BACKGROUND

There is a great need for risk stratification in patients with chronic cholestatic diseases in order to allow for more personalized care and adapted management as well as for well-designed therapeutic trials. Novel tools for monitoring primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) patients have been recently proposed. In addition, major insight has been gained into bile acid (BA) physiology during the last decade including the role of BAs as metabolic modulators and the gut-liver axis. As a consequence, alongside drugs targeting immune response or fibrotic processes, a number of novel anti-cholestatic agents have undergone pre-clinical and clinical evaluation and have shown promising results although none has been approved yet.

CONCLUSIONS

Biochemical non-response to ursodeoxycholic acid (UDCA) (mainly defined by bilirubin and alkaline phosphatase levels at 1 year) is a strong prognostic <em>factor</em> in PBC whereas present biochemical surrogates are far from robust in PSC. By contrast, liver stiffness measurement by vibration-controlled transient elastography (VCTE) is a very promising tool in both PBC and PSC. Novel therapeutic approaches include (i) agonists of nuclear receptors, especially farnesoid X receptor (FXR), pregnane X receptor (PXR), glucocorticoid receptor (GR) and peroxisome proliferator-activated receptor α (PPARα) that are transcriptional modifiers of bile formation; (ii) agonists of TGR5, a BA membrane receptor expressed in various tissues; (iii) inhibitors of the ileal apical sodium BA transporter; (iv) derivatives of the FXR-induced <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> from the ileum that suppresses hepatic BA synthesis and (v) norUDCA, a side chain shortened UDCA derivative with specific physicochemical and therapeutic properties. The most advanced clinical evaluation (PBC patients) relates to agonists for PPARα, FXR and GR/PXR most often in combination with UDCA, namely fibrates, obeticholic acid (OCA) and budesonide, respectively. Existing results look promising even though some side effects are worrisome such as pruritus in OCA-treated patients. Results of large well-designed studies are eagerly awaited.

CONCLUSIONS

Major advances in the management of cholestatic liver diseases are in progress and promising times for these patients seem likely in the near future.

Dear Editor, Scleroderma associated with neoplasia is rare, with only a small number of cases reported. We describe 4 patients with paraneoplastic scleroderma who were treated at the I. Department of Dermatovenereology, St. Anna Hospital, during the period between 2004 and 2014. The patients were diagnosed with cholangiogenic carcinoma, endometrial carcinoma, prostatic adenocarcinoma, and adenoma of the suprarenal gland. In the case of concurrent scleroderma and tumor, four situations may occur: they can develop independently of each other; scleroderma may be induced by the tumor; the tumor can develop in the scleroderma; or the tumor can be induced by immunosuppressive therapy. Sclerotization of the skin was described in association with lung cancer, carcinoid, plasma cell dyscrasia, cancer of the ovary, cervix, breast, esophagus, stomach, nasopharynx, melanoma, and sarcoma (1,2,5,7,10). Symptoms may be induced by substances secreted by the tumor (hormones, cytokines, etc.) (9). Tumorous cells further induce cytotoxic and autoantibody response. Scleroderma is characterized by immunological dysregulation, vasculopathy, and hyperproduction of the extracellular matrix by activated <em>fibroblasts</em>. Endothelial, inflammatory, and mesenchymal cells produce cytokines, chemokines, and <em>growth</em> <em>factors</em> e.g. Interleukin-1 (IL1), Interleukin-6 (IL6), tumor necrosis <em>factor</em> alpha (TNF α), collagen alpha 1, connective tissue <em>growth</em> <em>factor</em> (CTGF) (3), and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). This <em>factor</em> is also produced by lung cancer cells (4). The clinical picture of scleroderma and paraneoplastic scleroderma is similar. Diffuse thickening of the skin and/or sclerodermatous plaques can be seen. The histological picture is consistent with scleroderma. Capillaroscopy changes, antinuclear antibodies (ANA), sclerodactyly, and Raynaud phenomenon suggest the diagnosis of systemic scleroderma (SS) (4). Our patients did not fulfill enough of the criteria for SS. Both diffuse and localized scleroderma was seen in 3 patients and generalized localized scleroderma in one case. All patients had a histological picture consistent with scleroderma, negative ANA and ENA antibodies (Table 1, Figure 1). A 66-year-old woman presented with a 10 months history of sclerodermatous plaques on her neck, trunk, and upper and lower extremities. The skin on her breasts and cheeks was diffusely indurated. Examination showed thrombocytopenia, elevated transaminases, Cancer antigen <em>19</em>-9 (Ca <em>19</em>-9), thyroid stimulating hormone (TSH), and anti-thyroid peroxidase antibodies, dysmotility of the lower part of esophagus, hepatosplenomegaly, cholecystolithiasis, and benign polyps of colon. She was given prednisone 40 mg/day but did not return for follow up. After 6 months she was diagnosed with cholangiogenic carcinoma with metastatic disease and died shortly afterwards. A 74-year-old woman had localized scleroderma on the trunk for three years. She was treated with procaine penicillin for positive borrelia Immunoglobulin M (IgM) antibodies. Her condition worsened suddenly with confluent scleroderma plaques on her trunk, extremities, and genital region, and vasoneurosis on her lower extremities; she was started on prednisone 35 mg/day. Examination revealed endometrial cancer. The patient underwent a hysterectomy, adnexectomy, and radiotherapy with curative effect. Scleroderma patches softened with residual hyperpigmentation, and prednisone was stopped two years later. A 80-year-old man had a month-long history of diffuse thickening and toughening of the skin on the forearms and lower legs and scleroderma patches on the thighs and shins. Examination revealed prostate adenocarcinoma, and therapy with antiandrogen bicalutamide and prednisone 15 mg/day was started. Two years after the diagnosis he continues with bicalutamide treatment, prednisone 5 mg q.a.d. and has residual toughening of the skin on his lower legs. A 62-year-old woman with seronegative rheumatoid arthritis presented with diffusely tough skin on her extremities and trunk, present for 2 months. Examination revealed cervicitis with a benign endometric polyp, cholecystolithiasis, borderline pulmonary hypertension, and a hormonally inactive suprarenal adenoma. She was given prednisone 40 mg/day and penicillamine with effect. In the 3rd year of therapy she has residual induration of her lower legs and a scleroderma plaque in the lumbar region. She is monitored for her suprarenal adenoma. Two patients had scleroderma at the same time as a malignant tumor; in one patient the localized scleroderma worsened rapidly at the time of the tumor diagnosis, and in one patient a clinically silent adenoma was found. Adrenal tissue can secrete molecules such as serotonine or bFGF involved in fibroplasia (3,6). One patient died of a metastatic disease, two patients after the successful treatment of the tumor, and the patient with suprarenal adenoma experienced softening of the skin and regression of scleroderma. Although paraneoplastic scleroderma is often classified as a pseudoscleroderma, we regard neoplasia as a distinct triggering impulse for scleroderma. Recently, an association between RNA polymerase I/III antibodies in systemic scleroderma and cancer was suggested (8). Such studies may confirm the true link between scleroderma and malignancy. These patients are characterized by older age, sudden onset, diffuse thickening of the skin, and/or generalized morphea with a concurrent neoplastic process. In the case of a successful tumor treatment, skin changes regress.

Epidermal <em>growth</em> <em>factor</em> receptor signaling plays an important role in lung maturation. The authors hypothesized that specific protein kinase C (PKC) isoforms are expressed and activated by epidermal <em>growth</em> <em>factor</em> (EGF) and transforming <em>growth</em> <em>factor</em> alpha (TGFalpha) (EGF receptor [EGFR] ligands) in fetal lung <em>fibroblasts</em>. The authors found 4 PKC isoforms expressed in gestational day <em>19</em> (d<em>19</em>) fetal rat lung <em>fibroblasts</em>, and focused on PKCalpha because of its developmental expression pattern. PKCalpha immunolocalization in d17, d<em>19</em>, and d21 fetal lung <em>fibroblasts</em> was similar to EGFR. PKCalpha expression decreased with lung maturation. EGF, but not TGFalpha, stimulated PKCalpha activation and membrane translocation. Further studies of PKCalpha functions in fetal lung development are clearly needed.

Fatty acid synthase (FAS) plays a central role in fatty acid synthesis and its expression is under nutritional and hormonal control. We have investigated insulin-like <em>growth</em> <em>factor</em>-I (IGF-I) regulation of FAS by transfecting into 3T3-L1 <em>fibroblasts</em> chimeric genes comprising the 5'-flanking region of the FAS gene linked to a luciferase (LUC) reporter gene. First, the basal promoter activity of the 5' serial deletions from nucleotides -318 to -<em>19</em> of the FAS gene were compared. Deletions of the promoter sequences from -136 to -<em>19</em> resulted in a step-wise decrease in the promoter activity, with the -67 LUC and -<em>19</em> LUC plasmids retaining 40% and 16% of the luciferase activity of -136 LUC. Regulatory sequences important for the FAS basal promoter activity in 3T3-L1 <em>fibroblasts</em> are, therefore, located within the -136 to -<em>19</em> region. Treatment with 10 mM IGF-I also increased luciferase activity 1.8 +/- 0.2-, 1.8 +/- 0.3- and 2.5 +/- 0.1-fold in 3T3-L1 <em>fibroblasts</em> transiently transfected with -136 LUC, -110 LUC and -67 LUC plasmids, respectively. Deletion of sequences from -67 to -<em>19</em> resulted in the loss of responsiveness to IGF-I. Physiological doses of insulin (10 nM), however, did not increase luciferase activity in 3T3-L1 <em>fibroblasts</em> transfected with any of the above plasmids. Only upon treatment with pharmacological doses of insulin (1 microM), probably through IGF-I receptor, did luciferase activity increase 4.3 +/- 0.4-, 3.2 +/- 0.4- and 3.5 +/- 0.5-fold when transfected with -136 LUC, -110 LUC and -67 LUC plasmids, respectively; there was no increase with -<em>19</em> LUC. The half-maximal effect of IGF-I on FAS promoter activity was observed at 3 nM and a maximal effect was reached at 10 nM. These results indicate that the increased promoter activities observed are probably mediated through the IGF-I receptor. Furthermore, sequences responsible for IGF-I regulation of the FAS gene are located within the proximal promoter between nucleotides -67 and -<em>19</em> of the FAS gene.

We examined microsatellite instability (MSI) at 10 loci of dinucleotide repeats using the polymerase chain reaction (PCR) in patients with myelodysplastic syndrome (MDS). Bone marrow DNA was obtained from 45 patients repeatedly during the disease course and <em>fibroblast</em> DNA was also collected from <em>19</em> of them as a normal control. Three of the <em>19</em> patients showed an alteration at more than three loci, when the allele length was compared between their <em>fibroblast</em> DNA and the initial marrow DNA. On the other hand, none of the 45 patients showed an alteration when the initial sample was compared with the latest one. One of the three patients with MSI had refractory anemia and two refractory anemia with ring sideroblasts and none of them showed disease progression, complex chromosome abnormality, karyotypic evolution, or mutation of N-RAS or TP53. Moreover, a frameshift mutation within 10 repeating adenines of transforming <em>growth</em> <em>factor</em> beta type II receptor gene, which was recently recognized as a critical target of MSI, was not found in any of the patients including the three with MSI. These findings suggest that MSI is an early but infrequent genetic event and is independent of other critical genetic aberrations in the development of MDS.

BACKGROUND

A deficiency in the ileal hormone <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) has been described in patients with bile acid diarrhoea (BAD), but fasting FGF<em>19</em> levels have insufficient diagnostic power. We assess whether single postprandial sampling of FGF<em>19</em> has greater discriminative value than fasting FGF<em>19</em> for detection of BAD and we evaluate the reproducibility of fasting FGF<em>19</em>.

METHODS

Twenty-six patients consecutively referred to Se homocholic acid retention test (SeHCAT) were included. Serum FGF<em>19</em> was measured after an overnight fast and again 1 h postprandially and again in the fasting state 1 week later.

RESULTS

Nine of 26 patients had SeHCAT less than 10% and fasting FGF<em>19</em> was lower [median 62 pg/ml, interquartile range (IQR): 47-67] than in the 17 diarrhoea controls with SeHCAT at least 10% (median 103 pg/ml, IQR: 77-135, P=0.006). Postprandial FGF<em>19</em> in BAD patients (61 pg/ml, IQR: 48-69) was similar to fasting values (P=0.59) and increased insignificantly in diarrhoea controls (137 pg/ml, IQR: 88-182; P=0.25). The difference in postprandial FGF<em>19</em> between patients with BAD and diarrhoea controls was highly significant (P<0.001).

CONCLUSIONS

The difference in serum FGF<em>19</em> between groups of patients with BAD and diarrhoea controls is amplified postprandially. Within each group, the difference between fasting and postprandial FGF<em>19</em> was not statistically significant. Further investigations are warranted on stimulated FGF<em>19</em> response to elucidate its role in BAD.

Here, we report that male heparan sulfate 6-O-sulfotransferase-2 (Hs6st2) knockout mice showed increased body weight in an age-dependent manner even when fed with a normal diet and showed a phenotype of impaired glucose metabolism and insulin resistance. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the expression of mitochondrial uncoupling proteins Ucp1 and Ucp3 was reduced in the interscapular brown adipose tissue (BAT) of male Hs6st2 knockout mice, suggesting reduced energy metabolism. The serum level of thyroid-stimulating hormone was significantly higher and that of thyroxine was lower in the knockout mice. When cultures of brown adipocytes from wild-type and Hs6st2 knockout mice isolated and differentiated in vitro were treated with FGF<em>19</em> (<em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em>) or FGF21 in the presence or the absence of heparitinase I, phosphorylation of p42/p44 mitogen-activated protein (MAP) kinase was reduced. Heparan sulfate (HS) 6-O-sulfation was reduced not only in BAT but also in the thyroid tissue of the knockout mice. Thus, 6-O-sulfation in HS seems to play an important role in mediating energy metabolism by controlling thyroid hormone levels and signals from the FGF<em>19</em> subfamily proteins, and the alteration of the HS composition may result in metabolic syndrome phenotypes such as altered glucose and insulin tolerance.

In diabetic cardiomyopathy, mitochondrial fatty acid oxidation dominates over mitochondrial glucose oxidation, leading to metabolic disturbances. <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) acts as a metabolic regulator and may have a cardioprotective role on diabetic cardiomyopathy. In this study, we investigated the effects of FGF<em>19</em> on energy metabolism. FGF<em>19</em> treatment of diabetic hearts exhibited higher glucose uptake and lower lipid profiles, suggesting changes in energy metabolism. The protective effects of FGF<em>19</em> prevented ventricular dysfunction in diabetic hearts and improved mitochondrial function by the upregulation of PGC-1α expression. On the other side, knockdown of PGC-1α by siRNA attenuated the effects of FGF<em>19</em> on the enhancement of mitochondrial function and energy efficiency. Taken together, these results show that FGF<em>19</em> exhibited improved mitochondrial efficiency, which might be associated with higher cardiac contractility in diabetic hearts. It is also of note that modulation of PGC-1α, which is responsible for the activation by FGF<em>19</em>, may be a therapeutic target for diabetic cardiomyopathy.

The disulfide arrangement of yeast derived human insulin-like <em>growth</em> <em>factor</em> I (yIGF-I) was determined using a combination of Staphylococcus aureus V8 protease mapping, fast-atom-bombardment mass spectrometry as well as amino acid sequence and composition analysis. Three disulfide bridges were found between the following cysteine residues: Cys6-Cys48, Cys47-Cys52 and Cys18-Cys61. IGF-I isolated from human plasma (pIGF-I) was found to have an identical disulfide configuration. A yeast-derived isomeric form of IGF-I (yisoIGF-I) exhibited an altered disulfide arrangement: Cys6-Cys47, Cys48-Cys52 and Cys18-Cys61. Radioreceptor analysis of pIGF-I and yIGF-I showed high specific activity, 20,000 U/mg. However, yisoIGF-I demonstrated a severely reduced ability to bind to the IGF-I receptor (<em>19</em>%) and was less potent in provoking a mitogenic response in Balb/C 3T3 <em>fibroblasts</em> (50% at doses 10-100 ng/ml). The data demonstrate the importance of correct disulfide arrangement in IGF-I for full biological activity.

OBJECTIVE

To investigate the possibility of the human bone marrow multipotent adult progenitor cells (hMAPCs) to differentiate into hepatocytes with hepatocyte growth factor (HGF)/ fibroblast growth factor-4 (FGF-4) in vitro.

METHODS

(1) Obtaining the hMAPCs. Bone marrow was obtained from volunteers and then centrifuged through density gradient centrifugation methods. The collected mononuclear cells were cultured through adheret culture to get mesenchymal stem cells (MSCs). The hMAPCs were obtained through collecting and isolating the MSCs by magnetic activated cell sorting (MACS) through depletion selection by use of CD45 and GlyA microbeads. (2) Differentiation of the hMAPCs with HGF+FGF-4. Group A: HGF (20 ng/ml) + FGF-4 (10 ng/ml) induced hMAPCs; group B (positive control group): L-02 human hepatocytes(cell lines); and group C (negative control group): the undifferentiated hMAPCs. (3) The expressions of albumin (Alb), alpha fetoprotein (AFP), cytokeratin-18 (CK-18), and cytokeratin-19 (CK-19) were detected with immunocytochemistry to identify the characteristics of the differentiated cells at different times and the ratio of the positive cells was determined. (4) ALB, AFP, CK-18, and CK-19 expressions of the differentiated cells were detected by RT-PCR assay to investigate the mRNA transcriptions of characteristic hepatic proteins. (5) Alb expressions of the differentiated cells at different times were detected by Western blot on the 21st and 35th days.

RESULTS

(1) The results of immunocytochemistry. The staining of Alb, CK18 were essentially positive in group A. As an early marker of immature hepatocytes, AFP staining was positive on the 7th day but negative in later differentiating periods in group A. (2) The results of RT-PCR. On the 7th day, the differentiated hMAPCs expressed AFP mRNA but were negative in later differentiating periods. On the contrary, the mRNA of Alb and CK-18 were positive at all times. (3) The results of Western blot assay. Alb protein was positive on the 21st day and 35th day.

CONCLUSIONS

Under some definite inducing conditions hMAPCs can differentiate into hepatocyte-like cells. They may serve as a potential cell source for liver engineering.

BACKGROUND

Bile acid malabsorption (BAM) is one possible explanation for chronic diarrhea. BAM may be idiopathic, or result from ileal resection or inflammation including Crohn's disease, or may be secondary to other conditions, including cholecystectomy, peptic ulcer surgery, and chronic pancreatitis. No "gold standard" exists for clinical diagnosis of BAM, but response to treatment with a bile acid sequestrant (BAS) is often accepted as confirmation. The SeHCAT (tauroselcholic [selenium-75] acid) test uses a radiolabeled synthetic bile acid and provides a diagnostic test for BAM, but its performance against "trial of treatment" is unknown. <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF-<em>19</em>) and 7-alpha-hydroxy-4-cholesten-3-one (C4) also offer potential new biomarkers of BAM.

OBJECTIVE

This protocol describes a multicenter prospective study to evaluate the diagnostic accuracy of SeHCAT and 2 biomarkers in predicting BAM as assessed by trial of treatment.

METHODS

Participating gastroenterology centers should have a minimum workload of 30 SeHCAT patients per annum. Patients should not be pregnant, on medication that could confound follow-up, or have any severe comorbidity. All eligible patients attending a gastrointestinal appointment will be invited to participate. On attending the SeHCAT test, blood and fecal samples will be collected for analysis of FGF-<em>19</em> by enzyme-linked immunosorbent assay and for C4 and fractionated bile acids by liquid chromatography-mass spectrometry. A capsule containing radiolabeled SeHCAT will be administered orally and a scan performed to measure SeHCAT activity. Patients will return on day 7 to undergo a second scan to measure percentage SeHCAT retention. The test result will be concealed from clinicians and patients. BAS will be dispensed to all patients, with a follow-up gastroenterologist appointment at 2 weeks for clinical assessment of treatment response and adherence. Patients responding positively will continue treatment for a further 2 weeks and all patients will have a final follow-up at 8 weeks. The diagnostic accuracy of the SeHCAT test and biomarkers will be analyzed at different thresholds using sensitivity, specificity, positive and negative predictive value, likelihood ratios, and area under the curve in a sample of 600 patients. Multivariable logistic regression models will be used to assess the association between presence of BAM and continuous SeHCAT retention levels after adjustment for confounders.

RESULTS

Funding is being sought to conduct this research.

CONCLUSIONS

The SeHCAT test for diagnosis of BAM has been in common use in the United Kingdom for more than 30 years and an evidence-based assessment of its accuracy is overdue. The proposed study has some challenges. Some forms of BAS treatment are unpleasant due to the texture and taste of the resin powder, which may negatively affect recruitment and treatment adherence. Trial of treatment is not as "golden" a standard as would be ideal, and itself warrants further study.

Transforming <em>growth</em> <em>factor</em>-beta receptor II (TGF-βRII) is an attractive target for anti-scarring therapy in wound healing because it attenuates excessive TGF-β which has pleiotropic effects on the immune system. In the present study, the cDNA of rabbit TGF-βRII (rTGF-βRII) was amplified from rabbit peripheral blood by RT-PCR. The open reading frame of rTGF-βRII encodes a protein consisting of 567 amino acids, which contains a predicted transmembrane domain and a Serine/Threonine protein kinase domain similar to other identified mammalian TGF-βRIIs. The amino acid sequences of the biologically active, soluble rTGF-βRII and mouse, rat, human and chicken counterparts are 81%, 81%, 89% and 61%, respectively, identical. Recombinant soluble rTGF-βRII (rsTGF-βRII) fused with His tag was efficiently expressed in Escherichia coli BL21 (DE3) expression host strain. This fusion protein's molecular weight of ∼ <em>19</em> kDa was identified by SDS-PAGE and Western blotting. In vitro, purified rsTGF-βRII was able to inhibit the proliferation of keloid rabbit <em>fibroblasts</em> and decrease the level of collagen. These findings indicate that rTGF-βRII plays an important role in inhibiting the proliferation of keloid rabbit <em>fibroblasts</em> and provides the basis for investigations on the role of TGF-βRII in this important domestic species.

Epithelial denudation is one of the characteristics of chronic asthma. To restore its functions, the airway epithelium has to rapidly repair the injuries and regenerate its structure and integrity. Mesenchymal stem cells (MSCs) have the ability to differentiate into many cell lineages. However, the differentiation of MSCs into epithelial cells has not been fully studied. Here, we examined the differentiation of MSCs into epithelial cells using three different media compositions with various <em>growth</em> supplementations. The MSCs were isolated from porcine bone marrow by density gradient centrifugation. The isolated MSCs were CD11(-) CD34(-) CD45(-) CD44(+) CD90(+) and CD105(+) by immunostaining and flow cytometry. MSCs were stimulated with EpiGRO (Millipore), BEpiCM (ScienCell) and AECGM (PromoCell) media for 5 and 10 days, and epithelial differentiation was assessed by qPCR (keratin 14, 18 and EpCAM), fluorometry (cytokeratin 7-8, cytokeratin 14-15-16-<em>19</em> and EpCAM), western blot analysis (pancytokeratin, EpCAM) and flow cytometry (cytokeratin 7-8, cytokeratin 14-15-16-<em>19</em> and EpCAM). The functional marker MUC1 was also assessed after 10 days of air-liquid interface (ALI) culture in optimized media. Cells cultured in BEpiCM containing <em>fibroblast</em> <em>growth</em> <em>factor</em> and prostaglandin E2 showed the highest expression of the epithelial markers: CK7-8 (85.90%); CK-14-15-16-<em>19</em> (10.14%); and EpCAM (64.61%). The cells also expressed functional marker MUC1 after ALI culture. The differentiated MSCs when cultured in BEpiCM medium ex vivo in a bioreactor on a decellularized trachea for 10 days retained the epithelial-like phenotype. In conclusion, porcine bone marrow-derived MSCs demonstrate commitment to the epithelial lineage and might be a potential therapy for facilitating the repair of denuded airway epithelium.

Insulin-like <em>growth</em> <em>factor</em> (IGF)-stimulated lung <em>fibroblast</em> proliferation may be regulated by locally produced IGF-binding proteins (IGFBPs) during lung development. Recent evidence has shown that many <em>growth</em> <em>factors</em> participate in the regulation of cell proliferation by regulating IGFBPs. Because platelet-derived <em>growth</em> <em>factor</em>-BB (PDGF-BB) is highly expressed during lung development and is known to regulate IGFBP-4 production by lung cells, we examined the mechanisms by which PDGF-BB regulates ICFBP-4 production using primary cultures of <em>19</em>-day gestation rat lung <em>fibroblasts</em>. Exposure of fetal rat lung <em>fibroblasts</em> to PDGF-BB increased IGFBP-4 mRNA transcript abundance by 3.6- and 2.4-fold at 18 and 40 hours, respectively. Addition of Rp-adenosine-3'-5'-cyclic monophosphothioate triethylamine (rp-cAMPS), a competitive inhibitor of protein kinase A, blunted the PDGF-BB-stimulated increase in conditioned medium (CM) IGFBP-4 and the increase in IGFBP-4 mRNA. Proteolysis of IGFBP-4 was detected in aliquots of cell-free CM from cells exposed to SFM for 48 hours. IGFBP-4 proteolysis was inhibited by EDTA and 1,10-phenanthroline and was accentuated by the addition of IGF-I and IGF-II and, to a lesser extent, by des(1-3)IGF-I. Exposure of cells to PDGF-BB for 48 hours resulted in an inhibition of IGFBP-4 proteolysis that was associated with a decrease in the concentration of IGF-I in CM. These studies demonstrate that PDGF-BB increases the accumulation of ICFBP-4 in fetal rat lung <em>fibroblasts</em> CM through increased production and by inhibiting IGF-mediated IGFBP-4 proteolysis.

OBJECTIVE

This study was undertaken to evaluate the efficacy and safety of closing the fetoscopy access site in a midgestational rabbit model by using a commercially available bioactive membrane.

METHODS

Fetoscopy was performed in a total of 100 gestational sacs in 20 does at midgestation (23 days, term = 31 days). In 50 cases (group 1), the fetoscopic access port was closed with a 5-mm patch of biocompatible matrix derived from porcine small intestine containing growth factors (transforming growth factor-beta and fibroblast growth factor-beta). Fifty sacs served as positive controls (group 2) and 55 unoperated fetuses were used as negative controls (group 3). At 30 days of gestation, a second-look laparotomy was performed. Outcome parameters were fetal weight, fetal lung weight, fetal lung-to-body weight ratio, and microscopy of the plugging site.

RESULTS

Membrane integrity after fetoscopy was restored in 28 of the 40 (70%) of cases in group 1 versus 13 of the 32 (41%) in group 2 (P =.012). Birth weights were comparable (group 1: 30.65 +/- 5.68 g; group 2: 29.70 +/- 5.05 g; group 3: 29.52 +/- 6.25 g; NS), but fetal lung weight (group 1: 0.964 +/- 0.20 g; group 2: 0.798 +/- 0.17 g; P <.01) and fetal lung-to-body weight ratio (group 1: 0.032 +/- 0.0067; group 2: 0.027 +/- 0.0082; P <.05) were significantly higher in the study group. In group 1, cellular proliferation was significantly increased. Polymorphonuclear infiltration was observed in 19 of the 40 (48%) cases in group 1 versus 5 of the 32 (16%) cases in group 2 (P <.05). In one treated sac, a fibrous band joining the two fetal legs without constriction was present.

CONCLUSIONS

The use of a bioactive membrane improved fetal membrane repair rates and decreased incidence of pulmonary hypoplasia in the rabbit but increased polymorphonuclear infiltration. In one amniotic sac, a situation comparable to amniotic band syndrome was documented.

The two main subsets of nonalcoholic fatty liver disease (NAFLD) include: (1) nonalcoholic fatty liver (NAFL), the more common and non-progressive subtype; and (2) nonalcoholic steatohepatitis (NASH), the less common subtype, which has the potential to progress to advanced liver damage. Current treatment strategies have focused on lifestyle management of modifiable risk <em>factors</em>, namely weight, and on the optimization of the management of individual components of metabolic syndrome. Various hypothetical pathogenic mechanisms have been proposed, leading to the development of novel drugs with the potential to effectively treat patients with NASH. Numerous clinical trials are ongoing, utilizing these experimental drugs and molecules targeting specific mechanistic pathway(s) to effectively treat NASH. Some of these mechanistic pathways targeted by experimental pharmacologic agents include chemokine receptor 2 and 5 antagonism, inhibition of galectin-3 protein, antagonism of toll-like receptor 4, variation of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em>, agonism of selective thyroid hormone receptor-beta, inhibition of apoptosis signal-regulating kinase 1, inhibition of acetyl-coenzyme A carboxylase, agonism of farnesoid X receptor, antibodies against lysl oxidase-like-2, and inhibition of inflammasomes. Emerging data are promising and further updates from ongoing clinical trials are eagerly awaited.

Recent studies suggest that peptide <em>growth</em> <em>factors</em> play a functional role in cardiac muscle. To test whether embryonic cardiac muscle is a target for regulation by basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and platelet-derived <em>growth</em> <em>factor</em>, we analyzed the effects of these peptides on the expression of the intermediate filaments desmin and vimentin at the subcellular level during development. Sodium dodecyl sulfate-gel electrophoresis, immunoblotting and fluorescence-activated cell sorting analysis were used to study the effect of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and platelet-derived <em>growth</em> <em>factor</em> on cultures of chick cardiomyocytes during development. Cytoplasmic and cytoskeletal concentrations of desmin and vimentin were dependent on the stage of embryonic development and on the type of <em>growth</em> <em>factor</em> added to the culture. The most significant finding was the increase in desmin expression in the cytoplasmic and cytoskeletal compartments after treatment with basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (10 ng/ml) of chick heart cells at Hamburger and Hamilton stage <em>19</em>. In more mature stages, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> did not modify the levels of desmin expression. However, this <em>factor</em> led to a progressive deceleration in the rate of increase in vimentin expression. Platelet-derived <em>growth</em> <em>factor</em> increased vimentin expression in all stages studied, the greatest increases appearing in early stages of heart development. Our findings support the hypothesis that basic <em>fibroblast</em> <em>growth</em> <em>factor</em> plays a role in cardiomyocyte differentiation during the early stages of development, whereas platelet-derived <em>growth</em> <em>factor</em> has a dedifferentiating effect.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) is a potent mitogen and angiogenic <em>factor</em> normally absent from the adult circulation. We have previously shown that it appears in normal maternal serum and that circulating FGF-2 levels are elevated in pregnancies complicated by diabetes. This study was performed to determine whether serum FGF-2 is more abundant in pregnant diabetic women with retinopathy than in those without. Serum was collected monthly between 14-30 weeks gestation and every 2 weeks from then until delivery (35-38 weeks) from 36 women with type 1 diabetes. FGF-2 was extracted by heparin-Sepharose affinity chromatography and quantified by specific RIA. Patients were divided according to the White classification of diabetes. In 17 women without retinopathy (White groups B, C, and D0), immunoreactive FGF-2 was detectable at 14 weeks (mean +/- SEM, 154 +/- 39 pmol/L), was maximal after 26 weeks (306 +/- 38 pmol/L), after which values steadily declined to term (212 +/- 48 pmol/L). In <em>19</em> women with simplex or proliferative retinopathy (White groups D+ and R), circulating levels of FGF-2 were significantly greater between 22-32 weeks gestation (22 weeks, 480 +/- 102 vs. 239 +/- 38 pmol/L; P < 0.05). Serum FGF-2 was significantly correlated with hemoglobin A1c levels at 22, 30, and 34 weeks gestation. The mean birth weight of the infants did not significantly differ between groups. Macroalbuminuria was absent in all patients, and creatinine clearance and blood pressure did not significantly differ between the two groups. The results suggest that serum FGF-2 is substantially elevated in pregnant diabetic women with retinopathy in second and early third trimesters. It is unlikely that in these patients this was primarily due to altered FGF-2 clearance, but may relate to excessive production by the utero-placental compartment. The high circulating levels of FGF-2 may be causally related to the development of diabetic retinopathy.

OBJECTIVE

To investigate the role of calcineurin (CaN) in the lung fibroblast proliferation and collagen synthesis induced by basic fibroblast growth factor (bFGF).

METHODS

We used Western blot and immunohistochemical methods for investigating the content and distribution of calcineurin in the lung tissue. Calcineurin activity in different tissues was measured using (32)P-labelled substrate. In the primary culture of lung fibroblasts, (3)H-thymidine ((3)H-TdR) and (3)H-proline incorporation methods were used to study the effect of cyclosporin A (CsA), an inhibitor of calcineurin, on the lung fibroblast DNA and collagen synthesis stimulated by bFGF.

RESULTS

We found that calcineurin was expressed in lung tissue and has phosphatase activity (7.1 +/- 2.0 pmol Pi/mg pr/min). CsA (10(-8) - 10(-6) mol/L) inhibited lung fibroblast (3)H-TdR incorporation induced by bFGF in a dose-dependent manner, with the inhibitory rates by 20%, 46% and 66% (P < 0.01). CsA (10(-7) - 10(-6) mol/L) inhibited (3)H-proline incorporation in lung fibroblasts stimulated by bFGF, with the inhibitory rates by 21% and 37% (P < 0.01). In a culture medium, CsA (10(-8) - 10(-6) mol/L) inhibited (3)H-proline secretion induced by bFGF in a dose-dependent manner, with the inhibitory rates by 19%, 29% (P < 0.05) and 56% (P < 0.01). CsA (10(-7) mol/L) could inhibit calcineurin activity by 44% in lung fibroblasts (P < 0.01).

CONCLUSIONS

Calcineurin is expressed in lung tissue and has phosphatase activity. It is involved in the bFGF stimulated lung fibroblast DNA and collagen synthesis.

Bile acids (BA) are pleiotropic hormones affecting glucose and lipid metabolism. The physiochemical properties of different BA species affect their enterohepatic dynamics and their affinity for bile acid receptors. The BA pool composition is altered in patients with type 2 diabetes and obesity. In this study we used a 2-week very-low-calorie diet (VLCD) to investigate the effects of weight loss on BA pool composition and postprandial dynamics.

We performed mixed meal tests in obese, insulin resistant subjects before and after the VLCD. We measured postprandial plasma levels of glucose, insulin, BA and the BA-induced enterokine <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>).

The VLCD decreased weight by 4.5 ± 2.3 kg (p < 0.0001) within 14 days. Weight loss increased peak postprandial deoxycholate (DCA) levels (median [IQR]: 0.90 [0.90] vs. 1.25 [1.35] μmol/L; p = 0.045*). Other BA species, glucose, insulin and FGF<em>19</em> levels and prandial excursions were not significantly affected. The VLCD decreased resting and postprandial energy expenditure by 7 and 11% respectively.

VLCD induced weight loss increased postprandial DCA peak levels and decreased resting energy expenditure in obese insulin resistant subjects.

Epidermal <em>growth</em> <em>factor</em> (EGF) receptor (EGFR) regulates development of cell-cell communication in fetal lung, but the signal transduction mechanisms involved are unknown. We hypothesized that, in late-gestation fetal rat lung, phospholipase C-gamma (PLC-gamma) expression and activation by EGF is cell specific and developmentally regulated. PLC-gamma immunolocalized to cuboidal epithelium and mesenchymal clusters underlying developing saccules. PLC-gamma protein increased from day 17 to day <em>19</em> and then decreased. In cultured fetal lung <em>fibroblasts</em>, EGF stimulated PLC-gamma phosphorylation 2.6-fold (day 17), 10.8-fold (day <em>19</em>), and 4.2-fold (day 21). EGF stimulated (3)H-labeled diacylglycerol production in <em>fibroblasts</em> (beginning on day 18 in female and on day <em>19</em> in male rats), but not in type II cells at any time during gestation. EGFR blockade abrogated the observed stimulation of PLC-gamma phosphorylation by EGF. In conclusion, PLC-gamma expression and activation by EGF in fetal lung are cell specific, corresponding to the development of EGFR expression. EGF induces diacylglycerol production in a cell- and gestation-specific manner. PLC-gamma activation by EGFR in fetal lung <em>fibroblasts</em> may be involved in EGF control of lung development.

BACKGROUND Cytokeratin <em>19</em> (CK<em>19</em>) is a typical epithelial marker. In this study, we determined whether epidermal <em>growth</em> <em>factor</em> (EGF) or basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) could enhance CK<em>19</em> expression in adipose-derived stem cells (ADSCs), thereby inducing the differentiation of ADSCs into epithelial-like cells. MATERIAL AND METHODS ADSCs were isolated from perinephric fat, and the expression of CD29, CD90, and CD105 was confirmed. Following isolation, ADSCs were cultured in static medium or medium containing EGF or bFGF. RESULTS Flow cytometry revealed that EGF and bFGF could alter mesenchymal stem cell markers as well as the cell cycle of ADSCs. Western blotting and immunofluorescence revealed that after 14 days, EGF treatment enhanced the expression of CK<em>19</em> in ADSCs. CONCLUSIONS Our findings offer important insight for the clinical use of ADSCs in the generation of epithelial-like cells in the future.

A cholesterol-deficient <em>growth</em> medium for human skin <em>fibroblasts</em> was prepared by adding to Eagle's Minimum Essential Medium a bovine serum treated with ultracentrifugation to remove bulk lipoproteins followed by silicic acid adsorption to remove residual lipoproteins and cholesterol. Cell <em>growth</em> was slow, but the daily cell doublings could be increased by 76% by including 7.5 micrograms purified cholesterol/ml in the medium. Cell <em>growth</em> in cholesterol-deficient culture medium could be increased to that seen with medium containing 15% untreated fetal bovine serum by the inclusion of the following <em>growth</em> <em>factors</em>: epidermal <em>growth</em> <em>factor</em> (EGF), cortisol, non-essential amino acids, insulin, transferrin and selenium. Cholesterol increased the proliferation of these rapidly-<em>growing</em> cultures by <em>19</em>%. No effect of cholesterol was observed in transformed L-cell mouse <em>fibroblasts</em>.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23), a bone-derived hormone, participates in the hormonal bone-parathyroid-kidney axis, which is modulated by PTH, 1,25-dihydroxyvitamin D, plasma phosphate (Pi), and diet. Inappropriately high serum FGF23, seen in certain genetic and acquired disorders, results in urinary phosphate wasting and impaired bone mineralization. This study investigated the impact of FGF23 gene variation on phosphate homeostasis and bone health. The study included 183 children and adolescents (110 girls) aged 7-<em>19</em> years (median 13.2years). Urine and blood parameters of calcium and phosphate homeostasis were analyzed. Bone characteristics were quantified by DXA and peripheral quantitative computed tomography (pQCT). Genetic FGF23 variation was assessed by direct sequencing of coding exons and flanking intronic regions. Nine FGF23 polymorphisms were detected; three of them were common: rs3832879 (c.212-37insC), rs7955866 (c.716C>T, p.T239M) and rs11063112 (c.2185A>T). Four different haplotypes and six different diplotypes were observed among these three polymorphisms. The variations in FGF23 significantly associated with plasma PTH and urinary Pi excretion, even after adjusting for relevant covariates. FGF23 variations independently associated with total hip BMD Z-score, but not with other bone outcomes. In instrument analysis, genetic variance in FGF23 was considered a weak instrument as it only induced small variations in circulating FGF23, PTH and Pi concentrations (F statistic less than 10). The observed associations between FGF23 variations and circulating PTH, and Pi excretion and total hip BMD Z-scores suggest that FGF23 polymorphisms may play a role in mineral homeostasis and bone metabolism.