Translational imaging of the fibroblast activation protein (FAP) using the new ligand [68Ga]Ga-OncoFAP-DOTAGA.

PURPOSE: The fibroblast activation protein (FAP) is an emerging target for molecular imaging and therapy in cancer. OncoFAP is a novel small organic ligand for FAP with very high affinity. In this translational study, we establish [68Ga]Ga-OncoFAP-DOTAGA (68Ga-OncoFAP) radiolabeling, benchmark its properties in preclinical imaging, and evaluate its application in clinical PET scanning. METHODS: 68Ga-OncoFAP was synthesized in a cassette-based fully automated labeling module. Lipophilicity, affinity, and serum stability of 68Ga-OncoFAP were assessed by determining logD7.4, IC50 values, and radiochemical purity. 68Ga-OncoFAP tumor uptake and imaging properties were assessed in preclinical dynamic PET/MRI in murine subcutaneous tumor models. Finally, biodistribution and uptake in a variety of tumor types were analyzed in 12 patients based on individual clinical indications that received 163 ± 50 MBq 68Ga-OncoFAP combined with PET/CT and PET/MRI. RESULTS: 68Ga-OncoFAP radiosynthesis was accomplished with high radiochemical yields. Affinity for FAP, lipophilicity, and stability of 68Ga-OncoFAP measured are ideally suited for PET imaging. PET and gamma counting-based biodistribution demonstrated beneficial tracer kinetics and high uptake in murine FAP-expressing tumor models with high tumor-to-blood ratios of 8.6 ± 5.1 at 1 h and 38.1 ± 33.1 at 3 h p.i. Clinical 68Ga-OncoFAP-PET/CT and PET/MRI demonstrated favorable biodistribution and kinetics with high and reliable uptake in primary cancers (SUVmax 12.3 ± 2.3), lymph nodes (SUVmax 9.7 ± 8.3), and distant metastases (SUVmax up to 20.0). CONCLUSION: Favorable radiochemical properties, rapid clearance from organs and soft tissues, and intense tumor uptake validate 68Ga-OncoFAP as a powerful alternative to currently available FAP tracers.

An atlas of bloodstream-accessible bone marrow proteins for site-directed therapy of acute myeloid leukemia.

The concept of arming antibodies with bioactive payloads for a site-specific therapy of cancer has gained considerable interest in recent years. However, a successful antibody-based targeting approach critically relies on the availability of a tumor-associated target that is not only preferentially expressed in the tumor tissue but is also easily accessible for antibody therapeutics coming from the bloodstream. Here, we perfused the vasculature of healthy and acute myeloid leukemia (AML)-bearing rats with a reactive ester derivative of biotin and subsequently quantified the biotinylated proteins to identify AML-associated bone marrow (BM) antigens accessible from the bloodstream. In total, >1400 proteins were identified. Overall, 181 proteins were >100-fold overexpressed in AML as compared with normal BM. Eleven of the most differentially expressed proteins were further validated by immunohistochemistry and confocal microscopic analyses, including novel antigens highly expressed in AML cells (for example, adaptor-related protein complex 3 ß2) and in the leukemia-modified extracellular matrix (ECM) (for example, collagen-VI-α-1). The presented atlas of targetable AML-associated BM proteins provides a valuable basis for the development of monoclonal antibodies that could be used as carriers for a site-specific pharmacodelivery of cytotoxic drugs, cytokines or radionuclides to the BM in AML.

Different tissue distribution properties for glycosylation variants of fusion proteins containing the p40 subunit of murine interleukin-12.

Antibody-based fusion proteins are gaining increasing importance for therapeutic applications, but the impact of glycosylation on in vivo biopharmaceutical performance is not always completely understood. In this article, we have analyzed biochemical and pharmaceutical properties of fusion proteins, consisting of the F8 antibody (specific to the EDA domain of fibronectin, a marker of tissue remodeling and of angiogenesis) and of the p40 subunit of interleukin-12, an inhibitor of inflammation. The corresponding fusion protein (F8-IL12p40), which inhibits colitis development in mice, is a glycosylated protein with suboptimal disease targeting properties in vivo Since the protein was extensively glycosylated, as evidenced by PNGase F treatment and mass spectrometric analysis, we mutated four asparagine residues in various combinations. The corresponding proteins exhibited similar biochemical and antigen-binding properties, but differences in thermal stability and bioactivity. Asparagine mutations did not lead to recovery of disease targeting performance in vivo, as evidenced by quantitative biodistribution studies with radioiodinated protein preparations in tumor-bearing mice. By contrast, an almost complete recovery of targeting was achieved with an enzymatically deglycosylated protein preparation. These findings reinforce the concept that different glycostructures can have an impact on tissue distribution properties.

Community-acquired pneumonia and positive urinary antigen tests: Factors associated with targeted antibiotic therapy.

BACKGROUND: The use of rapid microbiological tests is supported by antimicrobial stewardship policies. Targeted antibiotic therapy (TAT) for community-acquired pneumonia (CAP) with positive urinary antigen test (UAT) has been associated with a favorable impact on outcome. We aimed to determine the factors associated with TAT prescription. PATIENTS AND METHODS: We conducted a retrospective multicenter study including all patients presenting with CAP and positive UAT for Streptococcus pneumoniae or Legionella pneumophila from January 2010 to December 2013. Patients presenting with aspiration pneumonia, coinfection, and neutropenia were excluded. CAP severity was assessed using the Pneumonia Severity Index (PSI). TAT was defined as the administration of amoxicillin for pneumococcal infection and either macrolides or fluoroquinolones (inactive against S. pneumoniae) for Legionella infection. RESULTS: A total of 861 patients were included, including 687 pneumococcal infections and 174 legionellosis from eight facilities and 37 medical departments. TAT was prescribed to 273 patients (32%). Four factors were found independently associated with a lower rate of TAT: a PSI score≥4 (OR 0.37), Hospital A (OR 0.41), hospitalization in the intensive care unit (OR 0.44), and cardiac comorbidities (OR 0.60). Four other factors were associated with a high rate of TAT: positive blood culture for S. pneumoniae (OR 2.32), Hospitals B (OR 2.34), E (OR 2.68), and H (OR 9.32). CONCLUSION: TAT in CAP with positive UAT was related to the hospitals as well as to patient characteristics.

F8-SIP mediated targeted photodynamic therapy leads to microvascular dysfunction and reduced glioma growth.

The extra domain A (ED A) of fibronectin has been identified as a tumor vessel specific neovascular marker in glioma. Antibody based vascular targeting against ED A of fibronectin allows precise accumulation of photosensitizer in glioma microvasculature and thereby promises to overcome drawbacks of current photodynamic therapy (PDT) for glioma treatment. Our aim was to characterize microcirculatory consequences of F8-small immunoprotein (SIP) mediated PDT by intravital microscopy (IVM) and to analyze the effects on glioma growth. For IVM SF126 glioma cells were implanted into dorsal skinfold-chamber of nude mice. PDT was performed after intravenous injection of photosensitizer (PS)-coupled F8-SIP or PBS (n = 4). IVM was performed before and after PDT for 4 days. Analysis included total and functional (TVD, FVD) vessel densities, perfusion index (PI), microvascular permeability and blood flow rate (Q). To assess tumor growth SF126 glioma cells were implanted subcutaneously. PDT was performed as a single and repetitive treatment after PS-F8-SIP injection (n = 5). Subcutaneous tumors were treated after uncoupled F8-SIP injection as control group (n = 5). PDT induced microvascular stasis and thrombosis with reduced FVD (24 h: 115.98 ± 0.7 vs. 200.8 ± 61.9 cm/cm(2)) and PI (39 ± 11 vs. 70 ± 10 %), whereas TVD was not altered (298 ± 39.2 vs. 278.2 ± 51 cm/cm(2)). Microvascular dysfunction recovered 4 days after treatment. Microvascular dysfunction led to a temporary reduction of glioma growth in the first 48 h after treatment with complete recovery 5 days after treatment. Repetitive PDT resulted in sustained reduction of tumor growth. F8-SIP mediated PDT leads to microvascular dysfunction and reduced glioma growth in a preclinical glioma model with recovery of microcirculation 4 days after treatment. Repetitive application of PDT overcomes microvascular recovery and leads to prolonged antiglioma effects.

Severe community-acquired pneumonia and positive urinary antigen test for S. pneumoniae: amoxicillin is associated with a favourable outcome.

Positive urinary antigen tests (UAT) for pneumococcal infection in community-acquired pneumonia (CAP) may lead to targeted antibiotic therapy. We report an audit aimed at defining the link between mortality and targeted therapy. We conducted a retrospective multicentre audit of patients with severe CAP for whom a UAT was positive for S. pneumoniae. Patients admitted from January 2010 to December 2013 to 8 medical centres (from A to H) were included. Co-morbidities were defined by the specific treatment administered before hospital care, or if the diagnosis was newly established during the hospital stay. We used the Pneumonia Severity Index (PSI) to assess disease severity. Only patients with PSI > 90 were included. Antibiotic treatments and the PSI were extracted from patients' charts. Amoxicillin had to be prescribed as a targeted antibiotic treatment or at the time of antibiotic reassessment. A total of 389 patients were included. The mean (±STD) PSI score was 128 ± 29; 38.9% of the patients had a class 5 PSI score. Intensive care was required for 36.6% of the patients. Amoxicillin was initially prescribed in 47 cases (12.1%) and in 34 cases after reassessment (8.7%). In logistic regression analysis, we found three parameters associated with mortality: being hospitalised in institution D, class 5 PSI score, and metastatic cancer. In contrast, three antibiotic regimens were protective factors, including targeted therapy: OR = 0.09, p < 0.001. In the context of severe CAP with positive UAT for S. pneumoniae, targeted therapy was associated with a reduction in mortality.

Targeted ED-B fibronectin SPECT in vivo imaging in experimental atherosclerosis.

AIM: The extracellular matrix protein ED-B fibronectin (ED-B) is upregulated in inflammatory atherosclerotic lesions. However, functional in vivo imaging of ED-B-containing plaques has not been explored. This study evaluated whether [(99m)Tc]-conjugated AP39 ([(99m)Tc]-AP39), a single-chain antibody specific to ED-B, can be used for in vivo detection of atherosclerotic plaques in Western diet (WD)-fed, apolipoprotein E-deficient (apoE-/-) mice as compared to wildtype (WT) control mice. METHODS: Using SPECT, 12-month-old WD-fed apoE-/- and WT mice were studied 4 hours after injecting [(99m)Tc]-AP39 (148 MBq). Subsequently, mice were sacrificed, thoracic aortas measured in a g-counter, and plaques analyzed using histology, immuno-histochemistry, autoradiography, and morphometry. RESULTS: In vivo [(99m)Tc]-AP39-SPECT imaging of apoE-/- mice demonstrated a significant signal activity in the plaque-ridden thoracic aorta (52.236 ± 40.646 cpm/cm³) that co-localized with the aortic arch and the supra-aortic arteries in MRI scans. Low signal activity (9.468 ± 4.976 cpm/cm³) was observed in WT mice. In apoE-/- mice, the strongest signals were detected in the aortic root, aortic arch and along the abdominal aorta. Autoradiography analysis of aortas from apoE-/- mice confirmed the in vivo observation by demonstrating signal localization in atherosclerotic plaques. The size of autoradiography-positive plaque areas correlated significantly with the size of ED-B-positive (r=0.645, P=0.044) or macrophage-infiltrated (r=0.84, P<0.002) plaques. A significant correlation was found between the sizes of ED-B-positive and macrophage-infiltrated plaque areas (r=0.93, P<0.01). CONCLUSION: [(99m)Tc]-AP39-SPECT in vivo imaging detects inflammatory plaque lesions in WD-fed apoE-/- mice.

Survival benefit of transplantation for recurrence of hepatocellular carcinoma after liver resection.

BACKGROUND: Liver transplantation (LT) for hepatocellular carcinoma (HCC) can be used for tumor recurrence after liver resection (LR) both for initially transplant-eligible patients as conventional salvage therapy (ST) and for non-transplant-eligible patients (beyond Milan criteria) with a goal of downstaging (DW). The aim of this study was to compare the intention-to-treat (ITT) survival rates of patients who are listed for LT, according to these two strategies. METHODS: We analyzed a prospective database of 399 consecutive patients who underwent hepatic resection for HCC from 2002 to 2011 to identify patients included in the waiting list for tumor recurrence. Intention-to-treat (ITT) survivals were compared with those of patients resected for HCC within and beyond Milan criteria in the same period and not included in the LT waiting list. RESULTS: The study group consisted of 42 patients, 28 in the ST group (within Milan) and 14 in the DW group (beyond Milan). The 5-year ITT survival rate was similar between the 2 groups, being 64% for ST and 60% for DW (P=.84). Twenty-five patients (15 ST and 10 DW) underwent LT, 13 (10 ST and 3 DW) were still awaiting LT, 4 (3 ST and 1 DW) dropped out of the waiting list because of tumor progression, and 7 (5 ST [33%] and 2 DW [20%]) had tumor recurrence. The 5-year ITT survival of ST patients was similar to that of 252 in-Milan HCC patients resected only (P=.3), whereas 5-year ITT survival of DW patients was significantly higher (P<.01) than that of 105 beyond-Milan HCC patients resected only. CONCLUSIONS: LR seems to be a safe and effective therapy both as alternative to transplantation and as downstaging strategy for intermediate-advanced HCC. The survival benefit of salvage LT, however, seems to be higher in the 2nd than in the 1st group.

Measured anatomical distributions of solar UVR on strawberry production workers in Italy.

The exposure of outdoor workers to solar ultraviolet radiation (UVR) has been known for some time. Measurements of erythemally weighted UVR are usually related to days of exposure and to the anatomical distribution of UVR on the human body. The aim of this research is to describe the UVR exposure of workers during a strawberry production cycle in order to assess the UVR hazard and to identify the highest exposure in the anatomical distribution of UVR on the body during an entire agricultural production cycle. This research was carried out on the experimental farm (Azienda Agraria Didattico-Sperimentale) of Università Politecnica delle Marche in Agugliano, Italy. A spectrometer was used to measure UVR, and electronic dosimeters were used to record UVR exposure. The measurements were carried out on all working days of the strawberry production cycle in 2012 during daily peak UVR levels. The daily UVR exposure geometric mean and the percentage ambient UVR in the strawberry production cycle were calculated and analyzed to assess the hazard for workers during the entire production cycle. The nape of the neck was the anatomical site most exposed to UVR. The mean daily UVR exposure on the nape of the neck was higher than 1.50 SED, the minimum value required to produce perceptible erythema in unacclimatized white skin, and a maximum value of 2.29 SED was measured. Real-time exposure data suggest that it may be useful to remind workers of the risks associated with UVR exposure.

Extracorporeal photopheresis increases neutrophilic myeloid-derived suppressor cells in patients with GvHD.

Extracorporeal photopheresis (ECP) is beneficial in patients with T-cell-mediated disorders, including GvHD, but the underlying immunological mechanisms are incompletely understood. Myeloid-derived suppressor cells (MDSCs) are innate immune cells characterized by their capacity to suppress T-cell proliferation. We quantified MDSCs by flow cytometry in peripheral blood from patients after BMT with GvHD before and after ECP treatment, patients after BMT but without GvHD and age-matched healthy controls. MDSC functionality was analyzed using T-cell proliferation, cytokine release and arginase activity. GvHD patients showed increased baseline percentages of neutrophilic MDSCs (PMN-MDSCs) compared with healthy controls and patients after BMT without GvHD. ECP treatment in GvHD patients rapidly increased circulating percentages of PMN-MDSCs. Functionally, PMN-MDSCs efficiently dampened Th1 and Th17 responses and were paralleled by an increase of cellular and extracellular arginase activity. Following ECP longitudinally over 16 weeks, two GvHD responder subgroups were identified, with group one continuously increasing PMN-MDSCs and group two with stable or decreasing PMN-MDSCs over time. This study demonstrates for the first time that ECP increases T-cell-dampening PMN-MDSCs in GvHD patients, a finding that should be confirmed in larger series of GvHD patients.

Trypan blue/giemsa staining to assess sperm membrane integrity in salernitano stallions and its relationship to pregnancy rates.

Aim of this study was to test the reliability of Trypan blue/Giemsa staining to evaluate sperm membrane integrity, acrosomal intactness and morphology in stallion to verify whether it could be applied in vitro as useful tool for sperm fertilizing ability. Fertility data on inseminated mares were collected to evaluate the relationship of sperm quality to pregnancy rates. Forty-one ejaculates were collected from 3 stallions of Salernitano Horse Breed and evaluated for gross appearance, volume, visual motility and membrane integrity with Trypan blue/Giemsa staining and thirty-five mares were inseminated during the breeding season from April to July. Differences among stallions were found in volume, sperm concentration (p < 0.05) and visual motility (p < 0.01). A decrease in sperm motility, concentration (p < 0.05) and total sperm number was found in June-July (p < 0.01). Live sperm with intact acrosome (LSIA) and proximal droplets (PD) were lower (p < 0.01) in June-July, while acrosome reacted sperm (ARS) percentage increased (p < 0.05). No fertility differences were found among stallions with an average fertility per cycle of 44.6% and a pregnancy rate of 68.6%. Higher percentages of LSIA were found in the ejaculates used to inseminate mares that became pregnant vs those used in mares not pregnant (p < 0.05). The significance of LSIA as test variable to verify the reliability of Trypan blue/Giemsa staining was confirmed by Receiver operating characteristic ROC analysis and the sensitivity of the test was 85% at a cut-off value of 48% LSIA. Trypan blue-Giemsa showed to be an accurate method that can be applied on field to evaluate sperm membrane integrity and to identify poor-quality ejaculates.

The antibody-based targeted delivery of TNF in combination with doxorubicin eradicates sarcomas in mice and confers protective immunity.

BACKGROUND: Soft-tissue sarcomas are a group of malignancies of mesenchymal origin, which typically have a dismal prognosis if they reach the metastatic stage. The observation of rare spontaneous remissions in patients suffering from concomitant bacterial infections had triggered the clinical investigation of the use of heat-killed bacteria as therapeutic agents (Coley's toxin), which induced complete responses in patients in the pre-chemotherapy era and is now known to mediate substantial elevations in serum TNF levels. METHODS: We designed and developed a novel immunocytokine based on murine TNF sequentially fused to the antibody fragment F8 (specific to extra-domain A of fibronectin). The antitumor activity was studied in two syngeneic murine sarcoma models. RESULTS: The L19 antibody (specific to extra-domain B of fibronectin) has shown by SPECT imaging procedures to selectively localise on sarcoma in a patient with a peripheral nerve sheath tumour, and immunohistochemical analysis of human soft-tissue sarcoma samples showed comparable antigen expression of EDA and EDB. The antibody-based pharmacodelivery of TNF by the fusion protein 'F8-TNF' to oncofetal fibronectin in sarcoma-bearing mice leads to complete and long-lasting tumour eradications when administered in combination with doxorubicin, the first-line drug for the treatment of sarcomas in humans. Doxorubicin alone did not display any therapeutic effect in both tested models of this study. The cured mice had acquired protective immunity against the tumour, as they rejected subsequent challenges with sarcoma cells. CONCLUSION: The findings of this study provide a rationale for the clinical study of the fully human immunocytokine L19-TNF in combination with doxorubicin in patients with soft-tissue sarcoma.

Phase I/II study of the tumour-targeting human monoclonal antibody-cytokine fusion protein L19-TNF in patients with advanced solid tumours.

PURPOSE: L19-TNF is an armed antibody that selectively targets human TNF to extra domain B-fibronectin on tumour blood vessels. We performed a phase I/II first-in-man trial with L19-TNF monotherapy in metastatic solid cancer patients to study safety and signs of clinical activity. METHODS: Six cohorts of patients were treated with increasing (1.3-13 µg/kg) doses of intravenous L19-TNF on day 1, 3, and 5 of repeated 3-weekly cycles, and 12 colorectal cancer patients were treated at 13 µg/kg. PK, antibody formation, changes in lymphocyte subsets, 5-HIAA plasma levels as well as safety and clinical activity were analysed. RESULTS: Thirty-four patients received at least one L19-TNF dose. The serum half-life of L19-TNF at 13 µg/kg was 33.6 min, and maximum peak serum concentration was 73.14 µg/L. Mild chills, nausea and vomiting but no haemato- or unexpected toxicity were observed. Grade 3 lumbar pain in bone metastasis was the only dose-limiting toxicity found in one patient. Objective tumour responses were not detected. Transient stable disease occurred in 19 of 31 evaluable patients. CONCLUSIONS: Intravenous L19-TNF on day 1, 3, and 5 of a 3-weekly schedule was safe up to 13 µg/kg, but did not result in objective tumour responses. The maximally tolerated dose (MTD) was not reached, allowing for further dose escalation of L19-TNF possibly in combination with chemotherapy.

Liver autotransplantation for the treatment of unresectable hepatic metastasis: an uncommon indication-a case report.

Ex situ ex vivo liver surgery represents a method to expand the surgical indications to treat otherwise unresectable liver tumors. We report the case of a 38-year old woman with hepatic metastasis from a pancreatoblastoma that was judged to be unresectable due to the involvement of the three hepatic veins. To treat the primary tumor, she underwent a pancreaticoduodenectomy, adjuvant chemotherapy, and thermal ablation of a liver metastasis. After appropriate preoperative study and with the permission of the ethics committee, she underwent ex situ ex vivo liver resection. The hepatectomy was performed by removing the whole liver en bloc with the retrohepatic vena cava. The inferior vena cava was reconstructed by interposition of a prosthetic graft. The ex situ ex vivo hepatic resection, a left hepatic lobectomy included the lesion in segments 1-5-7-8. The two hepatic veins were reconstructed using patches of saphenous vein. The organ was preserved continuously for 6 hours using hypothermic perfusion with 4°C Celsior solution. The liver was then reimplanted performing an anastomosis between the reconstructed hepatic veins and the caval prostheses. The patient was discharged at postoperative day 22 and is currently disease-free at 8 months after surgery and 44 months after the initial diagnosis. Ex situ, ex vivo liver surgery offers an additional option for patients with both primary and secondary liver tumors considered to be unresectable using traditional surgical approaches.

Subnormothermic machine perfusion for non-heart-beating donor liver grafts preservation in a Swine model: a new strategy to increase the donor pool?

We previously reported that subnormothermic machine perfusion (sMP; 20°C) is able to improve the preservation of livers obtained from non-heart-beating donors (NHBDs) in rats. We have compared sMP and standard cold storage (CS) to preserve pig livers after 60 minutes of cardiac arrest. In the sMP group livers were perfused for 6 hours with Celsior at 20°C. In the CS group they were stored in Celsior at 4°C for 6 hours as usual. To simulate liver transplantation, both sMP- and CS-preserved livers were reperfused using a mechanical continuous perfusion system with autologus blood for 2 hours at 37°C. At 120 min after reperfusion aspartate aminotransferase levels in sMP versus CS were 499 ± 198 versus 7648 ± 2806 U/L (P < .01); lactate dehydrogenase 1685 ± 418 versus 12998 ± 3039 U/L (P < .01); and lactic acid 4.78 ± 3.02 versus 10.46 ± 1.79 mmol/L (P < .01) respectively. The sMP group showed better histopathologic results with significantly less hepatic damage. This study confirmed that sMP was able to resuscitate liver grafts from large NHBD animals.

Dipeptidylpeptidase--IV, a key enzyme for the degradation of incretins and neuropeptides: activity and expression in the liver of lean and obese rats.

Given the scarcity of donors, moderately fatty livers (FLs) are currently being considered as possible grafts for orthotopic liver transplantation (OLT), notwithstanding their poor tolerance to conventional cold preservation. The behaviour of parenchymal and sinusoidal liver cells during transplantation is being studied worldwide. Much less attention has been paid to the biliary tree, although this is considered the Achille's heel even of normal liver transplantation. To evaluate the response of the biliary compartment of FLs to the various phases of OLT reliable markers are necessary. Previously we demonstrated that Alkaline Phosphatase was scarcely active in bile canaliculi of FLs and thus ruled it out as a marker. As an alternative, dipeptidylpeptidase-IV (DPP-IV), was investigated. This ecto-peptidase plays an important role in glucose metabolism, rapidly inactivating insulin secreting hormones (incretins) that are important regulators of glucose metabolism. DPP-IV inhibitors are indeed used to treat Type II diabetes. Neuropeptides regulating bile transport and composition are further important substrates of DPP-IV in the enterohepatic axis. DPP-IV activity was investigated with an azo-coupling method in the liver of fatty Zucker rats (fa/fa), using as controls lean Zucker (fa/+) and normal Wistar rats. Protein expression was studied by immunofluorescence with the monoclonal antibody (clone 5E8). In Wistar rat liver, DPP-IV activity and expression were high in the whole biliary tree, and moderate in sinusoid endothelial cells, in agreement with the literature. Main substrates of DPP-IV in hepatocytes and cholangiocytes could be incretins GLP-1 and GIP, and neuropeptides such as vasoactive intestinal peptide (VIP) and substance P, suggesting that these substances are inactivated or modified through the biliary route. In lean Zucker rat liver the enzyme reaction and protein expression patterns were similar to those of Wistar rat. In obese rat liver the patterns of DPP-IV activity and expression in hepatocytes reflected the morphological alterations induced by steatosis as lipid-rich hepatocytes had scarce activity, located either in deformed bile canaliculi or in the sinusoidal and lateral domains of the plasma membrane. These findings suggest that bile canaliculi in steatotic cells have an impaired capacity to inactivate incretins and neuropeptides. Incretin and/or neuropeptide deregulation is indeed thought to play important roles in obesity and insulin-resistance. No alteration in enzyme activity and expression was found in the upper segments of the biliary tree of obese respect to lean Zucker and Wistar rats. In conclusion, this research demonstrates that DPP-IV is a promising in situ marker of biliary functionality not only of normal but also of fatty rats. The approach, initially devised to investigate the behaviour of the liver during the various phases of transplantation, appears to have a much higher potentiality as it could be further exploited to investigate any pathological or stressful conditions involving the biliary tract (i.e., metabolic syndrome and cholestasis) and the response of the biliary tract to therapy and/or to surgery.

B domain containing Tenascin-C: a new urine marker for surveillance of patients with urothelial carcinoma of the urinary bladder?

BACKGROUND: ECM remodelling during tumorigenesis entails the re-occurrence of different Tn-C(L) splicing variants. In patients with urothelial carcinoma of the urinary bladder (UBC), B and C domain containing Tenascin-C (B(+) and C(+) Tn-C) urine levels were shown to be increased in case of muscle invasiveness. Thus, the present study was aimed at examining the ability of B(+) and C(+) Tn-C as potential urinary surveillance markers of UBC patients. METHODS: Urine levels of B(+) and C(+) Tn-C were determined by ELISA in 35 UBC patients during a 2 year follow-up period after therapy and related to clinical diagnosis and histological stage in 4 defined groups representing typical courses of disease. RESULTS: B(+) Tn-C levels showed significant differences between cases of tumour progression or regression. The urine levels of B(+) Tn-C could be used to discriminate between cases without tumour recurrence and such with tumour existence (cut-off value: 0.8 ng/ml) or between non-muscle invasive and muscle invasive tumour growth (cut-off value: 3.5 ng/ml). CONCLUSIONS: Progression of UBC with time is accompanied by significant changes in urinary levels of B(+) Tn-C. Urinary B(+) Tn-C can therefore be suggested as a valuable urine surveillance marker in UBC follow-up care.