BACKGROUND

Mesenchymal stem cells (MSCs) offer a novel therapeutic option in the treatment of acute myocardial infarction. MSCs are able to differentiate into myogenic cells after 5-azacytitdine treatment. However, 5-azacytidine might have genotoxic effects. Recently, it was reported that combined treatment with bone morphogenetic protein-2(BMP-2) and fibroblast growth factor-4(FGF-4) caused cardiac differentiation in non-precardiac mesoderm explants. Therefore, we investigated whether MSCs treated with combined BMP-2 and FGF-4 showed evidence of myogenic differentiation in vitro, and whether these cells resulted in sustained engraftment, myogenic differentiation, and improved cardiac function after implantation in infarcted myocardium.

RESULTS

In vitro study: MSCs were treated with BMP-2 + FGF-4 (GF-MSCs) and myogenic phenotype was evaluated immunohistochemically. Cell growth curve was used to compare MSC proliferative capacity between the growth factors and 5-azacytidine treatments. In vivo study: two weeks after coronary artery occlusion, GF-MSCs (n=15), MSCs (n=5) labelled with PKH26 were injected into infarcted myocardium. Control animals (n=5) received a culture medium into the infarcted myocardium. Two weeks after implantation, some engrafted GF-MSCs or MSCs expressed sarcomeric-alpha-actinin and cardiac myosin heavy chain, as was observed in culture. Echocardiography showed that the GF-MSC group had a better (p < 0.05) left ventricular performance than the other groups.

CONCLUSIONS

GF-MSCs induced myogenic differentiation in vitro. Moreover, GF-MSCs engrafted into the infarcted myocardium increased myogenic differentiation, prevented dilation of the infarcted region, and eventually improved heart function.

BACKGROUND

Epidermal growth factor (EGF)-related proteins, such as transforming growth factor-alpha (TGF-alpha), control cancer cell growth through hormonal pathways (i.e., autocrine [hormone acts on cell that produces it] and paracrine [hormone acts on nearby cells] pathways). Overexpression of TGF-alpha and/or its receptor (EGFR) has been detected in human cancers. The blockade of EGFR activation by the use of anti-EGFR monoclonal antibodies (MAbs) has been proposed as a potential anticancer therapy. The type I cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKAI) is generally overexpressed in human cancer cells and is involved in neoplastic transformation. Inhibition of PKAI by selective cAMP analogues, such as 8-chloro-cAMP (8-CI-cAMP), induces growth inhibition in various human cancer cell lines.

OBJECTIVE

On the basis of our previous observations of a cooperative anti-proliferative effect of anti-EGFR Mab 528 and 8-Cl-cAMP in human cancer cell lines in vitro, we evaluated the anticancer activity in vivo of the combination of an anti-EGFR MAb (MAb C225) and 8-Cl-cAMP.

METHODS

Athymic mice were injected subcutaneously with 10(7) human colon carcinoma GEO cells. After 7 days, when established tumor xenografts of 0.30-0.35 cm3 were detectable, 10-15 mice per group were treated intraperitoneally twice weekly with different doses of 8-Cl-cAMP and/or MAb C225. Cancer cell expression of various growth factors was evaluated by immunohistochemical analysis in tumors obtained from control and treated mice. Data were evaluated for statistical significance using the Student's t test and the Mantel-Cox logrank test. All P values represent two-sided tests of statistical significance.

RESULTS

A 5-week treatment with low doses of 8-Cl-cAMP (0.5 mg/dose) and MAb C225 (0.25 mg/dose) blocked GEO tumor growth (compared with that in control mice; P < .00001) and suppressed cancer cell production of autocrine growth factors, such as TGF-alpha, amphiregulin, and CRIPTO, and of angiogenic (promotes new blood vessel formation) factors, such as vascular endothelial growth factor and basic fibroblast growth factor, with no signs of toxicity. Control and 8-Cl-cAMP (0.5 mg/dose)-treated mice died within 9-10 weeks after tumor cell injection. In MAb C225 (0.25 mg/dose)-treated mice, GEO tumors resumed a growth rate comparable to that in control animals within 3 weeks following the end of treatment and the mice died between 11 and 20 weeks after tumor cell injection. GEO tumor growth was significantly delayed in the MAb C225 plus 8-Cl-cAMP treatment group (P < .00001) and was accompanied by a prolonged survival of mice (P < .00001) as compared with the control group.

CONCLUSIONS

Long-term treatment with a combination of agents that selectively inhibit two intracellular signal-transduction enzymes, such as the PKAI serine-threonine kinase and the EGFR tyrosine kinase, has anticancer activity in vivo, reflected by suppression of tumor proliferation and angiogenesis, with no signs of toxicity.

CONCLUSIONS

Since these inhibitors of intracellular mitogenic (growth-stimulating) signaling have a different mechanism(s) of action and do not antagonize the effects of cytotoxic therapy, a combination of anti-EGFR MAb C225 and 8-Cl-cAMP should be investigated as a nontoxic, long-term treatment for cancer patients following chemotherapy.

OBJECTIVE

Bile acid (BA) synthesis is regulated by negative feedback end-product inhibition, initiated by farnesoid X receptors (FXRs) in liver and gut. Studies on cholic acid (CA)-free Cyp8b1(-/-) mice have concluded that CA is a potent suppressor of BA synthesis. Cyp8b1(-/-) mice have increased BA synthesis and an enlarged BA pool, a phenotype shared with bile-duct-ligated, antibiotics-administered and with germ-free mice. Studies on such mice have concluded BA synthesis is induced due to reduced hormonal signalling by <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)<em>15</em> from intestine to liver. A mutual finding in these models is that potent FXR-agonistic BAs are reduced. We hypothesized that the absence of the potent FXR agonist deoxycholic acid (DCA) may be important for the induction of BA synthesis in these situations.

METHODS

Two of these models were investigated, antibiotic treatment and Cyp8b1(-/-) mice and their combination. Secondary BA formation was inhibited by ampicillin (AMP) given to wild-type and Cyp8b1(-/-) mice. We then administered CA, chenodeoxycholic acid (CDCA) or DCA to AMP-treated Cyp8b1(-/-) mice.

RESULTS

Our data show that the phenotype of AMP-treated wild-type mice resembles that of Cyp8b1(-/-) mice with fourfold induced Cyp7a1 expression, increased intestinal apical sodium-dependent BA transporter expression and increased hepatic BA levels. We also show that reductions in the FXR-agonistic BAs CDCA, CA, DCA or lithocholic acid cannot explain this phenotype; instead, it is likely due to increases in levels of α- and β-muricholic BAs and ursodeoxycholic acid, three FXR-antagonistic BAs.

CONCLUSIONS

Our findings reveal a potent positive feedback mechanism for regulation of BA synthesis in mice that appears to be sufficient without endocrine effects of FGF<em>15</em> on Cyp7a1. This mechanism will be fundamental in understanding BA metabolism in both mice and humans.

OBJECTIVE

Bile acids may play a role in the pathogenesis of IBS. We investigated the potential effects of bile acids entering the colon and its role in the symptom pattern in IBS.

METHODS

We measured 75Se-labelled homocholic acid-taurine (75SeHCAT) retention, and serum levels of 7α-hydroxy-4-cholesten-3-one (C4) and fibroblast growth factor (FGF) 19 in patients with IBS (n=141) and control subjects (75SeHCAT n=29; C4 and FGF19 n=435). In patients with IBS stool frequency and form, as well as GI symptom severity were registered, and in a proportion of patients colonic transit time and rectal sensitivity were measured (n=66). An 8-week open-label treatment with colestipol was offered to patients with 75SeHCAT <20%, and the effect of treatment was evaluated with IBS severity scoring system and adequate relief of IBS symptoms.

RESULTS

Compared with controls, patients with IBS had lower 75SeHCAT values (p=0.005), higher C4c levels (C4 corrected for cholesterol) (p<0.001), but similar FGF19 levels. Abnormal 75SeHCAT retention (<10%) was seen in 18% of patients, whereas 23% had elevated C4c levels. Patients with IBS with 75SeHCAT retention <10% had more frequent stools, accelerated colonic transit time, rectal hyposensitivity, a higher body mass index, higher C4c and lower FGF19 levels. Colestipol treatment improved IBS symptoms (IBS severity scoring system 220±109 vs. 277±106; p<0.01), and 15/27 patients fulfilled criteria for treatment response (adequate relief ≥50% of weeks 5-8).

CONCLUSIONS

Increased colonic bile acid exposure influences bowel habit and colonic transit time in patients with IBS. A high response rate to open label treatment with colestipol supports this, but placebo-controlled studies are warranted.

Cardiac <em>fibroblasts</em> are mainly responsible for the synthesis of major extracellular matrix proteins in the heart, including fibrillar collagen types I and III and fibronectin. In this report we show that these cells, when stimulated by transforming <em>growth</em> <em>factor</em> beta 1 (TGF-beta 1), acquire certain myocyte-specific properties. Cultured cardiac <em>fibroblasts</em> from adult rabbit heart were treated with TGF-beta 1 (10-<em>15</em> ng/ml) for different periods of time. Northern hybridization analysis of total RNA showed that cells treated with TGF-beta 1 for 24 hr expressed mRNA corresponding to sarcomeric actin mRNA. Immunofluorescence staining and light microscopy showed that cultured cardiac <em>fibroblasts</em> treated with TGF-beta 1 became stained with a monoclonal antibody to muscle-specific actin. After treatment of quiescent cells with TGF-beta 1, cell proliferation (as measured by [3H]thymidine incorporation) was moderately increased (1.5-fold, P less than 0.001). NIH 3T3 cells and human skin <em>fibroblasts</em>, treated with TGF-beta 1, did not express sarcomeric actin mRNA. Treatment of cardiac <em>fibroblasts</em> with the mitogenic agent phorbol 12-myristate 13-acetate or with norepinephrine, angiotensin II, or interleukin 1 beta did not induce myocyte-specific actin mRNA. Cultured cardiac <em>fibroblasts</em> at the subconfluent stage, when exposed to TGF-beta 1 in the presence of 10% fetal bovine serum, gave rise to a second generation of slowly <em>growing</em> cells that expressed muscle-specific actin filaments. Our findings demonstrate that cardiac <em>fibroblasts</em> can be made to differentiate into cells that display many characteristics of cardiac myocytes. TGF-beta 1 seems to be a specific inducer of such conversion.

Farnesoid X receptor (FXR), a metabolic nuclear receptor, plays critical roles in the maintenance of systemic energy homeostasis and the integrity of many organs, including liver and intestine. It regulates bile acid, lipid, and glucose metabolism, and contributes to inter-organ communication, in particular the enterohepatic signaling pathway, through bile acids and <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>15</em>/19 (FGF-<em>15</em>/19). The metabolic effects of FXR are also involved in gut microbiota. In addition, FXR has various functions in the kidney, adipose tissue, pancreas, cardiovascular system, and tumorigenesis. Consequently, the deregulation of FXR may lead to abnormalities of specific organs and metabolic dysfunction, allowing the protein as an attractive therapeutic target for the management of liver and/or metabolic diseases. Indeed, many FXR agonists have been being developed and are under pre-clinical and clinical investigations. Although obeticholic acid (OCA) is one of the promising candidates, significant safety issues have remained. The effects of FXR modulation might be multifaceted according to tissue specificity, disease type, and/or energy status, suggesting the careful use of FXR agonists. This review summarizes the current knowledge of systemic FXR biology in various organs and the gut⁻liver axis, particularly regarding the recent advancement in these fields, and also provides pharmacological aspects of FXR modulation for rational therapeutic strategies and novel drug development.

Small-cell lung cancer (SCLC) comprises about 13-<em>15</em>% of all lung cancers, and more than 29 400 new cases have been diagnosed in the United States in the year 2012. SCLC is a biologically complex tumor typically occurring in heavy smokers. Its medical treatment has almost remained unchanged over the last decades and selected treatment options have not been established so far, mainly due to the lack of targetable genetic alterations. In this study we analyzed a cohort of 307 SCLC samples for <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 (FGFR1) amplification using a dual color FISH probe. FGFR1 status was correlated with clinical data. FGFR1 amplifications were observed in 5.6% of evaluable pulmonary SCLCs. Most of them (93%) fulfilled the criteria for high-level amplification and only one case showed low-level amplification. Amplification patterns were homogenous in the entire tumor area without occurrence of any 'hot spot' areas. FGFR1 amplification status was not associated with age, sex, stage, smoking status or overall survival. FGFR1 amplification analysis by FISH analysis in SCLC is, under respect of certain technical issues, applicable in the routine clinical setting. However, the FGFR1 amplification patterns in SCLC differs strongly from the previously described FGFR1 amplification pattern in squamous cell carcinoma of the lung, as positive SCLC harbor mostly homogeneous high-level amplifications. We provide evidence that an estimated number of 1640 newly diagnosed FGFR1-positive SCLC cases in the United States annually could benefit from targeted therapy. Therefore, we recommend including SCLC in the screening for ongoing clinical trials with FGFR1 inhibitors.

OBJECTIVE

The purpose of this study was to evaluate the clinical activity and toxicity of recombinant human Interleukin (IL)-12 in patients with relapsed and refractory non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (HD).

METHODS

Forty-two previously treated patients (32 patients with NHL and 10 patients with HD) were enrolled on the study. Patients were treated with either intravenous (n = 11) or subcutaneous (n = 31) administration of IL-12. The patients had received a median of three prior treatment regimens, and 16 patients had undergone prior autologous stem cell transplantation.

RESULTS

All patients were assessable for toxicity, and 39 of 42 (93%) patients were assessable for response. Six of 29 (21%) patients with NHL had a partial or complete response, whereas none of the 10 patients with HD responded. Furthermore, <em>15</em> patients had stable disease that lasted for up to 54 months. Progression-free survival in patients with indolent NHL, aggressive NHL, and HD was 6, 2, and 2.5 months, respectively. Treatment was well tolerated, and the most common toxicity was flu-like symptoms. Reversible grade 3 hepatic toxicity was observed in three patients requiring dose reduction. IL-12 therapy increased the median number of peripheral blood CD8 T lymphocytes from 423/microl to 576/microl (P = 0.0019). Furthermore, IL-12 therapy decreased serum vascular endothelial <em>growth</em> <em>factor</em> and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> concentrations in 37% of the patients.

CONCLUSIONS

The ability of recombinant human IL-12 therapy to increase the number of circulating CD8+ cells and induce clinical remissions in patients with relapsed NHL warrants further investigation of the drug.

OBJECTIVE

Fibroblast growth factor (FGF)-19 and FGF-21 are novel metabolic regulators that improve insulin resistance and obesity in rodents. The aim of the study was to assess the effects of laparoscopic sleeve gastrectomy (LSG) on serum concentrations of FGF-19 and FGF-21 along with circulating bile acids and other relevant hormonal and biochemical parameters.

METHODS

Seventeen females with obesity undergoing LSG and 15 lean healthy females were included into the study. Anthropometric and biochemical parameters, serum concentrations of FGF-19 and -21, insulin, adiponectin, leptin, C-reactive protein, resistin, amylin (total), ghrelin (active), glucagon-like peptide 1 (GLP-1, active), glucose-dependent insulinotropic peptide (GIP, total), peptide YY (PYY, total), pancreatic polypeptide (PP), and bile acids, and mRNA expression of selected adipokines and inflammatory markers in bioptic samples of subcutaneous fat were assessed at baseline and 6, 12, and 24 months after LSG.

RESULTS

LSG markedly decreased body weight, BMI, waist circumference, and insulin levels and improved systemic inflammation and lipid levels. FGF-19 concentrations increased and FGF-21 concentrations decreased after LSG along with increased adiponectin and decreased leptin, amylin, and ghrelin levels. GLP-1, GIP, PP, and circulating bile acids were not affected by LSG. PYY decreased significantly 24 months after surgery only. mRNA expression analysis in subcutaneous fat showed markedly reduced proinflammatory state.

CONCLUSIONS

Our results indicate that increased FGF-19 and decreased ghrelin concentrations could have partially contributed to the improvement of systemic inflammation and some metabolic parameters after LSG, while changes of FGF-21 are rather secondary because of weight loss.

The expression in human <em>fibroblasts</em> of the beta 2-interferon (IFN-beta 2) gene, which is now recognized to be identical to the gene encoding B-cell differentiation <em>factor</em> BSF-2, is enhanced by several cytokines that affect cell <em>growth</em> (tumor necrosis <em>factor</em>, interleukin 1, platelet-derived <em>growth</em> <em>factor</em>, and beta 1-interferon). We have examined the possibility that IFN-beta 2 gene expression is regulated through activation, by diacylglycerol, of the protein kinase C pathway. The synthetic diacylglycerols 1,2-dioctanoylglycerol (diC8) and 1-oleoyl-2-acetylglycerol strongly enhanced IFN-beta 2, but not IFN-beta 1, gene expression in human <em>fibroblasts</em> (FS-4 strain). An increase in IFN-beta 2 mRNA level was detected within <em>15</em> min after addition of diC8 (290 microM) to FS-4 cells and was maximal approximately 20 hr later. An increase in IFN-beta 2 gene transcription was detected within 5 min of addition of diC8, and the rate of transcription was near-maximal by <em>15</em>-30 min. The enhancement of IFN-beta 2 gene expression by diC8, interleukin 1, or tumor necrosis <em>factor</em> was not prevented by H8, a preferential inhibitor of cAMP- and cGMP-dependent protein kinases, but was blocked by H7, an inhibitor of protein kinase C as well as of cyclic nucleotide-dependent protein kinases. diC8 was found to protect FS-4 cells from the cytopathic effect of vesicular stomatitis virus; this protection was blocked by polyclonal or monoclonal antibodies that neutralize IFN-beta, suggesting that the antiviral effect was due to the secretion of IFN-beta 2 by the diC8-treated <em>fibroblasts</em>. The calcium ionophore A23187 (1-10 microM) also elicited an increase in the level of IFN-beta 2 mRNA in FS-4 <em>fibroblasts</em>; appropriate combinations of A23187 and diC8 had at least an additive effect in enhancing IFN-beta 2 mRNA levels. These results show that protein kinase C-activating or [Ca2+]-elevating agents rapidly increase the expression of the IFN-beta 2 gene in human <em>fibroblasts</em>.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) receptors trigger a wide variety of cellular responses as diverse as cell migration, cell proliferation and cell differentiation. However, the molecular basis of the specificity of these responses is not well understood. The C. elegans FGF receptor EGL-<em>15</em> similarly mediates a number of different responses, including transducing a chemoattractive signal and mediating an essential function. Analysis of the migration-specific alleles of egl-<em>15</em> has identified a novel EGL-<em>15</em> isoform that provides a molecular explanation for the different phenotypic effects of lesions at this locus. Alternative splicing yields two EGL-<em>15</em> proteins containing different forms of a domain located within the extracellular region of the receptors immediately after the first IG domain. Neither of these two domain forms is found in any other FGF receptor. We have tested the roles of these EGL-<em>15</em> receptor isoforms and their two FGF ligands for their signaling specificity. Our analyses demonstrate different physiological functions for the two receptor variants. EGL-<em>15</em>(5A) is required for the response to the FGF chemoattractant that guides the migrating sex myoblasts to their final positions. By contrast, EGL-<em>15</em>(5B) is both necessary and sufficient to elicit the essential function mediated by this receptor.

We investigated the expression of the protooncogene c-fos in 3T3-L1 <em>fibroblasts</em> and adipocytes in response to a variety of <em>growth</em>-promoting agents in normal cells and in cells preincubated with phorbol esters to deplete them of protein kinase C. There was a rapid accumulation of c-fos mRNA in <em>fibroblasts</em> and adipocytes treated with phorbol 12-myristate 13-acetate, platelet-derived <em>growth</em> <em>factor</em>, <em>fibroblast</em> <em>growth</em> <em>factor</em>, fetal calf serum, bombesin, and insulin, especially in the adipocytes. Phorbol 12-myristate 13-acetate pretreatment abolished the increase in c-fos mRNA due to additional phorbol 12-myristate 13-acetate treatment and decreased but did not eliminate the ability of platelet-derived <em>growth</em> <em>factor</em>, <em>fibroblast</em> <em>growth</em> <em>factor</em>, fetal calf serum, bombesin, and insulin to stimulate c-fos mRNA. These data suggested that c-fos mRNA could be induced in serum-deprived 3T3-L1 <em>fibroblasts</em> and adipocytes by at least two separate pathways, one involving protein kinase C and the other independent of protein kinase C. In the very insulin-sensitive 3T3-L1 adipocytes, insulin rapidly and transiently increased c-fos expression (c-fos mRNA appeared by <em>15</em> min and disappeared after 60 min) via interaction with its own cellular receptor, rather than by interacting with receptors for one of the insulin-like <em>growth</em> <em>factors</em>. Cycloheximide treatment in combination with insulin or phorbol 12-myristate 13-acetate resulted in superinduction of c-fos mRNA. We conclude that insulin can rapidly stimulate c-fos mRNA accumulation in 3T3-L1 adipocytes and that part of the <em>growth</em> <em>factor</em>-stimulated increase in this mRNA that occurs in protein kinase C-deficient cells may be due to activation of a pathway similar or identical to that activated by insulin.

BACKGROUND

Fibroblast growth factor 23 (FGF23), a bone-derived phosphaturic hormone, is elevated in chronic kidney disease (CKD). There are scarce data on the levels of its essential co-receptor klotho, and longitudinal changes in FGF23 levels are also unknown.

METHODS

We examined FGF23 and soluble klotho (s-klotho) levels over 1 year in 154 children with CKD Stages 1-5 (CKD1-5), were on dialysis or who have received a transplantation.

RESULTS

In children with CKD1-5 and who were receiving dialysis, FGF23 correlated inversely with the estimated glomerular filtration rate (eGFR) (P < 0.001), whereas a decrease in s-klotho was observed with a lower eGFR (P = 0.01). There was no correlation between FGF23 and serum phosphate (P) or parathyroid hormone (PTH) in our cohort wherein 89 and 66%, respectively, had normal levels. FGF23 increased by 6-fold over a 12-month period in children with eGFR of 15-29 mL/min/1.73 m(2), with an overall 5% annual increase in the CKD1-5 and dialysis cohort. High FGF23 levels were seen with high calcium (Ca) levels (P < 0.001). FGF23 levels were high when 25-hydroxyvitamin D [25(OH)D] and 1,25-dihydroxyvitamin D [1,25(OH)(2)D] were deficient (P = 0.05 and P < 0.001, respectively). s-klotho levels correlated positively with 25(OH)D (P < 0.001) and negatively with PTH (P = 0.04) and age (P = 0.03). Multivariate regression analysis demonstrated a strong relationship between FGF23 and eGFR, whereas the association between s-klotho and eGFR as observed in univariate analysis was lost following the adjustment of confounders. In transplanted patients, FGF23 correlated with eGFR (P = 0.02) and 25(OH)D (P = 0.05).

CONCLUSIONS

This study shows increasing FGF23 and reduced s-klotho levels with progressive renal failure even in a population of children with well-controlled P levels. Novel associations between FGF23 and serum Ca as well as 25(OH)D warrant further investigation.

BACKGROUND

Thalidomide is a putative antiangiogenesis agent with activity in several hematologic malignancies.

METHODS

Forty-four patients who had myelofibrosis with myeloid metaplasia received treatment with thalidomide in a Phase II clinical trial at a dose of 200 mg daily with escalation by 200 mg weekly until the best tolerated dose (maximum, 800 mg) was reached.

RESULTS

Seventeen of 41 evaluable patients (41%) who received treatment for at least <em>15</em> days had a response. A complete response (without reversal of bone marrow fibrosis) was achieved in 4 patients (10%), a partial response was achieved in 4 patients (10%), and hematologic improvements in anemia, thrombopenia, and/or splenomegaly were observed in 9 patients (21%). Improvements in anemia occurred in 7 of 35 patients (20%) with hemoglobin levels <10.0 g/dL, and improvements in thrombopenia occurred in 5 of 24 patients (21%) with platelet counts <100 x 10(9)/L. Five of 24 patients (21%) became transfusion-independent. Major or minor regression of splenomegaly was noted in 9 of 29 evaluable patients (31%), and complete regression was noted in 5 patients. Responders had a lower baseline median vascular endothelial <em>growth</em> <em>factor</em> levels (77.9 pg/mL vs. 97.7 pg/mL; P <.01) and higher median basis <em>fibroblast</em> <em>growth</em> <em>factor</em> levels (60.8 pg/mL vs. 37.4 pg/mL; P <.01) compared with nonresponders. Nine patients (22%) had deterioration that was attributed to thalidomide (resolved after withdrawal) with either progressive cytopenias or excessive proliferation. Two patients developed Grade 3 neutropenia with recovery and resumed therapy with dose reductions, and both later achieved a complete response. Dose-related toxicities included fatigue (50%), constipation (48%), rash or pruritus (37%), sedation (35%), peripheral edema (29%), tremors (23%), peripheral neuropathy (22%), and orthostasis (16%).

CONCLUSIONS

Thalidomide warrants further evaluation in patients with MMM, particularly in combination regimens, along with the investigation of newer analogs.

Acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) belong to a family of mitogenic polypeptides that are involved in cellular proliferation and differentiation. In this study we investigated the potential role of aFGF and bFGF in chronic pancreatitis (CP), a fibrotic condition associated with acinar cell dedifferentiation and atrophy, and <em>fibroblast</em>ic proliferation. By immunohistochemistry, aFGF and bFGF were abundant in pancreatic ductal and acinar cells in pancreatic tissues from CP patients. Immunoblotting with the same highly specific monoclonal antibodies demonstrated a marked increase in aFGF and bFGF in pancreatic homogenates from CP patients by comparison with the normal pancreas. Northern blot analysis indicated that, by comparison with normal controls, 16 of 21 CP tissues exhibited a 14-fold increase in aFGF mRNA levels, and 19 of 21 CP tissues exhibited a <em>15</em>-fold increase in bFGF mRNA levels. In situ hybridization confirmed that this overexpression occurred in ductal and acinar cells, and indicated that both mRNA moieties colocalized with their respective proteins. These findings suggest that aFGF and bFGF may either be involved in the pathobiological mechanisms that occur in CP, or that their overexpression may be the consequence of other perturbations that occur in this disorder.

Reactive oxygen species modulate major cellular functions by mechanisms which are still poorly understood. Recently, H2O2 has been reported to stimulate the activity of the mitogen-activated protein kinases (MAPKs) ERK and JNK, and the expression of the proto-oncogenes c-fos and c-jun. As their expression is enhanced by H2O2 in astrocytes, we studied whether these MAPKs were stimulated by H2O2 in primary cultured astrocytes. The result was positive, a maximum of stimulation being reached with 200 microM H2O2 (0.3 pmol H2O2/cell) for both ERK and JNK. ERK was previously reported to stimulate cytosolic phospholipase A2 phosphorylation and activity. H2O2 stimulated the release of arachidonic acid in astrocytes, as already reported in other cell types. We found also that cPLA2 phosphorylation was increased by H2O2. Moreover, the stimulation by H2O2 of ERK and JNK was decreased by phospholipase A2 activity inhibitors. When astrocytes were incubated first with eicosatetraynoic acid, a structural analogue competing in arachidonic acid metabolism, the stimulation of JNK by H2O was also inhibited, suggesting the involvement of arachidonic acid metabolites. Cyclooxygenase or cytochrome P450 monooxygenase inhibitors failed in decreasing the MAPK stimulation by H2O2, whereas lipoxygenase inhibitors completely abolished that of JNK. Mitogenicity has been reported to be stimulated by H2O2 in other cell types. Although ERK was strongly and durably stimulated by 200 microM H2O2 in astrocytes, at the same extent as by mitogenic <em>growth</em> <em>factors</em>, basal thymidine incorporation rate was decreased by more than 80% after 12-<em>15</em> h. Moreover, the stimulation of thymidine incorporation induced by basic <em>fibroblast</em> <em>growth</em> <em>factor</em> was transiently abolished by H2O2. Furthermore, H2O2 likely induced the expression of CL100/PAC1/MKP-1, a dual specificity phosphatase which has been implicated in ERK and JNK inactivation in the nucleus. Finally, the prior treatment of astrocytes with MK886, a 5-lipoxygenase-activating protein inhibitor, prevented JNK from stimulation, but did not prevent thymidine incorporation from inhibition, both induced by H2O2. These results strongly suggest an involvement of arachidonic acid and/or its metabolites in the stimulation of both ERK and JNK following the oxidative stress evoked by H2O2, which induced a cell cycle arrest probably independent of the stimulation of JNK.

Indomethacin delays healing of experimental gastric ulcers. We investigated whether inhibition of gastric acid secretion by omeprazole or stimulation of angiogenesis by basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) may reverse this delay. Rats with gastric ulcers induced by cryoprobe were treated subcutaneously with either placebo, indomethacin (2 x 0.5 mg/kg), bFGF (2 x 100 micrograms/kg), omeprazole (1 x 40 mumol/kg), indomethacin plus omeprazole, or indomethacin plus bFGF given daily for 8, 10, <em>15</em>, and 22 days. Ulcer size, epithelial cell proliferation, angiogenesis, and maturation of granulation tissue were sequentially quantified. Omeprazole significantly accelerated ulcer healing in an early phase (days 3-8). In contrast, bFGF accelerated healing in a late phase (days 10-<em>15</em>). Indomethacin significantly delayed ulcer healing in late phase and decreased prostaglandin generation, cell proliferation, angiogenesis, and maturation of granulation tissue. Despite stimulation of angiogenesis, bFGF did not reverse indomethacin-induced delay in ulcer healing. In contrast, omeprazole reversed indomethacin-induced effects on angiogenesis, cell proliferation, maturation of granulation tissue, and ulcer healing rate.

In an attempt to isolate novel regulatory and/or tumor suppressor genes, we identified cDNAs whose abundance is low in NIH 3T3 cells and further decreased following the expression of the activated oncogene, v-src. The transcription of one such gene, 322, is suppressed at least <em>15</em>-fold in src-, ras-, and fos-transformed cells and 3-fold in myc-transformed cells but is unaffected in raf-, mos-, or neu-transformed cells. Activation of a ts-v-src allele in confluent 3Y1 <em>fibroblasts</em> resulted in an initial increase in 322 mRNA levels after 1 to 2 h followed by a rapid decrease to suppressed levels after 4 to 8 h. Morphological transformation was not detected until 12 h later, indicating that the accumulation of 322 transcripts is regulated by v-src and not as a consequence of transformation. Addition of fetal calf serum to starved subconfluent NIH 3T3 or 3Y1 <em>fibroblasts</em> resulted in a similar biphasic regulation of 322, indicating that 322 transcription is responsive to mitogenic <em>factors</em>. Sequence analysis of a putative full-length 322 cDNA clone (5.4 kb) identified a large open reading frame (ORF) encoding a 148.1-kDa product. In vitro transcription and translation of the 322 cDNA from a T7 promoter resulted in a 207-kDa product whose electrophoretic mobility on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel was unaffected by digestion with endoglycosidase F. The discrepancy in predicted versus measured molecular weights may result from the high percentage of acidic residues (roughly 20% Glu or Asp) in the 322 ORF product. Comparison of the 322 cDNA ORF with sequences in data banks indicates that this gene is novel. The 322 ORF product contains a potential Cys-1-His-3 Zn finger, at least five nuclear localization signals of the adenovirus E1a motif K(R/K)X(R/K), and alternating acidic and basic domains. Overexpression of the 322 cells resulted in the selection of rapidly <em>growing</em> cells which had lost the transduced 322 cDNA. Thus, 322 represent a novel src- and ras-regulated gene which encodes a potential regulator of mitogenesis and/or tumor suppressor.

Incubation of physiological concentrations of 125I-labeled insulin with liver membranes in the presence of anti-insulin IgG results in a 7- to <em>15</em>-fold increase in the specific binding of the hormone. The low-affinity/high-capacity binding sites are replaced by an apparently homogeneous class of high-affinity sites, and the nonlinear Scatchard plots are converted to linear plots without a change in the maximum number of binding sites. Similarly, the binding of insulin to receptors in 3T3 <em>fibroblasts</em> is increased substantially in the presence of anti-insulin antibody, and the biological activity of subactive concentrations of insulin is enhanced by antibody in these cells. However, the affinity of 125I-labeled epidermal <em>growth</em> <em>factors</em> (EGF) in <em>fibroblasts</em> is not affected by anti-EGF IgG. In adipocytes anti-insulin IgG in the same concentration range only inhibits the binding of insulin and suppresses insulin-mediated glucose oxidation. Monovalent Fab' fragments from anti-insulin IgG inhibit the binding of the hormone, indicating that the enhancement of binding in liver membranes and <em>fibroblasts</em> requires the bivalency of the antibody.

Syndecan-1 (Sdc1) plays a major role in wound healing and modulates inflammatory responses. Sdc1 expression is reduced in lesions of patients with ulcerative colitis. The aim of this study was to investigate the role of Sdc1 in murine dextran sodium sulfate (DSS)-induced colitis. DSS colitis was induced in Sdc1-deficient (knockout (KO)) and wild-type mice by oral administration of 3% DSS. KO mice exhibited a significantly increased lethality as compared with wild-type controls (61 versus 5%, P < 0.05). Impaired mucosal healing and prolonged recruitment of inflammatory cells in KO mice were accompanied by significant up-regulation of tumor necrosis <em>factor</em>-alpha, CC chemokine ligand 3/macrophage inflammatory protein-1alpha, and vascular cell adhesion molecule-1, as determined by histological correlation between 0 and <em>15</em> days after colitis induction, TaqMan low-density array analysis, and quantitative real-time PCR. Treatment from days 7 through 14 with enoxaparin, a functional analogue of the Sdc1 heparan sulfate chains, significantly reduced lethality of KO mice due to DSS-induced colitis, which was correlated with improved mucosal healing. In vitro, Sdc1-deficient polymorphonuclear cells displayed increased adhesion to endothelial cells and intercellular adhesion molecule-1, and enoxaparin reverted adhesion to wild-type levels. Small interfering RNA-mediated knockdown of Sdc1 expression resulted in reduced basic <em>fibroblast</em> <em>growth</em> <em>factor</em>-mediated mitogen-activated protein kinase signaling and reduced Caco-2 cell proliferation. We conclude that Sdc1 has a protective effect during experimental colitis. The modification of missing Sdc1 function by heparin analogues may emerge as a promising anti-inflammatory approach.

We determined whether cutaneous angiogenesis induced by exposure of mice to ultraviolet-B (UVB) radiation is associated with an imbalance between positive and negative angiogenesis-regulating molecules. Unshaved C3H/HeN mice were exposed to a single dose (<em>15</em> kJ per m2) of UVB. At various times, the mice were killed, and their external ears were processed for routine histology and immunohistochemistry. Antibodies against proliferating cell nuclear antigen and bromodeoxyuridine identified dividing cells. Antibodies against CD31/ PECAM-1 identified endothelial cells, and antibodies against basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), vascular endothelial <em>growth</em> <em>factor</em>/vascular permeability <em>factor</em>, and interferon-beta (IFN-beta) identified angiogenesis-regulating molecules. Epidermal hyperplasia was documented by 48 h and reached a maximum on day 7 after exposure to UVB. The expression of bFGF increased by 24 h, whereas the expression of IFN-beta decreased by 72 h after exposure to UVB. The expression of vascular endothelial <em>growth</em> <em>factor</em>/vascular permeability <em>factor</em> increased slightly after irradiation. The altered balance between bFGF and IFN-beta was associated with increased endothelial cell proliferation (bromodeoxyuridine + CD31 + cells) within existing blood vessels, leading to telangiectasia and new blood vessels. UV-induced epidermal hyperplasia and cutaneous angiogenesis were highest in IFN-alpha/beta receptor knockout mice. These results demonstrate that in response to UVB radiation, dividing keratinocytes produce a positive angiogenic molecule (bFGF) but not a negative angiogenic molecule (IFN-beta), and that this altered balance is associated with enhanced cutaneous angiogenesis.

Kit ligand (Kitl), the ligand for the Kit receptor tyrosine kinase, plays important roles in hematopoiesis, gametogenesis and melanogenesis. Kitl is synthesized as a membrane-anchored precursor that can be processed to produce the soluble <em>growth</em> <em>factor</em>. Here, we evaluated the role of ADAM (a disintegrin and metalloprotease) metalloproteases in ectodomain shedding of Kitl. We found that both ADAM17 and ADAM19 affect Kitl1 shedding, albeit in different ways. Overexpression of ADAM19 resulted in decreased levels of Endo-H-resistant mature Kitl1, thereby reducing the amount of Kitl that is shed from cells following stimulation with phorbol esters. ADAM17 was identified as the major phorbol-ester-stimulated sheddase of Kitl1, whereas ADAMs 8, 9, 10, 12 and <em>15</em> were not required for this process. ADAM17 also emerged as the major constitutive and phorbol-ester-stimulated sheddase of Kitl2 in mouse embryonic <em>fibroblasts</em>. Mutagenesis of the juxtamembrane domain of Kitl2 showed no stringent sequence requirement for cleavage by ADAM17, although two nonadjacent stretches of four amino acid residues were identified that are required for Kitl2 shedding. Taken together, this study identifies a novel sheddase, ADAM17, for Kitl1 and Kitl2, and demonstrates that ADAM19 can reduce ADAM17-dependent phorbol-ester-stimulated Kitl1 ectodomain shedding.

BACKGROUND

Chronic uremia is considered a proinflammatory state associated with high cardiovascular morbidity and mortality. The aim of the present study is to evaluate the potential relationship between the prevalence of coronary artery calcification (CAC) and selected factors that may be involved in the process of atherogenesis (lipid profile, acute-phase reactants, growth factors, and cytokines).

METHODS

The study group consisted of 43 patients (19 women, 24 men) with a mean age of 50.6 +/- 13.4 years treated with peritoneal dialysis (PD) for a median period of 15 months (range, 2 to 96 months). Only patients with sinus rhythm were included. CAC score (CaSc) was measured using multirow spiral computed tomography (MSCT). As parameters of lipid profile, total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides were assayed. C-reactive protein (CRP) and fibrinogen represented the level of acute-phase activation. Proinflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor-alpha [TNF-alpha]), leptin, and basic fibroblast growth factor (bFGF) also were measured.

RESULTS

Median CaSc equaled 17.9 Agatston units (range, 0 to 5,502 Agatston units). No calcification was detected in 20 subjects (46.5%; CaSc < 10 Agatston units). CaSc correlated with age (R = 0.57; P < 0.0001), body mass index (R = 0.42; P < 0.005), and serum leptin (R = 0.3; P < 0.05) and CRP levels (R = 0.38; P < 0.05). The correlation with PD therapy duration was borderline statistically significant (P = 0.063). Patients with the greatest values for CaSc >> 400 Agatston units) were characterized by significantly greater levels of IL-6, bFGF, and CRP compared with subjects with a CaSc less than 10 Agatston units (P < 0.05 for all). Patients with history of coronary artery disease (CAD) had significantly greater CaSc values (median, 778.6 versus 3.3 Agatston units; P < 0.001) compared with those without CAD. Serum triglyceride levels were significantly greater and HDL cholesterol levels were significantly lower in patients with CAD. The first group also was characterized by significantly greater serum TNF-alpha (P < 0.01) and CRP levels (P < 0.005). In multiple regression analysis, only age was independently associated with CaSc (beta = 0.45; P = 0.002).

CONCLUSIONS

Our results may suggest an association between CAC and chronic inflammation activity in the mentioned group of patients. To our knowledge, this is the first study reporting the prevalence of CAC in PD patients using the MSCT method. The association between CaSc results and classic, as well as inflammatory, risk factors for CAD found in this study should be interpreted with caution because of its method limitations (cross-sectional design, heterogeneity of study population, and small number of studied patients).

We have analysed expression of the first 7 members of the family of heparin-binding <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGFs) and their 4 high-affinity receptors (FGFRs) in human pancreatic carcinoma cell lines, both at the mRNA and protein levels. In cell lines expressing FGFRs, 2 typical patterns were observed: (i) expression of FGFR-I, -3 or -4 along with the expression of at least one FGF; (ii) co-expression of FGFR-3 and FGFR-4 in the absence of FGF expression. Using RT-PCR, transcripts representing multiple isoforms of both extracellular and intracellular domains of FGFR-I were detected in the cell line PT45. A novel extracellular domain variant of FGFR-I was predicted to encode the first immunoglobulin loop in a potentially secreted form. Protein expression of the splice variants of FGFR-I was confirmed by immunoprecipitation with specific antibodies in radiolabelled ligand cross-linking experiments. The type I carboxyl end and the alpha subtype extracellular domain were detected in the PANC-I cell line, while the type I carboxyl terminus and the gamma subtype extracellular domain were expressed in the PT45 cell line. Expression of FGF-2 in PT45 was also detected by immunoprecipitation using 3 different anti-FGF-2 antibodies. Apart from the 18-kDa product, higher molecular weight isoforms, namely 22- and 23-kDa isoforms, were expressed. In an assay of anchorage-independent <em>growth</em>, exogenous FGF-2 stimulated a maximum <em>15</em>-fold and 10-fold increase in colony formation by the cell lines MIA PACA-2 and PANC-I respectively. Treatment of monolayer cultures of the same cell lines did not promote <em>growth</em>. However, a specific neutralising antibody against FGF-2 reduced cell proliferation of MIA PACA-2 cells by 50%.