OBJECTIVE

Preclinical studies suggest antiangiogenesis strategies may be effective in the treatment of prostate cancer. In tumor models, the copper-chelating agent tetrathiomolybdate (TM) has been shown to be antiangiogenic. We evaluated the antitumor activity of TM in patients with hormone-refractory prostate cancer (HRPC).

METHODS

Nineteen patients with asymptomatic HRPC enrolled. Copper depletion was monitored using serum ceruloplasmin levels. Once the target ceruloplasmin level of 5-15 mg/dl was attained, patients underwent staging evaluation. Patients were reassessed every 12 weeks, and TM was continued until they developed evidence of disease progression or intolerable toxicity. Prostate-specific antigen and levels of vascular endothelial growth factor, basic fibroblast growth factor, interleukin (IL)-6 and IL-8 were measured at study entry, at the time of copper depletion, and monthly while on therapy.

RESULTS

Seventeen of 19 patients achieved copper deficiency on TM therapy. Of the 16 evaluable patients, 14 developed progressive disease, 1 discontinued therapy because of toxicity and 1 patient opted to discontinue therapy because of rising prostate-specific antigen level without objective evidence of progressive disease. Levels of vascular endothelial growth factor, IL-6 and IL-8, but not basic fibroblast growth factor, were elevated when compared to normal controls prior to TM therapy, but there was no significant change during therapy. There was no correlation between prostate-specific antigen and levels of angiogenesis factors.

CONCLUSIONS

Copper depletion with TM did not delay disease progression in patients with asymptomatic metastatic HRPC.

This study aimed to examine <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>19</em> (FGF-<em>19</em>) in type 2 diabetic (T2DM) patients with metabolic syndrome (MetS) and to evaluate the relationship between FGF-<em>19</em> and other cardiovascular risk <em>factors</em>, such as atherogenic index of plasma (AIP) and hsCRP. 26 T2DM patients with MetS and 12 healthy controls were enrolled in the study. Serum FGF-<em>19</em> levels were measured by sandwich ELISA, and compared with other cardiovascular risk <em>factors</em>; lipid profile, AIP, glucose, HbA1c, and hsCRP. AIP was calculated as log (TG/HDL-c). The median (1-3.quartile) FGF-<em>19</em> levels in T2DM patients with MetS and healthy controls were 122.90 (108.63-237.60) pg/ml and 293.45 (153.64-370.31) pg/ml, respectively (P=0.003). Patients were also grouped by body mass index (BMI) <30 kg/m(2) (n=13) and ≥30 kg/m(2) (n=13) with median (1-3.quartile) FGF-<em>19</em> values 168.70 (113.54-275.77) pg/mL and 115.89 (97.94-200.40) pg/mL, respectively (P=0.007). Significant negative correlations were found between FGF-<em>19</em> and BMI, triglyceride, log (TG/HDL-c), hsCRP, and HbA1c (r=-0.526, P=0.001; r=-0.327, P=0.05; r=-0.312, P=0.05; r=-0.435, P=0.006; r=-0.357, P=0.028, respectively). We showed that FGF-<em>19</em> levels are low in T2DM patients with MetS. The negative relationship between FGF-<em>19</em> and several known cardiovascular risk <em>factors</em> such as TG, log (TG/HDL-c), hsCRP and HbA1c in diabetic patients with MetS suggests that FGF-<em>19</em> can be used as a contributing marker.

Nonalcoholic fatty liver disease (NAFLD) affects a quarter of the adult population. A significant subset of patients are lean, but their underlying pathophysiology is not well understood. We investigated the role of bile acids (BAs) and the gut microbiome in the pathogenesis of lean NAFLD. BA and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) levels (a surrogate for intestinal farnesoid X receptor [FXR] activity), patatin-like phospholipase domain-containing 3 (PNPLA3) and transmembrane 6 superfamily member 2 (TM6SF2) variants, and gut microbiota profiles in lean and non-lean NAFLD were investigated in a cohort of Caucasian patients with biopsy-proven NAFLD (n = 538), lean healthy controls (n = 30), and experimental murine models. Patients with lean NAFLD had a more favorable metabolic and histological profile compared to those with non-lean NAFLD (P < 0.05 for all). BA levels were significantly higher in NAFLD with advanced compared to earlier stages of liver fibrosis. Patients with lean NAFLD had higher serum secondary BA and FGF<em>19</em> levels and reduced 7-alpha-hydroxy-4-cholesten-3-one (C4) levels (P < 0.05 for all). These differences were more profound in early compared to advanced stages of fibrosis (P < 0.05 for both). Lean patients demonstrated an altered gut microbiota profile. Similar findings were demonstrated in lean and non-lean murine models of NAFLD. Treating mice with an apical sodium-dependent bile acid transporter inhibitor (ASBTi) (SC-435) resulted in marked increases in fgf15, a shift in the BA and microbiota profiles, and improved steatohepatitis in the lean model. CONCLUSION: Differences in metabolic adaptation between lean and non-lean NAFLD patients, at least in part, explain the pathophysiology and provide novel options for therapy. This article is protected by copyright. All rights reserved.

Atypical <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGF) 21 and <em>19</em> play a central role in energy metabolism through the mediation of Klotho coreceptor. Contradictory findings are available about the association of FGF21 and FGF<em>19</em> with nonalcoholic fatty liver disease (NAFLD) in humans. We investigated the association of serum FGF21, FGF<em>19</em> and liver Klotho coreceptor with non-alcoholic steatohepatitis (NASH) and fibrosis in children with NAFLD. Serum FGF21 and FGF<em>19</em> were measured in 84 children with biopsy-proven NAFLD and 23 controls (CTRL). The hepatic expression of Klotho coreceptor was measured in 7 CTRL, 9 patients with NASH (NASH+) and 11 patients without NASH (NASH-). FGF21 and FGF<em>19</em> showed a tendency to decrease from CTRL (median FGF21 = <em>19</em>6 pg/mL; median FGF<em>19</em> = 201 pg/mL) to NASH- (FGF21 = 89 pg/mL; FGF<em>19</em> = 81 pg/mL) to NASH+ patients (FGF21 = 54 pg/mL; FGF<em>19</em> = 41 pg/mL) (p<0.001 for all comparisons) and were inversely associated with the probability of NASH and fibrosis in children with NAFLD. The hepatic expression of Klotho coreceptor was inversely associated with NASH (R(2) = 0.87, p<0.0001) and directly associated with serum FGF21 (R(2) = 0.57, p<0.0001) and FGF<em>19</em> (R(2) = 0.67, p<0.0001). In conclusion, serum FGF<em>19</em> and FGF21 and hepatic Klotho expression are inversely associated with hepatic damage in children with NAFLD and these findings may have important implications for understanding the mechanisms of NAFLD progression.

<AbstractText>X-linked hypophosphatemia (XLH) is characterized by excess <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23), hypophosphatemia, skeletal abnormalities, and <em>growth</em> impairment. We aimed to understand the burden of disease of XLH across the lifespan.</AbstractText><AbstractText>Responses were collected from adults with XLH and parents/caregivers of a child with XLH in an online survey, including multiple-choice and open-ended questions on demographics, disease manifestations, treatment history, assistive device use, and age-specific patient-reported outcomes (PROs).</AbstractText><AbstractText>Data were collected from 232 adults with XLH (mean age, 45.6 years; 76% female) and 90 parents/caregivers of a child with XLH (mean age, 9.1 years; 56% female). Mean age recalled for symptom onset was 3.2 years for adults and 1.3 years for children. When surveyed, nearly all children (99%) and 64% of adults were receiving oral phosphate, active vitamin D, or both. Prior participation in a trial investigating burosumab, a fully human monoclonal antibody against FGF23, was reported in 3% of children and 10% of adults; of these respondents, only one child reported current treatment with burosumab at the time of the survey. Both children and adults reported typical features of XLH, including abnormal gait (84% and 86%, respectively), bowing of the tibia/fibula (72% and 77%), and short stature (80% and 86%). Nearly all adults (97%) and children (80%) reported bone or joint pain/stiffness. Adults reported a history of fractures (n/N = 102/232; 44%), with a mean (SD) age at first fracture of 26 (16) years. Adults reported osteophytes (46%), enthesopathy (27%), and spinal stenosis (<em>19</em>%). Mean scores for PROs evaluating pain, stiffness, and physical function were worse than population norms. Analgesics were taken at least once a week by 67% of adults.</AbstractText><AbstractText>Despite the common use of oral phosphate and active vitamin D established in the <em>19</em>80s, children with XLH demonstrate a substantial disease burden, including pain and impaired physical functioning that persists, as demonstrated by similar responses reported in adults with XLH.</AbstractText>

Epithelial-to-mesenchymal transition (EMT) is a process for fully differentiated epithelial cells to undergo a phenotypic change to <em>fibroblasts</em> via diverse intracellular signaling pathways. While the pivotal role of <em>fibroblasts</em> in renal fibrosis is widely accepted, their origin remains undefined. In addition, although a large number of studies have provided evidence of EMT in human kidney diseases, specific signaling pathways leading to EMT have not yet been discovered in humans. To evaluate the origin of interstitial <em>fibroblasts</em> and signaling pathways involved in the EMT process, we analyzed the differential expression of EMT-related molecules in paraffin-fixed sections from <em>19</em> human fibrotic kidneys and 4 control kidneys. In human fibrotic kidneys, tubular epithelial cells (TECs) with intact tubular basement membrane (TBM) showed loss or down-regulation of an epithelial marker (E-cadherin), de novo expression of mesenchymal markers (vimentin and fibronectin), and significant up-regulation of inducers and mediators controlling the EMT process (transforming <em>growth</em> <em>factor</em>-β1 (TGF-β1), p-Smad2/3, β1-integrin, p38 mitogen-activated protein kinase (MAPK), WNT5B and β-catenin) in the areas of interstitial inflammation and fibrosis, compared with their expression in control kidneys. In conclusion, the type II EMT process in humans is thought to be an adaptive response of TECs to chronic injury and is regulated by interconnections of TGF-β/Smad, integrin/integrin-linked kinase (ILK) and wnt/β-catenin signaling pathways.

Oncogenic mutations in ras lead to constitutive activation of downstream signaling pathways that modulate the activities of transcription <em>factors</em>. In turn, these <em>factors</em> control the expression of a subset of genes responsible for neoplastic cell transformation. Recent studies suggest that transcription <em>factor</em> NF-kappa B contributes to cell transformation by inhibiting the cell death signal activated by oncogenic Ras. In this study, inhibition of NF-kappa B activity by forced expression of a super-repressor form of I kappa B alpha, the major inhibitor of NF-kappa B, markedly decreased the <em>growth</em> rate, saturation density and tumorigenicity of oncogenic H-Ras transformed rat embryo <em>fibroblasts</em>. Such clonally isolated cells overexpressing I kappa B alpha super-repressor not only were viable but also exhibited no sign of spontaneous apoptosis. Inhibition of NF-kappa B in these cells was functionally demonstrated by both the loss of cytokine induced DNA binding activity and a profoundly increased sensitivity to cell death in response to TNF-alpha treatment. In contrast, inhibition of NF-kappa B activity in non-transformed <em>fibroblasts</em> had minimal effect on <em>growth</em>, but rendered the cells resistant to a subsequent transformation by H-ras oncogene. Similar results were also obtained with rat intestinal epithelial cells harboring an inducible ras oncogene. Taken together, these findings suggest that NF-kappa B activity is essential for abnormal cell proliferation and tumorigenicity activated by the ras oncogene and highlight an alternative functional role for NF-kappa B in oncogenic Ras-mediated cell transformation that is distinct from its anti-apoptotic activity. Oncogene (2000) <em>19</em>, 841 - 849.

Fetal development and tumor progression both require a complex system of extracellular matrix (ECM) synthesis and breakdown, which is mediated by, for instance, the matrix metalloproteinases (MMPs). Human metalloelastase (MMP-12) is an MMP, the expression of which has so far been documented in macrophages associated with atherosclerosis, wound repair, and certain cancers. In this study we first examined the expression of MMP-12 during human fetal development. By in situ hybridization MMP-12 transcripts were detected in chondrocytes of hypertrophic cartilage in vertebrae of the spinal column, in ribs, and in extremities undergoing ossification, beginning at the gestational age of 8 weeks. Also, periosteal cells expressed MMP-12 at 11 weeks. No expression of MMP-12 mRNA could be noted in other fetal tissues, including the skin, lungs, intestine, kidney, and liver. Expression of MMP-12 mRNA could not be detected in adult normal cartilage or osteosarcomas, but in chondrosarcomas both macrophages (8 of <em>19</em> samples) (identified by CD68 immunostaining) and chondrosarcoma cells (8 of <em>19</em>) were positive. MMP-12 was also demonstrated in the tumors by western blotting and it was expressed in the same regions as MMP-13 mRNA. By immunostaining, MMP-12 mRNA colocalized with the protein in both fetal and chondrosarcoma specimens. Unlike basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and transforming <em>growth</em> <em>factor</em>-beta (TGF-beta), tumor necrosis <em>factor</em>-alpha (TNF-alpha) induced MMP-12 mRNA production in chondrosarcoma-derived HTB-94 cells. Our results suggest that MMP-12 plays an important role in ECM remodeling during fetal bone development and is induced when chondrocytes undergo malignant transformation.

Stem cell <em>factor</em> (SCF) of keratinocyte origin regulates melanocyte <em>growth</em> and survival. Deprivation of survival <em>factors</em> causes the apoptosis of melanocytes. Vitiligo often develops following physical trauma, even if this is minor. The exact mechanism of the Koebner phenomenon in vitiligo is unclear. Apoptosis of keratinocytes, which occurs more in depigmented suction-blistered epidermis than in the normally pigmented counterpart, could reduce levels of keratinocyte-derived <em>factors</em> such as SCF and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). Levels of SCF expression were examined in the depigmented and normally pigmented paired epidermis of <em>19</em> patients with vitiligo, and bFGF expression in six patients. The expression of SCF (p<0.001) and bFGF was usually reduced in the depigmented compared with the normally pigmented epidermis. Apoptosis of cultured normal human keratinocytes, which was induced by staurosporine, resulted in a concentration-dependent decrease in levels of SCF mRNA and protein. Normal human melanocytes proliferated more in medium containing SCF or keratinocyte (XB-2) feeder than in medium with neither. Deprivation of SCF or keratinocyte feeder in the culture medium induced a marked decrease in melanocytes as a result of apoptosis. Therefore, lower expression of keratinocyte-derived <em>factors</em>, including SCF, in vitiliginous keratinocytes, which could result from keratinocyte apoptosis, might be responsible for passive melanocyte death and may explain the Koebner phenomenon.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGF) regulate bone <em>growth</em>, but their expression in human cartilage is unclear. Here, we determined the expression of entire FGF family in human fetal <em>growth</em> plate cartilage. Using reverse transcriptase PCR, the transcripts for FGF1, 2, 5, 8-14, 16-<em>19</em>, and 21 were found. However, only FGF1, 2, 17, and <em>19</em> were detectable at the protein level. By immunohistochemistry, FGF17 and <em>19</em> were uniformly expressed within the <em>growth</em> plate. In contrast, FGF1 was found only in proliferating and hypertrophic chondrocytes whereas FGF2 localized predominantly to the resting and proliferating cartilage. In addition, only the 18 kD isoform of FGF2 was found in resting chondrocytes while proliferating chondrocytes also synthesized 22 kD and 24 kD FGF2, similar to in vitro cultivated chondrocytes. In cell <em>growth</em> experiments, FGF1, 2, and 17 but not FGF<em>19</em> inhibited the proliferation of FGFR3-expressing rat chondrosarcoma chondrocytes (RCS) with relative potency FGF2>>) FGF1 = FGF17. We conclude that FGF1, 2, 17, and <em>19</em> are the predominant FGF ligands present in developing human cartilage that are, with the exception of FGF<em>19</em>, experimentally capable of inhibiting chondrocyte proliferation.

Hepatocellular carcinoma (HCC), a primary malignancy of the liver, is the second leading cause of cancer mortality worldwide. <em>Fibroblast</em> <em>Growth</em> <em>Factor</em> <em>19</em> (FGF<em>19</em>) is one of the most frequently amplified genes in HCC patients. Moreover, mice expressing an FGF<em>19</em> transgene have been shown to develop HCC. However, the downstream signalling pathways that mediate FGF<em>19</em>-dependent tumorigenesis remain to be deciphered. Here we show that FGF<em>19</em> triggers a previously unsuspected, non-cell-autonomous program to activate STAT3 signalling in hepatocytes through IL-6 produced in the liver microenvironment. We show that the hepatocyte-specific deletion of Stat3, genetic ablation of Il6, treatment with a neutralizing anti-IL-6 antibody or administration of a small-molecule JAK inhibitor, abolishes FGF<em>19</em>-induced tumorigenesis, while the regulatory functions of FGF<em>19</em> in bile acid, glucose and energy metabolism remain intact. Collectively, these data reveal a key role for the IL-6/STAT3 axis in potentiating FGF<em>19</em>-driven HCC in mice, a finding which may have translational relevance in HCC pathogenesis.

Among matrix metalloproteinases (MMP), MMP-<em>19</em> displays unique structural features and tissue distribution. In contrast to most MMPs, MMP-<em>19</em> is expressed in normal human epidermis and down-regulated during malignant transformation and dedifferentiation. The contribution of MMP-<em>19</em> during tumor angiogenesis is presently unknown. In an attempt to give new insights into MMP-<em>19</em> in vivo functions, angiogenic response of mutant mice lacking MMP-<em>19</em> was analyzed after transplantation of murine malignant PDVA keratinocytes and after injection of Matrigel supplemented with basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. In situ hybridization and immunohistochemical analysis revealed that MMP-<em>19</em> is produced by host mesenchymal cells but not by endothelial capillary cells or CD11b-positive inflammatory cells. Based on a new computer-assisted method of quantification, we provide evidence that host MMP-<em>19</em> deficiency was associated with an increased early angiogenic response. In addition, increased tumor invasion was observed in MMP-<em>19</em>-/- mice. We conclude that, in contrast to most MMPs that promote tumor progression, MMP-<em>19</em> is a negative regulator of early steps of tumor angiogenesis and invasion. These data highlight the requirement to understand the individual functions of each MMP to improve anticancer strategies.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) was recently introduced as a novel metabolic regulator reversing diabetes mellitus, hepatic steatosis, hyperlipidemia, and adiposity. In the current study, we determined circulating FGF<em>19</em> levels in patients on chronic hemodialysis (CD) as compared to controls with a glomerular filtration rate (GFR) above 50 ml/min. FGF<em>19</em> was measured by ELISA in control (n=60) and CD (n=60) patients and correlated to clinical and biochemical measures of renal function, glucose, and lipid metabolism, as well as inflammation, in both groups. Median serum FGF<em>19</em> levels were 1.5-fold higher in CD patients (266.7 microg/l) as compared to subjects with a GFR above 50 ml/min (178.1 microg/l) (p=0.001). Furthermore, fasting glucose negatively and independently predicted circulating FGF<em>19</em> in controls (p<0.05). Moreover, adiponectin was a positive and C-reactive protein was a negative independent predictor of FGF<em>19</em> serum concentrations in CD patients. Taken together, we have demonstrated that circulating FGF<em>19</em> levels are significantly increased in end-stage renal disease. Furthermore, FGF<em>19</em> is associated with a beneficial metabolic profile in both control and CD patients.

BACKGROUND

The use of infliximab in the treatment of Crohn's disease patients with symptomatic stenosis is controversial.

OBJECTIVE

To explore the influence of this agent on intestinal fibrogenesis by measuring in infliximab-treated Crohn's disease patients the serum levels of basic fibroblast growth factor and vascular endothelial growth factor, two factors known to be involved in the process of intestinal wound healing and fibrosis in this condition.

METHODS

Serum levels of basic fibroblast growth factor and vascular endothelial growth factor were determined by enzyme-linked immunosorbent assay in 22 patients with steroid refractory or fistulizing Crohn's disease before, during (2 weeks) and after 12 weeks of treatment with infliximab, administered at week 0, 2 and 6 in a dose of 5 mg/kg.

RESULTS

A substantial improvement in 19 of the 22 Crohn's disease patients was accompanied by a rapid and durable reduction in basic fibroblast growth factor and vascular endothelial growth factor serum levels.

CONCLUSIONS

The action of infliximab in reducing serum basic fibroblast growth factor and vascular endothelial growth factor would seem to suggest a role of this agent in down-regulating the process of intestinal fibrogenesis in Crohn's disease.

BACKGROUND

Bile acids and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) have been suggested as key mediators of the improvements in glucose metabolism after Roux-en-Y gastric bypass (RYGB).

OBJECTIVE

To describe fasting and postprandial state total bile acid (TBA) and FGF<em>19</em> concentrations before and after RYGB and relate them to parameters of glucose metabolism, glucagon-like peptide-1, cholecystokinin, and cholesterol fractions.

METHODS

A prospective descriptive study was performed at the Department of Endocrinology, Hvidovre Hospital, Hvidovre, Denmark.

METHODS

Thirteen type 2 diabetic (T2D) patients and 12 normal glucose tolerant (NGT) subjects participated in the study.

METHODS

A 4-hour liquid meal test was performed before and 1 week, 3 months, and 1 year after RYGB.

METHODS

We measured fasting and postprandial TBA and FGF<em>19</em> concentrations.

RESULTS

Fasting TBA concentrations decreased in NGT subjects (P < .001) and were unchanged in T2D patients 1 week after surgery, but then increased gradually in both groups with time from surgery (ANOVA Ptime < .001). Area under the curve (AUC) TBA was decreased in NGT subjects 1 week after RYGB (before surgery, 567 mmol * min/L [interquartile range, 481-826]; 1 wk, 4<em>19</em> [381-508]; P = .009) and was unchanged in T2D patients (894 [573-1002]; 695 [349-1147]; P = .97) but then increased with time from surgery in both groups (Ptime < .001). Fasting FGF<em>19</em> concentrations were unchanged acutely after RYGB (NGT, 140 pg/mL [100-162], 134 [1<em>19</em>-204], P = .42; T2D, 162 [130-<em>19</em>6], 154 [104-164], P = .68) and remained unchanged throughout the follow-up period. AUC FGF<em>19</em> increased gradually with time after surgery (Ptime < .001), resembling the changes seen with AUC TBA. One week after RYGB, glucose metabolism improved, low-density lipoprotein-cholesterol and high-density lipoprotein-cholesterol decreased, and cholecystokinin and glucagon-like peptide-1 secretion increased, whereas FFA concentrations were unchanged.

CONCLUSIONS

TBA and FGF<em>19</em> do not explain acute changes in glucose metabolism, cholesterol fractions, and gut hormone secretion after RYGB.

The serum levels of several cytokines were determined in 94 patients with Adamantiades-Behçet's disease (ABD), aged 36.1+/-11.0 years, during the active stage (n = 75) and the inactive stage (n = <em>19</em>) of the disease. A group of 75 healthy individuals matched for age and sex served as controls. Cytokine levels were determined using commercially available ELISA kits. Of the 75 patients with active disease and <em>19</em> with inactive disease, 38 (51%) and 4 (21%), respectively, and 23 healthy controls (31%) were found to have detectable levels of interleukin 8 (IL-8) in their serum (P < 0.05). Also, increased IL-8 serum levels were found in patients with active disease (median 12 pg/ml, P = 0.010) compared to patients with inactive disease (< or = 10 pg/ml) and to healthy controls (< or = 10 pg/ml). In particular, patients with oral aphthous ulcers (n = 51, 34 pg/ml) and neurological features (n = 4, 71 pg/ml) exhibited increased IL-8 levels. In contrast, there was no correlation between disease activity and the serum levels of IL-1alpha, IL-1beta, tumor necrosis <em>factor</em> alpha (TNF-alpha), soluble intercellular adhesion molecule-1 or basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). In a second set of experiments, the involvement of dermal microvascular endothelial cells in IL-8 secretion was investigated. Immortalized human dermal microvascular endothelial cells (HMEC-1 cells) were maintained for 4 h in vitro with serum from 18 ABD patients or with IL-1beta, a known stimulator of IL-8 synthesis, TNF-alpha or their combination at five- to tenfold higher concentrations than those found in the serum of ABD patients. Increased IL-8 secretion was found after incubation with ABD patients' serum (median 20 pg/ml), but IL-1beta, TNF-alpha and IL-1beta + TNF-alpha failed to induce IL-8 secretion by HMEC-1 cells (< or = 1-1.2 pg/ml) in biologically relevant concentrations. Our study showed increased IL-8 serum levels in ABD patients with active oral and neurological manifestations. Human microvascular endothelial cells may, at least partially, be responsible for the enhanced IL-8 secretion in the active stage of the disease.

METHODS

Human lumbar anulus tissue and cultured human lumbar anulus cells were used in retrospective studies of the immunocytochemical localization of the vitamin D receptor (VDR) in disc tissue, and of the in vitro effects of the active metabolite of vitamin D, 1,25(OH)2D3, on anulus cell proliferation, cytokine, and proteoglycan (PG) production. 24,25-D3 was also analyzed. Studies were approved by the authors' Human Subjects Institutional Review Board. Discs were obtained from surgical specimens and from control donors.

OBJECTIVE

To determine if human anulus cells express the VDR in vivo, and to test the effect of in vitro exposure to 1,25(OH)2D3 and 24,25-D3 on anulus cell proteoglycan and cytokine production in 3-dimensional culture.

BACKGROUND

Intragenic polymorphisms in the VDR gene have been associated with disc degeneration. 1,25(OH)2D3 has well-recognized effects on calcium homeostasis and bone mineralization, and is a negative growth regulator of a variety of normal and tumor cells. Its effects on human disc cells, however, are unexplored.

METHODS

Immunocytochemistry was performed on human lumbar disc anulus tissue from 19 subjects; human disc cells were cultured to test the effect of 1,25(OH)2D3 on proliferation of anulus cells from 5 subjects. A paired experimental design was used to determine proteoglycan production in control or 1,25(OH)2D3-treated cells, or in control or 24,25-D3-treated cells using the dimethylmethylene blue assay. A paired experimental design was also used to identify differences in cytokine production in conditioned media from control or 1,25(OH)2D3-treated cells, or in control or 24,25-D3-treated cells using ELISA assays.

RESULTS

Immunocytochemistry documented expression of the VDR in anulus cells. Young donor discs (aged newborn, 15 years) showed positive localization in all cells of the outer anulus, and some inner anulus cells. In adults (mean age, 38.9 years), some, but not all anulus cells, showed positive localization. Exposure to 10M 1,25(OH)2D3 in monolayer significantly reduced cell proliferation in vitro (P = 0.03). PG production in 3-dimensional was unchanged from control in both 1,25(OH)2D3- and 24,25-D3-treated cells. Cytokine production differed, however. 1,25(OH)2D3-treated cells showed significantly decreased production of vascular endothelial <em>growth</em> factor (VEGF) (P = 0.01), monocyte chemoattractant protein-1 (MCP-1) (P = 0.0006), angiogenin (P = 0.002), and thrombopoietin (P = 0.03) compared with controls. 24,25-D3-treated cells showed significantly elevated vascular endothelial <em>growth</em> factor-D (P = 0.01), beta-fibroblast <em>growth</em> factor (0.03), and significantly decreased interleukin-8, interferon-gamma, leptin, MCP-1, and TIMP-2 (tissue inhibitor of metalloproteinases-2) compared with controls (P <or= 0.01).

CONCLUSIONS

Data suggest that 1,25(OH)2D3 and 24,25-D3 may play roles as regulators of cell proliferation and production of specific cytokines in the lumbar anulus.

BACKGROUND

We previously described increased levels of growth and differentiation factor 15 (GDF-15) in skeletal muscle and serum of patients with mitochondrial diseases. Here we evaluated GDF-15 as a biomarker for mitochondrial diseases affecting children and compared it to fibroblast-growth factor 21 (FGF-21). To investigate the mechanism of GDF-15 induction in these pathologies we measured its expression and secretion in response to mitochondrial dysfunction.

METHODS

We analysed 59 serum samples from 48 children with mitochondrial disease, 19 samples from children with other neuromuscular diseases and 33 samples from aged-matched healthy children. GDF-15 and FGF-21 circulating levels were determined by ELISA.

RESULTS

Our results showed that in children with mitochondrial diseases GDF-15 levels were on average increased by 11-fold (mean 4046pg/ml, 1492 SEM) relative to healthy (350, 21) and myopathic (350, 32) controls. The area under the curve for the receiver-operating-characteristic curve for GDF-15 was 0.82 indicating that it has a good discriminatory power. The overall sensitivity and specificity of GDF-15 for a cut-off value of 550pg/mL was 67.8% (54.4%-79.4%) and 92.3% (81.5%-97.9%), respectively. We found that elevated levels of GDF-15 and or FGF-21 correctly identified a larger proportion of patients than elevated levels of GDF-15 or FGF-21 alone. GDF-15, as well as FGF-21, mRNA expression and protein secretion, were significantly induced after treatment of myotubes with oligomycin and that levels of expression of both factors significantly correlated.

CONCLUSIONS

Our data indicate that GDF-15 is a valuable serum quantitative biomarker for the diagnosis of mitochondrial diseases in children and that measurement of both GDF-15 and FGF-21 improves the disease detection ability of either factor separately. Finally, we demonstrate for the first time that GDF-15 is produced by skeletal muscle cells in response to mitochondrial dysfunction and that its levels correlate in vitro with FGF-21 levels.

Co-expression of several members of the matrix metalloproteinase (MMP) family is characteristic of human malignant tumours. To investigate the role of stromelysin-2 (MMP-10) in <em>growth</em> and invasion of skin tumours, we studied cutaneous carcinomas with high metastatic capacity (squamous cell carcinomas, SCCs), only locally destructive tumours (basal cell carcinomas, BCCs) and pre-malignant lesions (Bowen's disease and actinic keratosis) using in situ hybridization. Expression of MMP-10 was compared with that of stromelysin-1 (MMP-3) and of MT1-MMP, the expression of which has been shown to correlate with tumour invasiveness. MMP-10 was expressed in 13/21 SSCs and 11/<em>19</em> BCCs only in epithelial laminin-5 positive cancer cells, while premalignant lesions were entirely negative. MT1-MMP mRNA was detected in <em>19</em>/21 SCCs both in epithelial cancer cells and stromal <em>fibroblasts</em> and in 14/18 BCCs only in <em>fibroblasts</em>. The level of MMP-10 was upregulated in a cutaneous SCC cell line (UT-SCC-7) by transforming <em>growth</em> <em>factor</em>-alpha and keratinocyte <em>growth</em> <em>factor</em>, and by interferon-gamma in combination with transforming <em>growth</em> <em>factor</em>-beta1 and tumour necrosis <em>factor</em>-alpha both in UT-SCC-7 and HaCaT cells. Our results show that MMP-10 expression does not correlate with the invasive behaviour of tumours as assessed by their histology and MT1-MMP expression, but may be induced by the wound healing and inflammatory matrix remodelling events associated with skin tumours.

OBJECTIVE

To characterize the expression and function of interleukin (IL) <em>19</em>, a recently described T-helper 2 anti-inflammatory IL, on endothelial cell (EC) pathophysiological features.

RESULTS

The expression and effects of anti-inflammatory ILs on EC activation and development of angiogenesis are uncharacterized. We demonstrate by immunohistochemistry and immunoblot that IL-<em>19</em> is expressed in inflamed, but not normal, human coronary endothelium and can be induced in cultured human ECs by serum and basic fibroblast growth factor. IL-<em>19</em> is mitogenic and chemotactic, and it promotes EC spreading. IL-<em>19</em> activates the signaling proteins STAT3, p44/42, and Rac1. In functional ex vivo studies, IL-<em>19</em> promotes cordlike structure formation of cultured ECs and enhances microvessel sprouting in the mouse aortic ring assay. IL-<em>19</em> induces tube formation in gelatinous protein (Matrigel) plugs in vivo.

CONCLUSIONS

To our knowledge, these data are the first to report expression of the anti-inflammatory agent, IL-<em>19</em>, in ECs; and the first to indicate that IL-<em>19</em> is mitogenic and chemotactic for ECs and can induce the angiogenic potential of ECs.

Neovascularization of the atherosclerotic plaque is responsible for its weakening and consequently for the complications of vascular disease. Macrophages are a source of <em>growth</em> <em>factors</em> that can modulate angiogenesis. In this study, we analyzed the effect of oncostatin M (OSM) on angiogenesis, as it could be involved in the development of atherosclerosis. The effect of OSM was compared with those of leukemia inhibitory <em>factor</em> (LIF) and interleukin-6 (IL-6). On human dermal microvasculature endothelial cells (HMEC-1s), OSM (22.5 to 112.5 pmol/L) induced a dose-dependent increase in cell proliferation greater than that induced by the classic angiogenic <em>factors</em> vascular endothelial <em>growth</em> <em>factor</em> (VEGF; 543 pmol/L) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF; 1.1 nmol/L). LIF (<em>19</em> to 475 pmol/L) induced only a 30% increase in cell proliferation, and IL-6 had no effect. Furthermore, in a modified Boyden-chamber model, OSM, LIF, and IL-6 were chemoattractant for HMEC-1s. In a tridimensional gel of fibrin, OSM increased tube formation and tube length, which were already noticeable by day 3. LIF and IL-6 induced a weaker effect that was only obvious by day 10. The angiogenic effect of OSM was also demonstrated in vivo in a rabbit corneal model: OSM was more potent than LIF, the length of the neovessels being longer with OSM than with LIF, whereas IL-6 was without effect. We tested <em>factors</em> that could be involved in the proliferative effect of OSM on HMEC-1s. OSM induced only a slight increase in the urokinase receptor and a 60% increase in VEGF secretion, whereas it does not modify IL-8 secretion or bFGF levels. The effect of OSM seems to depend on endothelial cell origin and cell species: OSM (up to 112.5 pmol/L) did not induce human umbilical vein endothelial cell proliferation and even had a small inhibitory effect (17%) on calf pulmonary artery endothelial cells. In conclusion, OSM induces an angiogenic effect on capillary endothelial cells, which could be, at least in part, implicated in pathological processes such as atherosclerosis or tumor <em>growth</em>.

Different strategies are being investigated for treatment of spinal cord injuries, one of the most promising being application of neurotrophic <em>factors</em>, which have been shown to prevent neuronal death and stimulate regeneration of injured axons. Ex vivo gene therapy has emerged as the leading delivery method at the site of the injury, and we have shown previously that encapsulating genetically engineered <em>fibroblasts</em> in an immunoprotective alginate capsule can permit implantation of the <em>factor</em>-secreting cells without need for immunosuppression. This strategy could be greatly enhanced by providing the sprouting neurons with a permissive substrate upon which to attach and grow. We report here studies on the modification of an alginate gel surface by either coating it with laminin or by covalent attachment of YIGSR peptide. Using NB2a neuroblastoma cells, we found that native alginate elicited minimal cell attachment ( approximately 1.5%); however, YIGSR-alginate conjugate elicited a fivefold increase in numbers of cells attached using peptide ratios of 0.5 and 1 mg/g alginate, ranging from 9.5% of the cells at the lower ratio, to about 44% at the higher. Only a further <em>19</em>% increase was obtained at an increased peptide density of 2 mg/g alginate ( approximately 63% over control). Laminin-coated gels showed approximately 60% cell attachment. However, laminin coating did not stimulate differentiation and neurite <em>growth</em>, whereas both numbers and lengths of out<em>growth</em>s increased with increasing peptide density on peptide-modified alginate. We demonstrate here the ability of the peptide-modified alginate gels to allow adhesion of NB2a neuroblastoma cells and to promote neurite out<em>growth</em> from these cells when attached to the peptide-modified alginate surface. Also, we show that the adhesion of NB2a neuroblastoma cells and neurite out<em>growth</em> from the attached cells is a function of the peptide density on the gel surface.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-23 is a bone-derived hormone that increases urinary phosphate excretion and inhibits hydroxylation of 25-hydroxyvitamin D. Recent studies suggest that <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 may be an early biomarker of CKD progression. However, its role in kidney function decline in the general population is unknown. We assessed the relationship between baseline (<em>19</em>90-<em>19</em>92) serum levels of intact <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 and incident ESRD in 13,448 Atherosclerosis Risk in Communities study participants (56.1% women, 74.7% white) followed until December 31, 2010. At baseline, the mean age of participants was 56.9 years and the mean eGFR was 97 ml/min per 1.73 m(2). During a median follow-up of <em>19</em> years, 267 participants (2.0%) developed ESRD. After adjustment for demographic characteristics, baseline eGFR, traditional CKD risk <em>factors</em>, and markers of mineral metabolism, the highest <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 quintile (>54.6 pg/ml) compared with the lowest quintile (<32.0 pg/ml) was associated with risk of developing ESRD (hazard ratio, 2.10; 95% confidence interval, 1.31 to 3.36; trend P<0.001). In a large, community-based study comprising a broad range of kidney function, higher baseline <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 levels were associated with increased risk of incident ESRD independent of the baseline level of kidney function and a number of other risk <em>factors</em>.

BACKGROUND

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>)-mediated activation of mitogen-activated protein kinase and the β-catenin pathway may be involved in the development and progression of hepatocellular carcinoma. This study aimed to elucidate the prognostic significance of FGF<em>19</em> protein expression in hepatocellular carcinoma patients.

METHODS

By immunohistochemistry, we investigated the expression of FGF<em>19</em> protein in tumor tissue from 281 hepatocellular carcinoma patients who underwent curative hepatectomy. Univariate and multivariate analyses were performed to evaluate its predictive value for tumor recurrence and survival of patients. The median follow-up period was 75.6 months.

RESULTS

FGF<em>19</em> protein expression was observed in 135 (48.0 %) of the 281 hepatocellular carcinomas. FGF<em>19</em> expression was significantly associated with larger tumor size (P < 0.001), and higher BCLC stage (P = 0.001). FGF<em>19</em> expression was correlated with the early recurrence (P < 0.001), but not with the late recurrence (P = 0.582). FGF<em>19</em> expression (P = 0.002), viral etiology (P = 0.028), and intrahepatic metastasis (P < 0.001) were independent predictors of early recurrence. Multivariate analyses of survival revealed that FGF<em>19</em> expression (P < 0.001), intrahepatic metastasis (P < 0.001), and liver cirrhosis (P = 0.0<em>19</em>) were independent predictors of shorter disease-free survival. FGF<em>19</em> expression (P = 0.005), larger tumor size (P = 0.038), major portal vein invasion (P = 0.048), intrahepatic metastasis (P < 0.001), lower albumin level (P = 0.024), and liver cirrhosis (P = 0.031) were independent predictors of shorter disease-specific survival.

CONCLUSIONS

FGF<em>19</em> protein expression might be an effective predictor of early recurrence and a marker for poor prognosis of hepatocellular carcinoma after curative hepatectomy, indicating that FGF<em>19</em> might be a potential preventive target in hepatocellular carcinoma patients.