enzymes.

Plasma liver-enzyme tests are widely used in the clinic for the diagnosis of liver diseases and for monitoring the response to drug treatment. There is considerable evidence that human genetic variation influences plasma levels of liver enzymes. However, such genetic variation has not been systematically assessed. In the present study, we performed a genome-wide association study of plasma liver-enzyme levels in three populations (total n = 7715) with replication in three additional cohorts (total n = 4704). We identified two loci influencing plasma levels of alanine-aminotransferase (ALT) (CPN1-ERLIN1-CHUK on chromosome 10 and PNPLA3-SAMM50 on chromosome 22), one locus influencing gamma-glutamyl transferase (GGT) levels (HNF1A on chromosome 12), and three loci for alkaline phosphatase (ALP) levels (ALPL on chromosome 1, GPLD1 on chromosome 6, and JMJD1C-REEP3 on chromosome 10). In addition, we confirmed the associations between the GGT1 locus and GGT levels and between the ABO locus and ALP levels. None of the ALP-associated SNPs were associated with other liver tests, suggesting intestine and/or bone specificity. The mechanisms underlying the associations may involve cis- or trans-transcriptional effects (some of the identified variants were associated with mRNA transcription in human liver or lymphoblastoid cells), dysfunction of the encoded proteins (caused by missense variations at the functional domains), or other unknown pathways. These findings may help in the interpretation of liver-enzyme tests and provide candidate genes for liver diseases of viral, metabolic, autoimmune, or toxic origin. The specific associations with ALP levels may point to genes for bone or intestinal diseases.

Tc-99m-MAA SPECT-derived tumor-to-normal liver ratios for partition model dosimetry in selective internal radiation therapy (SIRT)

Aim: When planning SIRT using 90Y microspheres, the partition model is used to refine the activity calculated by the body surface area (BSA) method to potentially improve the safety and efficacy of treatment. For this partition model dosimetry, accurate determination of mean tumor-to-normal liver ratio (TNR) is critical since it directly impacts absorbed dose estimates. This work aimed at developing and assessing a reliable methodology for the calculation of 99mTc-MAA SPECT/CT-derived TNR ratios based on phantom studies. Materials and methods: IQ NEMA (6 hot spheres) and Kyoto liver phantoms with different hot/background activity concentration ratios were imaged on a SPECT/CT (GE Infinia Hawkeye 4). For each reconstruction with the IQ phantom, TNR quantification was assessed in terms of relative recovery coefficients (RC) and image noise was evaluated in terms of coefficient of variation (COV) in the filled background. RCs were compared using OSEM with Hann, Butterworth and Gaussian filters, as well as FBP reconstruction algorithms. Regarding OSEM, RCs were assessed by varying different parameters independently, such as the number of iterations (i) and subsets (s) and the cut-off frequency of the filter (fc). The influence of the attenuation and diffusion corrections was also investigated. Furthermore, both 2D-ROIs and 3D-VOIs contouring were compared. For this purpose, dedicated Matlab© routines were developed in-house for automatic 2D-ROI/3D-VOI determination to reduce intra-user and intra-slice variability. Best reconstruction parameters and RCs obtained with the IQ phantom were used to recover corrected TNR in case of the Kyoto phantom for arbitrary hot-lesion size. In addition, we computed TNR volume histograms to better assess uptake heterogeneityResults: The highest RCs were obtained with OSEM (i=2, s=10) coupled with the Butterworth filter (fc=0.8). Indeed, we observed a global 20% RC improvement over other OSEM settings and a 50% increase as compared to the best FBP reconstruction. In any case, both attenuation and diffusion corrections must be applied, thus improving RC while preserving good image noise (COV<10%). Both 2D-ROI and 3D-VOI analysis lead to similar results. Nevertheless, we recommend using 3D-VOI since tumor uptake regions are intrinsically 3D. RC-corrected TNR values lie within 17% around the true value, substantially improving the evaluation of small volume (<15 mL) regions. Conclusions: This study reports the multi-parameter optimization of 99mTc MAA SPECT/CT images reconstruction in planning 90Y dosimetry for SIRT. In phantoms, accurate quantification of TNR was obtained using OSEM coupled with Butterworth and RC correction.

Brain metastasis: opportunity for drug development?

PURPOSE OF REVIEW: Brain metastases are a common clinical problem, and only limited treatment options exist. We review recent advances in medical brain metastasis research with a focus on the most common tumor types associated with secondary brain colonization: melanoma, breast cancer and lung cancer. We speculate on opportunities for drug development in patients with brain metastases, both as a treatment of established disease and as an adjuvant and prophylactic strategy. RECENT FINDINGS: BRAF inhibitors and the immunomodulatory anticytotoxic T-lymphocyte-associated antigen 4 antibody ipilimumab have shown clinically meaningful activity in melanoma patients with brain metastases. In breast cancer, current studies on drug treatment of brain metastases are mainly focusing on human epidermal growth factor receptor 2 targeting agents such as lapatinib. Emerging data seem to implicate a potential role of targeted agents including antiangiogenic compounds, pazopanib, and epithelial growth factor receptor inhibitors for prevention of brain metastasis formation in breast cancer or nonsmall cell lung cancer. SUMMARY: Novel drugs are beginning to enter clinical practice for selected patients with brain metastases. The promising findings from recent studies may fuel more research on brain metastases and their optimal drug treatment.

Hepatic amyloidosis increases liver stiffness measured by transient elastography.

Liver stiffness values in transient elastography (TE) have to be interpreted with caution. Steatosis, congestion, acute inflammation and extrahepatic cholestasis can indeed influence measurements. Obtained stiffness values in the cirrhotic range can also be present in the absence of fibrosis as in hepatic amyloidosis. Here we report two cases of systemic amyloidosis with hepatic involvement where high stiffness values were measured at TE. In fact, deposits of amyloid may increase the rigidity of the liver parenchyma resulting in higher liver stiffness values. Therefore, results of TE should always be interpreted in their clinical context and if inconsistent, the performance of a liver biopsy might be necessary.

Radiation-induced modifications of the tumor microenvironment promote metastasis.

Radiotherapy is successfully used to treat cancer. Emerging evidence, however, indicates that recurrences after radiotherapy are associated with increased local invasion, metastatic spreading and poor prognosis. Radiation-induced modifications of the tumor microenvironment have been proposed to contribute to increased aggressive tumor behavior, an effect also referred to as tumor bed effect, but the putative mechanisms involved have remained largely elusive. We have recently demonstrated that irradiation of the prospective tumor stroma impairs de novo angiogenesis through sustained inhibition of proliferation, migration and sprouting of endothelial cells. Experimental tumors growing within a pre-irradiated field have reduced tumor angiogenesis and tumor growth, increased hypoxia, necrosis, local invasion and distant metastasis. Mechanisms of progression involve adaptation of tumor cells to local hypoxic conditions as well as selection of cells with invasive and metastatic capacities. The matricellular protein CYR61 and integrin αVβ5 emerged as molecules that cooperate to mediate lung metastasis. Cilengitide, a small molecular inhibitor of αV integrins prevented lung metastasis formation. These results represent a conceptual advance to the understanding of the tumor bed effect and indicate that αV integrin inhibition might be a potential therapeutic approach for preventing metastasis in patients at risk for post-radiation recurrences.

Bioinformatics-driven identification and examination of candidate genes for non-alcoholic fatty liver disease.

ObjectiveCandidate genes for non-alcoholic fatty liver disease (NAFLD) identified by a bioinformatics approach were examined for variant associations to quantitative traits of NAFLD-related phenotypes.Research Design and MethodsBy integrating public database text mining, trans-organism protein-protein interaction transferal, and information on liver protein expression a protein-protein interaction network was constructed and from this a smaller isolated interactome was identified. Five genes from this interactome were selected for genetic analysis. Twenty-one tag single-nucleotide polymorphisms (SNPs) which captured all common variation in these genes were genotyped in 10,196 Danes, and analyzed for association with NAFLD-related quantitative traits, type 2 diabetes (T2D), central obesity, and WHO-defined metabolic syndrome (MetS).Results273 genes were included in the protein-protein interaction analysis and EHHADH, ECHS1, HADHA, HADHB, and ACADL were selected for further examination. A total of 10 nominal statistical significant associations (P<0.05) to quantitative metabolic traits were identified. Also, the case-control study showed associations between variation in the five genes and T2D, central obesity, and MetS, respectively. Bonferroni adjustments for multiple testing negated all associations.ConclusionsUsing a bioinformatics approach we identified five candidate genes for NAFLD. However, we failed to provide evidence of associations with major effects between SNPs in these five genes and NAFLD-related quantitative traits, T2D, central obesity, and MetS.