This review highlights the huge advances made in the understanding of Crohn's disease in the last <em>15</em> years. The pathogenic immune response in the gut wall is a highly polarised T helper cell type 1 response, probably directed against antigens of the commensal flora. There is marked over-expression of pro-inflammatory cytokines such as tumor necrosis <em>factor</em> (TNF)-alpha and increased production of matrix degrading enzymes by <em>fibroblasts</em> and macrophages, which are probably responsible for ulceration and fistula formation. Crohn's disease runs in families and the susceptibility genes identified so far are associated with innate recognition of microbial products (Nod2) or epithelial barrier function (OCTN cation transporter genes and DLG5). Endogenous healing pathways mediated by transforming <em>growth</em> <em>factor</em> (TGF)-beta1 are inhibited because mucosal inflammatory cells express Smad7, the endogenous intracellular inhibitor of TGF-beta signalling. This makes it unlikely that enteral feeds containing TFG-beta are therapeutic by means of direct anti-inflammatory effects, however TGF-beta may still be involved because it is a well known epithelial motogen and may promote mucosal healing, in synergy with changes in mucosal bacterial populations as a result of the change in the diet.

Successful approaches to tissue engineering smooth muscle tissues utilize biodegradable scaffolds seeded with autologous cells. One common problem in using biological scaffolds specifically is the difficulty of inducing cellular penetration and controlling de novo extracellular matrix deposition/remodeling in vitro. Our hypothesis was that small intestinal submucosa (SIS) exposed to specific mechanical stimulation regimes would modulate the synthesis of de novo collagen and elastin by bladder smooth muscle cells (BSMC) within the SIS matrix. We further hypothesized that the cytokines vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2), two key <em>growth</em> <em>factors</em> involved in epithelial mesenchymal signaling, will promote the cellular penetration into SIS necessary for mechanical stimulation. BSMC were seeded at 0.5 x 10(6) cells/cm(2) onto the luminal side of SIS specimens. VEGF (10 ng/mL) and FGF-2 (5 ng/mL) were added to each insert in the media every other day for up to 7 days in static culture. Following static culture, specimens were stretched strip-biaxially under <em>15</em>% peak strain at either 0.5 or 0.1 Hz for an additional 7 days. Following the culture period, specimens were assayed histologically and biochemically for cellular penetration, proliferation, elastin, collagen, and protease activity. Histological analyses demonstrated that in standard culture media, BSMC remained on the surface of the SIS while both FGF-2 and VEGF profoundly promoted in<em>growth</em> of the BSMC into the SIS. The penetration of the cells in response to these cytokines was confirmed using a Transwell assay. Following cellular penetration, BSMC produced significant amounts of elastic fibers under cyclic mechanical stretching at 0.1 Hz under <em>15</em>% stretch, as evidenced by colorimetric assay and histology using a Verhoeff-Van Gieson stain. Protease activity was assessed in the media and found to be statistically increased in static culture following FGF-2 treatment. These findings demonstrate, for the first time, the capability of BSMC to produce histologically apparent elastin fibers in vitro. Moreover, our results suggest that a strategy involving <em>growth</em> <em>factors</em> and controlled mechanical stimulation may be used to engineer functional, elastin-rich tissue replacements using decellularized biologically derived scaffolds.

Formation of bone-like tissue in culture by stromal bone marrow cells (SBMC) derived from young <em>growing</em> rats is dependent on dexamethasone (Dex) (Cell Tissue Res 254:317; 1988) and is significantly enhanced by basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) (J Bone Miner Res 8:919; 1993). The aim of this study was to examine the effect of maturation on the osteogenic potential and the response to Dex and bFGF of SBMC by using cultures derived from young <em>growing</em> (6 weeks old) and adult (9 months old) rats. SBMC cultures were grown in the presence of Dex (10(-8) or 10(-7) mol/L) at both P(0) and P(1) and either in the presence or absence of bFGF. The effect of Dex and bFGF on mineralized bone-like tissue (MBT) formation was assessed at P(1). The highest levels of mineralized tissue formation in P(1) subcultures in the absence of bFGF were obtained when cultures derived from young rats (6 weeks old) were treated with Dex 10(-7) and 10(-8) mol/L at P(0) and P(1), respectively, and when cultures derived from adult rats were exposed to Dex 10(-8) mol/L both at P(0) and P(1). Under these optimal Dex concentrations, the amount of MBT formed by adult rat-derived cultures was <em>15</em>-fold lower than that of young rat-derived ones. The addition of bFGF to P(0) cultures or to P(1) cultures grown under optimal Dex conditions enhanced MBT formation in P(1) cultures derived from both young and adult rats, but this effect was considerably more pronounced in the adult rat-derived cultures. The maximal levels of MBT formation were produced by cultures derived from adult rats treated with bFGF at both P(0) and P(1), whereas in cultures derived from young rats, the addition of bFGF at P(0) was not necessary for maximal MBT production. This stimulating effect of bFGF on MBT formation by adult rat-derived cultures was accompanied by a 2.2-, 1.8-, and 4.3-fold increase in proliferation, alkaline phosphatase activity, and Ca(2+) deposition rate, respectively. bFGF increased the level of glucocorticoid receptor by approximately 2. 3-fold in Dex-treated cultures derived from young animals. These results indicate that maturation is associated with a decrease in the proportion of osteoprogenitor cells in the stromal bone marrow and in their capacity to express the osteogenic phenotype. They further point to the significant role of bFGF in stimulating proliferation and osteogenic expression of stromal bone marrow osteoprogenitors derived from adult rats.

Unilateral pneumonectomy in rats causes compensatory <em>growth</em> of the remaining lung. During this <em>growth</em>, there are large increases in the cell numbers and in the rates of collagen and non-collagen protein production. We examined possible mechanisms by which these changes might occur. Assessment of the effect of bronchoalveolar lavage (BAL) fluid on <em>fibroblasts</em> in vitro demonstrated the presence of stimulatory activity for <em>fibroblast</em> replication in control animals. This activity was greatly increased two and six days postpneumonectomy (1<em>15</em> +/- 26% and 75 +/- 18% above control values, respectively), but had returned to normal by 14 days. Preliminary characterization suggests that the activity is heat labile and consists of at least two moieties with apparent molecular weights of 5-<em>15</em> kD and 70-220 kD. The activity was partially blocked by antibodies to insulin-like <em>growth</em> <em>factor</em>-1 (IGF-1), and levels of IGF-1 were increased by about 100% (p less than 0.001) two days after pneumonectomy compared with control values. Examination of BAL cells demonstrated an early influx of leucocytes into the remaining lung of pneumonectomized rats. At two days, about 25% of the lavageable cells were neutrophils, but macrophages were the predominant cell type at all times. The extravascular albumin space of the lung increased by about 65% (p less than 0.01), six days after pneumonectomy. The influx of circulatory proteins and cells are potential sources of the increased mitogenic activity observed in the lung.

Tumor necrosis <em>factor</em>-like weak inducer of apoptosis (TWEAK) could play a role in the regulation of interleukin (IL)-<em>15</em> and IL-18, cytokines crucial for angiogenesis and uterine natural killer (uNK) cell recruitment during embryo implantation. We therefore confirmed the endometrial presence of TWEAK/<em>fibroblast</em> <em>growth</em> <em>factor</em> inducible-14 (Fn-14) and documented simultaneously the cytotoxic KIR receptor (NKp46) of uNK cells in the human endometrium while TWEAK, Fn-14, IL-<em>15</em>, and IL-18 mRNA were quantified by real-time polymerase chain reaction in relation to the recruitment of CD56+ cells among fertile control women and patients who had failed to implant after assisted reproduction treatment.

alpha-Macroglobulins derived from plasma or secreted by macrophages are platelet-derived <em>growth</em> <em>factor</em> (PDGF) binding proteins that compete with cell-surface receptors on <em>fibroblasts</em> for PDGF binding. alpha 2-Macroglobulin (alpha 2M) derived from bovine plasma was tested for its ability to modulate the PDGF-induced proliferation of primary passage rat lung <em>fibroblasts</em> (RLFs) and a human skin <em>fibroblast</em> cell line (CRL <em>15</em>08). <em>Fibroblasts</em> were grown in 10% fetal bovine serum (FBS) for 24 hr, then washed with serum-free medium before adding serum-free defined medium (SFDM) containing insulin and transferrin. To this medium were added varying concentrations of human plasma-derived AB-PDGF and alpha 2 M, alone or in combination. Receptor-recognized alpha 2M was prepared by treatment with methylamine. Both native alpha 2M and the alpha 2M-methylamine (alpha 2M-MA) were tested for <em>growth</em> promoting activity in the absence or presence of PDGF. After 3 days, a concentration-dependent <em>growth</em> curve of <em>fibroblast</em> proliferation was demonstrated for PDGF alone, with near maximal stimulation reached at <em>15</em>-20 ng/ml PDGF. alpha 2M and alpha 2M-MA alone had no effect on cell proliferation. However, alpha 2M-MA concentrations above 32 micrograms/ml synergistically enhanced PDGF-stimulated proliferation greater than 100% in the presence of <em>15</em> ng/ml PDGF. Native alpha 2M enhanced PDGF-stimulated <em>growth</em> 80-100% above PDGF controls only at low concentrations (32-64 micrograms/ml alpha 2M). High concentrations of native alpha 2M (128-256 micrograms/ml) either had no effect on <em>growth</em> or were inhibitory to PDGF-stimulated <em>growth</em>, depending on the cell type tested. Rat lung <em>fibroblasts</em> were shown to secrete a <em>factor</em>(s) that inhibited the trypsin-binding capacity of native alpha 2M. We further demonstrated that early passage RLFs possess specific cell-surface receptors for [125I]-PDGF and [125I]-alpha 2M-MA, and preincubation of RLFs with alpha 2M-MA increased the specific binding of [125I]-PDGF to the cell surface of these <em>fibroblasts</em>. Considered together, these data support the view that receptor-recognized alpha 2M synergistically enhances the proliferative capacity of PDGF. We postulate that receptor-recognized alpha Ms enhance PDGF-stimulated <em>growth</em> by increasing the local concentration of PDGF at the cell surface, where the PDGF could be released in close proximity to its own receptors.

OBJECTIVE

The growth of ocular neovascularization is regulated by a balance between stimulating and inhibiting growth factors. Somatostatin affects angiogenesis by inhibiting the growth hormone-insulin-like growth factor axis and also has a direct antiproliferative effect on human retinal endothelial cells. The purpose of our study is to investigate the expression of somatostatin receptor (sst) subtypes and particularly sst subtype 2A (sst2A) in normal human macula, and to study sst2A in different stages of age-related maculopathy (ARM), because of the potential anti-angiogenic effect of somatostatin analogues.

METHODS

Sixteen eyes (10 enucleated eyes, 4 donor eyes, and 2 surgically removed choroidal neovascular [CNV] membranes) of 15 patients with eyes at different stages of ARM were used for immunohistochemistry. Formaldehyde-fixed paraffin-embedded slides were incubated with a polyclonal anti-human sst2A antibody. mRNA expression of five ssts and somatostatin was determined in the posterior pole of three normal human eyes by reverse transcriptase-polymerase chain reaction.

RESULTS

The immunohistochemical expression of sstA in newly formed endothelial cells and fibroblast-like cells was strong in fibrovascular CNV membranes. mRNA of sst subtypes 1, 2A, and 3, as well as somatostatin, was present in the normal posterior pole; sst subtypes 4 and 5 were not detectable.

CONCLUSIONS

Most early-formed CNV in ARM express sst2A. The presence of mRNA of sst subtype 2A was observed in normal human macula, and subtypes 1 and 3 and somatostatin are also present. sst2A receptors bind potential anti-angiogenic somatostatin analogues such as octreotide. Therefore, somatostatin analogues may be an effective therapy in early stages of CNV in ARM.

OBJECTIVE

Fibroadenomas are benign tumours composed of both glandular and fibrous tissue. The mechanisms regulating the growth of these tumours and the relation between the stromal and epithelial cells are poorly understood. Acidic fibroblast growth factor (aFGF) is a well known fibroblast activator, which acts through four specific cell surface receptors, among which, fibroblast growth factor receptor 4 (FGFR4) is highly specific. The aim of this study was to evaluate the distribution of aFGF and FGFR4 in specific cell types of fibroadenomas to understand their possible role in the growth of these breast lesions.

METHODS

Formalin fixed and paraffin wax embedded tissues from 15 fibroadenomas and peritumoral normal breasts were investigated for the expression of aFGF and FGFR4 using immunohistochemistry. The presence of aFGF mRNA was also investigated using in situ hybridisation.

RESULTS

Immunoreactivity for aFGF and FGFR4 was seen in epithelial cells, but it was lacking in myoepithelial cells of both normal tissues and fibroadenomas. Strong FGFR4 immunoreactivity was found in stromal fibroblasts, which were also weakly positive for aFGF. aFGF mRNA was detected in epithelial cells and in some stromal fibroblasts.

CONCLUSIONS

These results suggest a paracrine/autocrine modulation of epithelial and stromal cells of fibroadenomas through an aFGF-FGFR4 interaction. This interaction might regulate various cell functions and the growth of fibroadenomas.

The aim of the present study was to develop a three-dimensional (3D) collagen gel culture system for the in vitro <em>growth</em> and survival of buffalo preantral follicles with or without <em>growth</em> <em>factors</em>. Buffalo ovaries were collected from a local abattoir and preantral follicles were isolated through microdissection. Isolated preantral follicles were put either in collagen gel coated culture dish or embedded in a microdrop of collagen gel. The culture medium was TCM-199 fortified with fetal calf serum (10%), insulin transferin selenium solution (ITS, 1%), epidermal <em>growth</em> <em>factor</em> (EGF, 20 ng/ml) and follicle stimulating hormone (FSH, 0.5 microg/ml). Follicles were divided into three groups and cultured in the medium described above (group a, control), with addition of insulin like <em>growth</em> <em>factor</em> (IGF-I, 100 ng/ml, group b), or with addition of IGF-I and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF, 10 ng/ml, group c). Preantral follicles were incubated at 38.5 degrees C in 5% CO(2) and maximum humidity. Culture medium was replenished after every 72 h and spent medium was stored at -30 degrees C for hormone analysis. We found that the extracellular matrix of collagen gel maintained follicle viability and <em>growth</em> by providing surface interaction and increasing attachment of follicles. Preantral follicles embedded in collagen gel droplets had better antrum formation and development as compared to the whole surface coated culture method. Follicles cultured with IGF-I on collagen gel matrix showed a significantly (P<0.05) higher survival rate and larger mean diameter of follicles on day 10 of culture with improved <em>growth</em> and mucification as compared to the control group. However, follicles cultured in the combination of IGF-I with bFGF had decreased survival rate and smaller mean follicles diameter than the IGF-I group (b). Progesterone (P(4)) accumulation was greater on day 9 of culture in follicles cultured in IGF-I as compared to control; whereas, P(4) was markedly decreased in the combination of IGF-I with bFGF. Follicles of the control group could survive for up to 10-<em>15</em> days before degenerating, but follicles cultured with <em>growth</em> <em>factors</em> were able to survive up to 20 days and showed signs of early antrum formation. In summary, we have shown that collagen gel was a novel and efficacious 3D microenvironment for the extended culture of buffalo preantral follicles. Supplementation of culture medium with <em>growth</em> <em>factors</em> was found to be essential for antrum formation.

Mesenchymal stem cells (MSCs) are multipotent cells, which have the capability to differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, and marrow stroma. However, they lose the capability of multi-lineage differentiation after several passages. It is known that basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) increases <em>growth</em> rate, differentiation potential, and morphological changes of MSCs in vitro. In this report, we have used 2-DE coupled to MS to identify differentially expressed proteins at the cell membrane level in MSCs <em>growing</em> in bFGF containing medium. The cell surface proteins isolated by the biotin-avidin affinity column were separated by 2-DE in triplicate experiments. A total of <em>15</em> differentially expressed proteins were identified by quadrupole-time of flight tandem MS. Nine of the proteins were upregulated and six proteins were downregulated in the MSCs cultured with bFGF containing medium. The expression level of three actin-related proteins, F-actin-capping protein subunit alpha-1, actin-related protein 2/3 complex subunit 2, and myosin regulatory light chain 2, was confirmed by Western blot analysis. The results indicate that the expression levels of F-actin-capping protein subunit alpha-1, actin-related protein 2/3 complex subunit 2, and myosin regulatory light chain 2 are important in bFGF-induced morphological change of MSCs.

N-t-butyl hydroxylamine (NtBHA) delays senescence-dependent changes in human lung <em>fibroblasts</em> (IMR90) (Atamna et al., J. Biol. Chem. 275, 6741-6748). The current study examines the effect of NtBHA on mitochondria in old and young rats and human primary <em>fibroblasts</em> (IMR90). In NtBHA-treated rats, the age-dependent decline in food consumption and ambulatory activity was reversed without affecting body weight. The respiratory control ratio of mitochondria from liver of old rats improved after feeding NtBHA. These findings suggest that NtBHA improved mitochondrial function in vivo. The age-dependent increase in proteins with thiol-mixed disulfides was significantly lower in old rats treated with NtBHA. NtBHA was effective only in old rats; no significant effect was observed in young rats. In IMR90 cells, NtBHA delayed senescence-associated changes in mitochondria and cellular senescence induced by maintaining the cells under suboptimal levels of <em>growth</em> <em>factors</em>. Proteasomal activity was also higher in cells treated with NtBHA than in untreated cells. NtBHA accumulates in cells 10- to <em>15</em>-fold the extracellular concentration and is maintained by mitochondrial NADH. NtBHA is an antioxidant that is recycled by mitochondrial electron transport chain and prevents radical-induced toxicity to mitochondria.

The current study tests a hypothesis that nuclear receptor signaling is altered in chronic hepatitis C patients and that the altered pattern is specific to alcohol drinking history. The expression of a panel of more than 100 genes encoding nuclear receptors, coregulators, and their direct/indirect targets was studied in human livers. Gene expression pattern was compared between <em>15</em> normal donor livers and 23 hepatitis C virus (HCV) genotype 1-positive livers from patients without a drinking history (matched for age, sex, and body mass index). HCV infection increased the expression of nuclear receptors small heterodimer partner and constitutive androstane receptor (CAR) as well as genes involved in fatty acid trafficking, bile acid synthesis and uptake, and inflammatory response. However, the expression of retinoid X receptor (RXR) α, peroxisomal proliferator-activated receptor (PPAR) α and β as well as steroid regulatory element-binding protein (SREBP)-1c was decreased in HCV-infected livers. Gene expression pattern was compared in chronic hepatitis C patients with and without a drinking history. Alcohol drinking increased the expression of genes involved in fatty acid uptake, trafficking, and oxidation, but decreased the expression of genes responsible for gluconeogenesis. These changes were consistent with reduced fasting plasma glucose levels and altered expression of upstream regulators that include RXRα, PPARα, and CAR. The messenger RNA levels of <em>fibroblast</em> <em>growth</em> <em>factor</em> 21, interleukin-10, and fatty acid synthase, which are all regulated by nuclear receptors, showed independent correlation with hepatic HCV RNA levels.

CONCLUSIONS

Our findings suggest that those genes and pathways that showed altered expression could potentially be therapeutic targets for HCV infection and/or alcohol drinking-induced liver injury.

BACKGROUND

Transforming growth factor-beta (TGF-beta) is composed of a family of multifunctional polypeptide growth factors involved in embryogenesis, inflammation, regulation of immune response, angiogenesis, wound healing, and extracellular matrix formation. TGF-beta1 is the most common isoform found in human tissues. A role of TGF-beta in the pathogenesis of periodontal disease has been suggested. The aim of the present study was a comparative immunohistochemical evaluation of TGF-beta1 in normal keratinized gingiva and in the peri-implant soft tissues surrounding failing non-submerged implants.

METHODS

Twenty patients participated in this study. Ten biopsies from healthy keratinized mucosa and 10 biopsies from peri-implant soft tissues surrounding failing implants were obtained (one biopsy per patient). The biopsies were obtained from different patients.

RESULTS

In 5 cases of healthy mucosa, the stromal cells were positive between 1 to 5. In 7 cases, the epithelial layers were positive, between 1 and 18 cells. The superficial epithelial layer was negative in all cases. In 9 cases, there was a positivity of the vascular component, between 2 and 16 vessels. In failing implants, the stromal cells were positive in 6 cases, between 1 and 4. In all cases, cells of the epithelial layers were positive, between 15 and 40. The vascular component was positive in all cases, between 12 and 30 vessels. The differences between TGF-beta1 expression in the epithelium around healthy and failing implants were statistically significant (P < 0.0001). The differences between TGF-beta1 expression in the blood vessels in the soft tissues around healthy and failing implants were also statistically significant (P < 0.0001). No statistically significant difference was observed between the 2 groups in the TGF-beta1 expression in the stromal cells (P = 0.88).

CONCLUSIONS

TGF-beta1 may be one of the most important factors in the regulation of the infiltrate, and in the production of tissue repair with a stimulation of fibroblasts and endothelial cells.

The activation of protein kinase C and protein phosphorylation by tumor promoters were examined using quiescent cultures of BALB/3T3 and C3H/10T1/2 cells, because in these cells tumor promoters enhance chemically induced transformation and also induce DNA synthesis and ornithine decarboxylase. The cytosol and membrane fractions were partially purified, and the activity of protein kinase C was assayed. In quiescent cells, protein kinase C activity was found only in the cytosol fraction. Treatment with 100 ng of 12-O-tetradecanoylphorbol-13-acetate or teleocidin B per ml caused rapid translocation of protein kinase C from the cytosol to the membrane fraction. The activity in the cytosol disappeared almost completely after <em>15</em> min when the activity in the membrane reached a peak. The membrane activity gradually decreased to the control level after 6 h, while no activity reappeared in the cytosol within 6 h. Under these circumstances, a membrane protein with a molecular weight of 90,000 and pl of 4.0-4.4 (termed p90) was specifically phosphorylated, possibly by the activated protein kinase C, in both cell-free and intact-cell systems. On treatment of quiescent BALB/3T3 cells with 100 ng of 12-O-tetradecanoylphorbol-13-acetate, p90 phosphorylation increased 2-fold in 1 min, reaching a peak in <em>15</em> min of 3.4-fold the initial value. The phosphorylation of p90 increased with increase in the concentrations of 12-O-tetradecanoylphorbol-13-acetate between 0.1 and 10 ng/ml and reached a plateau at 10 ng/ml. p90 phosphorylation also occurred on exposure of the cells to non-phorbol ester tumor promoters (mezerein and teleocidin B) and <em>growth</em> <em>factors</em>, such as platelet-derived <em>growth</em> <em>factor</em> and <em>fibroblast</em> <em>growth</em> <em>factor</em>. p90 was not immunoprecipitated by antibody against the insulin receptor. Phosphorylation of p90 occurred at a serine residue. The present study suggests that activation of protein kinase C and phosphorylation of p90 by it are early events leading to tumor promotion.

OBJECTIVE

The capacity to replace lost neurons after insults is retained by several regions of adult mammalian brains. However, it is unknown how many neurons actually replace and mature into region-specific functional neurons to restore lost brain function. In this paper, the authors asked whether neuronal regeneration could be achieved efficaciously by growth factor treatment using a global ischemia model in rats, and they analyzed neuronal long-term maturation processes.

METHODS

Rat global ischemia using a modified 4-vessel occlusion model was used to induce consistent ischemic neuronal injury in the dorsolateral striatum. To potentiate the proliferative response of neural progenitors, epidermal growth factor and fibroblast growth factor-2 were infused intraventricularly for 7 days from Day 2 after ischemia. Six weeks after ischemia, the number of neurons was counted in the defined dorsolateral striatum. To label the proliferating neural progenitors for tracing studies, 5-bromo-2′-deoxyuridine (BrdU; 150 mg/kg, twice a day) was injected intraperitoneally from Days 5 to 7, and immunohistochemical studies were conducted to explore the maturation of these progenitors. Migration of the progenitors was further studied by enhanced green fluorescent protein retrovirus injection. The effect of an antimitotic drug (cytosine arabinoside) on the neuronal count was also evaluated for contribution to regeneration. To see electrophysiological changes, treated rats were subjected to slice studies by whole-cell recordings. Finally, the effect of neural regeneration was assessed by motor performance by using the staircase test.

RESULTS

Following epidermal growth factor and fibroblast growth factor-2 infusion into the lateral ventricles for 7 days beginning on Day 2, when severe neuronal loss in the adult striatum was confirmed (2.3% of normal controls), a significant increase of striatal neurons was observed at 6 weeks (~ 15% of normal controls) compared with vehicle controls (~ 5% of normal controls). Immunohistochemical studies by BrdU and enhanced green fluorescent protein retrovirus injection disclosed proliferation of neural progenitors in the subventricular zone and their migration to the ischemic striatum. By BrdU tracing study, NeuN- and BrdU-positive new neurons significantly increased at 6 and 12 weeks following the treatment. These accounted for 4.6 and 11.0% of the total neurons present, respectively. Antimitotic treatment demonstrated an approximately 66% reduction in neurons at 6 weeks. Further long-term studies showed dynamic changes of site-specific maturation among various neuronal subtypes even after 6 weeks. Electrophysiological properties of these newly appeared neurons underwent changes that conform to neonatal development. These regenerative changes were accompanied by a functional improvement of overall behavioral performance.

CONCLUSIONS

Treatment by growth factors significantly contributed to regeneration of mature striatal neurons after ischemia by endogenous neural progenitors, which was accompanied by electrophysiological maturation and improved motor performance. Recognition and improved understanding of these underlying dynamic processes will contribute to the development of novel and efficient regenerative therapies for brain injuries.

OBJECTIVE

We evaluated the effects of combined PPARg agonist with bacillus Calmette-Guérin in bladder cancer growth in vitro and in vivo, focusing on the tissue remodeling mechanisms induced by bacillus Calmette-Guérin.

METHODS

PPARs are a superfamily of nuclear receptors that are transcription factors activated by ligands. Activation of PPARg, the γ subtype, causes proliferation inhibition or differentiation of tumor cells. Previously, we reported that the inhibition of murine bladder tumor growth induced by bacillus Calmette-Guérin, which is the standard treatment for patients with nonmuscle invasive, high grade bladder cancer, increased PPARg expression in vitro and in vivo. In vitro the cell growth inhibition induced by bacillus Calmette-Guérin was enhanced by the PPARg agonist 15-d-PGJ2, raising the possibility that PPARg activation may be a therapeutic modality for this disease.

RESULTS

In MB49 cells bacillus Calmette-Guérin and 15-d-PGJ2 induced PPARg expression, nuclear translocation and transcriptional activity. In vivo bacillus Calmette-Guérin reduced tumor size, an effect that was partially reversed when bacillus Calmette-Guérin was combined with the PPARg agonist rosiglitazone. The same result was found when we analyzed the effect of the PPARg antagonist BADGE (Fluka Chemical, Buchs, Switzerland) combined with bacillus Calmette-Guérin. Analysis of the activation of macrophages and fibroblasts demonstrated that rosiglitazone inhibited the tissue remodeling mechanisms induced by bacillus Calmette-Guérin.

CONCLUSIONS

Results suggest that PPARg is involved in the antitumor action of bacillus Calmette-Guérin. However, exogenous PPARg agonists would not be a favorable therapeutic modality because they can inhibit the tissue remodeling needed for an overall satisfactory bacillus Calmette-Guérin response.

There are several <em>factors</em> like angiogenesis, lymphangiogenesis, genetic alterations, mutational <em>factors</em> that are involved in malignant transformation of potentially malignant oral lesions (PMOLs) to oral squamous cell carcinoma (OSCC). <em>Fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) is one of the prototypes of the large family of <em>growth</em> <em>factors</em> that bind heparin. FGF-2 induces angiogenesis and its receptors may play a role in synthesis of collagen. FGFs are involved in transmission of signals between the epithelium and connective tissue, and influence <em>growth</em> and differentiation of a wide variety of tissue including epithelia. The present study was undertaken to analyze expression of FGF-2 and its receptors FGFR-2 and FGFR-3 in 72 PMOLs, 108 OSCC and 52 healthy controls, and their role in risk assessment for malignant transformation of Leukoplakia (LKP) and Oral submucous fibrosis (OSMF) to OSCC. Immunohistochemistry was performed using antibodies against FGF-2, FGFR-2 and FGFR-3. IHC results were validated by Real Time PCR. Expression of FGF-2, FGFR-2 and FGFR-3 was upregulated from PMOLs to OSCC. While 90% (9/10) of PMOLs which showed malignant transformation (transformed) expressed FGF-2, only 24.19% cases (<em>15</em>/62) of PMOLs which were not transformed (untransformed) to OSCC expressed FGF-2. Similarly, FGFR-2 expression was seen in 16/62 (25.81%) of untransformed PMOLs and 8/10 (80%) cases of transformed PMOLs. FGFR-3 expression was observed in 23/62 (37.10%) cases of untransformed PMOLs and 6/10 (60%) cases of transformed PMOLs. A significant association of FGF-2 and FGFR-2 expression with malignant transformation from PMOLs to OSCC was observed both at phenotypic and molecular level. The results suggest that FGF-2 and FGFR-2 may be useful as biomarkers of malignant transformation in patients with OSMF and LKP.

Urothelial papillomas and low-grade urothelial carcinomas have shown a high incidence of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) mutations and are associated with a favorable prognosis. The association of FGFR3 mutations with inverted papillomas is less known. We analyzed 20 cases of inverted papilloma in the urinary tract. Mutations of FGFR3 (exons 7, 10, and <em>15</em>) and TP53 genes were evaluated by DNA sequencing in these cases. Point mutations of the FGFR3 gene were identified in 45% (9 of 20) of inverted papillomas with four cases exhibiting mutations at multiple exons. Seven cases had exon 7 mutations containing R248C, S249T, L259L, P260P, and V266M. Two cases had exon 10 and <em>15</em> mutations including A366D, H412H, E627D, D641N, and H643D; five cases had N653H. The most frequent mutation was identified at R248C. None of the inverted papillomas exhibited mutations in TP53. During a mean follow-up of 78 months, none had recurrence or developed urothelial carcinoma. These findings support the concept that low-grade and low-stage urothelial neoplasms arise in a background of molecular changes that are distinctly different from the molecular changes of high-grade and high-stage urothelial cancers.

Efficient in vitro and in vivo angiogenesis assays, to assess and compare anti-angiogenic activity are a prerequisite for the discovery and characterization of anti-angiogenic targets. Here we describe an optimized Matrigel plug assay based on subcutaneously implanted chambers and two fast and reproducible measuring techniques. Plexiglas ring/nylon net filter-chambers (0.2 ml) containing <em>growth</em> <em>factor</em>-reduced Matrigel and 300 ng basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) were subcutaneously implanted into the right flank of rats. Chamber angiogenesis was scored on day 5 and day 10 post-implantation by computer image analysis of the chamber, and by optical density reading at 4<em>15</em> nm of a PBS solution of the chamber content. bFGF significantly induced chamber angiogenesis and histological examination confirmed that numerous blood vessels were present in the bFGF-induced chambers. The anti-angiogenic control compound TNP-470 (10 mg/kg/d s.c.) completely inhibited the bFGF-induced angiogenesis. In contrast, the anti-inflammatory or immuno-suppressive compounds cyclosporin A (<em>15</em> mg/kg/d p.o.), indomethacin (1 mg/kg/d p.o.), and prednisolone (5 mg/kg/d p.o.) showed no anti-angiogenic activity, indicating that the bFGF-induced angiogenesis was not driven by an inflammatory response or by a foreign body reaction. Finally, two candidate anti-angiogenic compounds were tested in the assay. Continuous low-dose therapy with cyclophosphamide (25 mg/kg/d p.o.) significantly inhibited bFGF-induced angiogenesis, whereas 1alpha,25-dihydroxyvitamin D3 (0.5 micro g/kg/d p.o.) showed no significant anti-angiogenic activity. In conclusion, this in vivo chamber angiogenesis assay is a useful new tool for drug evaluation as well as research into anti-angiogenesis.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 (FGFR1) is a receptor tyrosine kinase promoting tumor <em>growth</em> in a variety of cancers, including glioblastoma. Binding of FGFs triggers the intracellular Ras/Raf/ERK signaling pathway leading to cell proliferation. Down-regulation of FGFR1 and, consequently, inactivation of its signaling pathways represent novel treatment strategies for glioblastoma. In this study, we investigated the internalization and endocytic trafficking of FGFR1 in the human glioma cell line U373. Stimulation with FGF-2 induced cell rounding accompanied by increased BrdU and pERK labeling. The overexpression of FGFR1 (without FGF treatment) resulted in enhanced phosphorylated FGFR1 suggesting receptor autoactivation. Labeled ligand (FGF-2-Cy5.5) was endocytosed in a clathrin- and caveolin-dependent manner. About 25 % of vesicles carrying fluorescently tagged FGFR1 represented early endosomes, <em>15</em> % transferrin-positive recycling endosomes and 40 % Lamp1-positive late endosomal/lysosomal vesicles. Stimulation with FGF-2 increased the colocalization rate in each of these vesicle populations. The treatment with the lysosomal inhibitor leupeptin resulted in FGFR1 accumulation in lysosomes, but did not enhance receptor recycling as observed in neurons. Analysis of vesicle distributions revealed an accumulation of recycling endosomes in the perinuclear region. In conclusion, the shuttling of receptor tyrosine kinases can be directly visualized by overexpression of fluorescently tagged receptors which respond to ligand stimulation and follow the recycling and degradation pathways similarly to their endogenous counterparts.

We have defined a chemical medium to grow an anchorage and highly serum-dependent Chinese Hamster Lung <em>fibroblast</em> cell line (CCl 39) in the absence of serum. This serum-free medium referred to as DM4, contains Transferrin (5 microgram/ml), Insulin (10 microgram/ml), Epidermal <em>Growth</em> <em>Factor</em> (10 ng/ml) and Thrombin (0.05 U/ml). Thrombin appeared to be a very potent mitogen for Chinese Hamster Lung cells. DM4 allows exponential <em>growth</em> of CCl 39 cells plated in serum-coated dishes without any lag and with a generation time of <em>15</em> hours. Continuous subculturing and clonal <em>growth</em> can be obtained by passaging the cells with Ca++, Mg++ free phosphate-buffered saline in fibronectin coated dishes. The entire definition of <em>growth</em> <em>factor</em> requirements for this anchorage dependent cell line is of great value to approach the mechanisms of escape of <em>growth</em> control by these cells.

Phosphatidylinositol (PI) breakdown represents a powerful system participating in the transduction mechanism of some neurotransmitters and <em>growth</em> <em>factors</em> and producing two second messengers, diacylglycerol and inositol trisphosphate. The transformation of PC12 neuroblastoma cells into neuron-like cells induced by nerve <em>growth</em> <em>factor</em> (NGF) is preceded by a rapid stimulation of PI breakdown; however, it was not known whether PI breakdown mediates actions of other members of the neurotrophin family. The present study analyzed the effects of NGF, brain-derived neurotrophic <em>factor</em> (BDNF), and neurotrophin-3 (NT-3) on PI breakdown in primary cultures of embryonic rat brain cells. Cultures were grown for 7 days; PI was then labeled by incubating cultures with myo-[3H]inositol, which then were exposed acutely to <em>growth</em> <em>factors</em>. BDNF and NT-3, but not NGF, elevated the levels of labeled inositol phosphates within 10-<em>15</em> min after addition to the cultures in a dose-dependent manner. ED50 values for BDNF and NT-3 were 12.4 and 64.5 ng/ml, respectively. Comparable effects were found in cultures of cortical, striatal, and septal cells. The actions of BDNF and NT-3 probably reflect actions on neurons, because no effects were seen in cultures of nonneuronal cells. In contrast, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> induced a marked stimulation of PI breakdown in cultures of nonneuronal cells. K252b, which selectively blocks neurotrophin actions by inhibiting trk-type receptor proteins, prevented the PI breakdown mediated by BDNF and NT-3. The findings suggest that rapid and specific induction of PI breakdown is involved in the signal transduction of BDNF and NT-3, and they provide evidence that cortical neurons are functionally responsive to BDNF and NT-3 during development.

The aim of this study was to show the temporal and spatial molecular responses in the rat stomach that follow absolute ethanol-induced acute mucosal injury. Intense signals for immediate early genes (IEG)/transcriptional <em>factors</em> such as c-fos, c-jun, and nerve <em>growth</em> <em>factor</em>-induced gene-A (NGFI-A) mRNAs were observed in the superficial mucosa and in the blood vessels from <em>15</em> min to 6 hr after administration, peaking at <em>15</em>-30 min. Signals for heat shock protein (HSP) 70 mRNA were also detected in the superficial mucosa, in the <em>fibroblasts</em> around gastric erosions, and in the blood vessels from <em>15</em> min to 6 hr (peak at 1-2 hr). The signals for cyclooxygenase-2 (COX-2) mRNA were up-regulated in the surface mucous cells that surround the erosions from 30 min to 6 hr (peak at 60-90 min). These findings suggest that IEG, HSP70, and COX-2 are involved in gastric mucosal restitution in different ways.

The majority of patients eligible for periodontal regenerative therapies are aged subjects. Since periodontal ligament cells (PDLC) are essential for periodontal regeneration, the aim of the present study was to determine the effect of cellular aging on PDLC, including genes associated with extracellular matrix metabolism and <em>growth</em>-associated <em>factors</em>. PDLC cultures were obtained from subjects aged <em>15</em> to 20 years and subjects aged more than 60 years. Proliferation, cell viability, mineralization assays, and mRNA levels were assessed for type I and III collagen, platelet-derived <em>growth</em> <em>factor</em> (PDGF)-1, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), metalloproteinase (MMP)-2 and-8, and tissue inhibitor of metalloproteinases (TIMP)-1 and-2. Data analysis demonstrated that aging negatively influenced cell proliferation and mineral nodule formation (p < 0.05). Gene expression analysis further showed that mRNA levels for bFGF, PDGF-1, and TIMP-2 were not affected by aging (p>> 0.05). In addition, mRNA levels for type I and III collagen were significantly lower in aged cells (p < 0.05), whereas MMP-2 and-8 and TIMP-1 mRNA levels were higher (p < 0.05). Within the limits of the present study, data analysis suggests that aging modulates important biological properties of periodontal ligament cells, diminishes the potential for mineral nodule formation, and favors extracellular matrix degradation.