OBJECTIVE

The goals of this study were to evaluate the cognitive outcome after surgery for synostotic frontal plagiocephaly and to compare the effects of early (< 1 year of age) and late >> or = 1 year of age) surgical correction on intellectual development.

METHODS

In a prospective study involving 220 patients, both preoperative and postoperative cognitive function were measured using developmental quotient (DQ) or intelligence quotient (IQ) determination. Preoperative intracranial pressure (ICP) was also measured, and the presence of the fibroblast growth factor receptor 3 (FGFR3) P250R mutation was assessed.

CONCLUSIONS

Preoperative DQ/IQ was not affected by patient age at the time of surgery. There was no statistically significant difference in mean postoperative IQ between patients who underwent surgery before 1 year of age and those who underwent surgery at 1 year of age or older. In the latter group, there was a statistically significant increase in the incidence of elevated ICP >> or = 15 mm Hg in 16%), but no effect on cognitive outcome was observed. Likewise, there was no statistically significant correlation between the presence of the FGFR3 mutation and postoperative IQ. The indication for surgical correction appears to be largely cosmetic.

The cytokine transforming <em>growth</em> <em>factor</em>-beta has multiple effects on a wide variety of cell types. These effects include modulation of <em>growth</em> and regulation of gene transcription. In the present work, we demonstrate that TGF-beta1 increases transcription of the latent transforming <em>growth</em> <em>factor</em>-beta binding protein-2 ( LTBP-2) gene in cultured human fetal lung <em>fibroblasts</em> leading to a significant increase in LTBP-2 mRNA steady state level. The stability of LTBP-2 mRNA was not appreciably altered. A corresponding increase in production of LTBP-2 protein accompanied the increase in mRNA. Through the use of specific inhibitors, we demonstrate that a member of the Ras super family and a protein kinase C, probably of the atypical (non-diacylglycerol, non-Ca++ dependent) class are likely to be components in the signaling pathway. However, phospholipases, G proteins and extracellular-signal regulated kinases do not appear to be involved. These results combined with previous findings on elastin regulation by TGF-beta1 (Kucich et al. (1997). Am. J. Respir. Cell Mol. Biol., 17: 10-<em>16</em>) demonstrate that TGF-beta1 can coordinately increase the steady state levels of mRNAs encoding components of the elastic fiber, but through diverse mechanisms. In contrast to LTBP-2, increased elastin expression is achieved by message stabilization. Furthermore, the TGF-beta1 signaling pathways differ and while the pathway leading to increased LTBP-2 transcription shares components with those modulating transcription of other genes, it is unlikely to be precisely congruent with any other previously described one.

OBJECTIVE

The aim of this study was to examine the effect of basic fibroblast growth factor (FGF-2) on osseointegration of dental implants with low primary stability in a beagle dog model.

METHODS

Customized titanium implants that were designed to have low contact with the existing bone were installed into the edentulous mandible of beagle dogs. To degrade the primary stability of the implants, the diameters of the bone sockets exceeded the implant diameters. FGF-2 (0.3%) plus vehicle (hydroxypropyl cellulose) or vehicle alone was topically applied to the sockets in the FGF-2 and control groups, respectively. In Study 1, the new bone area and length of new bone-to-implant contact (BIC) were evaluated at 4, 8, and 12 weeks after installation using histomorphometry and scanning electron microscopy. In Study 2, the implant stability quotient (ISQ) values were sequentially measured for 16 weeks using an Osstell system.

RESULTS

The histomorphometric analysis revealed that the new bone area and length of BIC in the FGF-2 group were significantly larger than those in the control group at 4 weeks. Electron microscopic observation showed intimate contact between the mature lamellar bone and the implant surfaces, osseointegration, in both groups. The ISQ values in the FGF-2 group were significantly increased from 6 to 16 weeks compared with those in the control group.

CONCLUSIONS

Taken together, our study demonstrates that FGF-2 promoted new bone formation around the dental implants and subsequent osseointegration, resulting in promotion of stability of implants with low primary stability.

The role of circulating <em>growth</em> <em>factors</em> in the pathogenesis of childhood HIV-1-associated nephropathy (HIVAN) is not clearly understood. In previous studies, we found a significant accumulation of <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) in the circulation and kidneys of children with HIVAN. The purpose of this study was to determine whether circulating FGF-2 may play a role in the pathogenesis of HIVAN by increasing the renal recruitment and attachment of HIV-infected mononuclear cells to renal epithelial cells. Using in vitro cell adhesion assays, we showed that FGF-2 increased the attachment of peripheral blood mononuclear cells (PBMCs) to fibronectin-coated tissue culture dishes by approximately threefold through a mechanism that involved the alpha5 integrin subunit. In addition, we found that FGF-2 induces a similar increase in the attachment of HIV-infected PBMCs and monocytes/macrophages to plastic tissue culture dishes and to monolayers of primary renal tubular epithelial cells harvested from the urine of HIV-infected children with renal disease. Finally, we injected <em>16</em> adult C57Bl6/J male mice with recombinant adenoviral vectors carrying either the LacZ gene or a secreted form of human FGF-2 (5 x 10(8)pfu/mouse) and demonstrated that high levels of circulating FGF-2 can increase the renal recruitment of circulating inflammatory cells and induce transient tubulointerstitial injury in vivo. These data suggest that FGF-2 may have an immunomodulatory role in the pathogenesis of HIVAN by recruiting HIV-infected cells in the kidney.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) is a mitogen found in CUG-initiated 21-25 kDa ("hi") or AUG-initiated <em>16</em>-18 kDa ("lo") forms. Previously we demonstrated that "hi"-but not "lo"-FGF-2 caused a distinct nuclear phenotype characterized by apparently condensed chromatin present as separate clumps in the nucleus of cardiac myocytes. In this manuscript we investigated whether these effects were related to apoptosis or mitosis and whether they reflected a direct effect of "hi" FGF-2 on chromatin. Myocytes overexpressing "hi" FGF-2 and presenting the clumped chromatin phenotype: (i) were not labeled above background with antibodies to phosphorylated histones H1 and H3 used as indicators of mitotic chromatin condensation; (ii) did not stain positive for TUNEL; (iii) their nuclear lamina, visualized by anti-laminB immunofluorescence, appeared intact; (iv) neither caspase inhibitors, nor Bcl-2 or "lo" FGF-2 overexpression prevented the manifestation of the compacted nuclear phenotype. Purified recombinant "hi" FGF-2 was more potent than "lo" FGF-2 in promoting the condensation/aggregation of chick erythrocyte chromatin partially reconstituted with histone H1 in vitro. We conclude that the DNA phenotype induced by "hi" FGF-2 in cardiac myocytes likely reflects a direct effect on chromatin structure that does not require the engagement of mitosis or apoptosis. By affecting chromatin compaction "hi" FGF-2 may contribute to the regulation of gene expression.

Twenty years ago, surgeons noted the ability of early-gestation fetal skin to heal in a scarless manner. Since that time, numerous investigators have attempted to elucidate the mechanisms behind this phenomenon. As a result of this effort, it is now well established that many animals undergo a transition late in development from scarless cutaneous healing to a scar-forming, adultlike phenotype. The authors have been interested in the role played by cytokines known to be involved in the adult wound-healing process and how they relate to scarless repair. They therefore asked the following question: Are genes for epidermal <em>growth</em> <em>factor</em> (EGF) and platelet-derived <em>growth</em> <em>factor</em>-B (PDGF-B) expressed differentially as a function of gestational age in fetal rat skin and dermal <em>fibroblasts</em>? To answer this question, skin from fetal Sprague-Dawley rats (N = 56) at time points that represented both the scarless and scar-forming periods of rat gestation was harvested. In addition, <em>fibroblasts</em> derived from fetal rat skin were cultured in vitro at similar times. These cells were expanded in culture and, when confluent, total ribonucleic acid from both <em>fibroblasts</em> and whole skin was extracted and subjected to Northern blot analysis with probes for EGF and PDGF-B. Results demonstrated that neither EGF nor PDGF-B gene expression changed markedly as a function of gestational age in fetal <em>fibroblasts</em> alone. In whole skin, however, both EGF and PDGF-B demonstrated a marked decrease in gene expression with increasing gestational age. Furthermore, the most striking decrease in gene expression for both cytokines came between <em>16</em> and 18 days of gestation-the transition point between scarless and scar-forming repair in the fetal rat. These data suggest that EGF and PDGF may play a role in the mechanism of scarless cutaneous repair. Moreover, it appears that fetal <em>fibroblasts</em> are not the cell type responsible for this differential gene expression. These results raise questions about the unique cytokine milieu likely to be present during the time of scarless healing and the cells that ultimately guide the mechanisms leading to skin regeneration.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is a bone cell mitogen that affects osteoblastic function by suppressing type I collagen synthesis. The investigators in this study examined whether bFGF also regulates interstitial collagenase and tissue inhibitors of metalloproteinases (TIMPs) in osteoblast-enriched cells isolated from 22-day fetal rat calvariae. After exposure to 600 pM bFGF, interstitial collagenase messenger RNA (mRNA) levels, as determined by Northern hybridization analysis, increased after 2 h and were maximally stimulated to approximately 13-fold at 6 h. Exposure of osteoblast-enriched cells to 0.06-6 nM bFGF increased collagenase mRNA in a dose-dependent manner, and bFGF also increased immunoreactive collagenase measured in the culture medium by Western blot analysis. The protein synthesis inhibitor cycloheximide, as well as two inhibitors of protein kinase C, staurosporine and sangivamycin, prevented the bFGF induction of collagenase transcripts, whereas indomethacin, an inhibitor of prostaglandin synthesis, decreased the effect of bFGF on collagenase mRNA levels by about 50%. After exposure to 600 pM bFGF, levels of TIMP 1 and TIMP 3 mRNAs were also maximally stimulated to approximately 6-fold at <em>16</em> h and 4-fold at 6 h. bFGF did not modify TIMP 2 expression. In conclusion, bFGF may modulate degradation of collagenous bone matrix by inhibiting collagen as well as stimulating collagenase and TIMPs by osteoblasts.

Patients frequently experience a loss of salivary function following irradiation (IR) for the treatment of an oral cavity and oropharyngeal cancer. Herein, we tested if transfer of <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF2) cDNA could limit salivary dysfunction after fractionated IR (7.5 or 9 Gy for 5 consecutive days to one parotid gland) in the miniature pig (minipig). Parotid salivary flow rates steadily decreased by <em>16</em> weeks post-IR, whereas blood flow in the targeted parotid gland began to decrease ~3 days after beginning IR. By 2 weeks, post-IR salivary blood flow was reduced by 50%, at which point it remained stable for the remainder of the study. The single preadministration of a hybrid serotype 5 adenoviral vector encoding FGF2 (AdLTR2EF1a-FGF2) resulted in the protection of parotid microvascular endothelial cells from IR damage and significantly limited the decline of parotid salivary flow. Our results suggest that a local treatment directed at protecting salivary gland endothelial cells may be beneficial for patients undergoing IR for oral cavity and oropharyngeal cancer.

Essential <em>factors</em> required for <em>growing</em> oocytes derived from bovine early antral follicles and their mechanisms of action are poorly understood. <em>Fibroblast</em> <em>growth</em> <em>factor</em> 7 (FGF7) is a member of the heparin-binding FGF family with a distinctive pattern of target-cell specificity. The effect of FGF7 on the stimulation of oocyte <em>growth</em> in a culture of cumulus-oocyte complexes with granulosa cells (COCGs, oocyte diameter; 90-100 microm) was investigated. The oocyte diameter of COCGs was increased significantly in the FGF7-containing medium (10 ng/ml; 117.2 +/- 3.2 microm, 50 ng/ml; 1<em>16</em>.5 +/- 3.5 microm) compared to the control (0 ng/ml; 110.5 +/- 2.8 microm) after <em>16</em> days. However, there was no stimulatory effect of FGF7 on the proliferation of cumulus-granulosa cells. The FGF7 receptor, <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2IIIb (FGFR2IIIb), was detected in cumulus-granulosa cells from COCGs. Messenger RNA expression of FGFR2IIIb was induced to cumulus-granulosa cells by FGF7. The mRNA expression levels of KIT ligand (KITLG), KIT (KIT), <em>growth</em> differentiation <em>factor</em> 9 (GDF9), and bone morphogenetic protein 15 (BMP15) in the cultured COCGs were determined in FGF7-treated (10 ng/ml) cultures using real time RT-PCR analysis. The levels of KITLG and KIT, but not GDF9 and BMP15 mRNA expression were stimulated by FGF7. Furthermore, neutralizing antibody for KIT attenuated the stimulatory action of FGF7 on the oocyte <em>growth</em>. These results strongly suggest that FGF7 may be an important regulator for oocyte <em>growth</em> and its action is mediated via the KIT/KITLG signaling pathway.

BACKGROUND

Angiogenesis is strongly influenced by vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF), whose production is also regulated by interferon (IFN)-gamma and interleukin (IL)-10. The aim of this study was to evaluate the modifications of serum VEGF, b-FGF, IFN-gamma and IL-10 levels in patients with inguinal hernia undergoing hernioplasty with the Lichtenstein technique (LH) using polypropylene mesh or with Bassini open conventional inguinal hernia repair (BH).

METHODS

Randomly, 16 patients underwent BH, and 16 were treated with the LH technique using polypropylene mesh. Blood samples were collected 24 h prior to surgery and then 6, 24, 48 and 168 h postoperatively. The serum concentrations of VEGF, b-FGF, IFN-gamma and IL-10 were evaluated.

RESULTS

In BH patients, a peak of VEGF synthesis at 6 h with a normalization of this parameter 24 h after surgery has been observed. In the same subjects, b-FGF synthesis increased after surgery reaching significant levels 48 h later. On the contrary, in LH patients, a decrease in the serum VEGF and b-FGF concentrations was detected after surgery and their increase afterwards. IL-10 was increased in both groups 6 h after operation and declined to preoperative levels 24 h afterwards. IFN-gamma enhanced in LH patients 6 h after surgery, whereas no modifications were detected in BH subjects.

CONCLUSIONS

This preliminary study shows that VEGF and b-FGF modifications, associated with alterations of cytokine secretion, are detectable in human undergoing hernioplasty, and suggests that they could somehow influence in the wound-healing process.

High-affinity tyrosine kinase A (trkA) neurotrophin receptors on neurons and nonneuronal cells elicit differentiation or survival functions in response to nerve <em>growth</em> <em>factor</em> (NGF), whereas the low-affinity neurotrophin (p75) receptor modulates trkA activity or can independently cause apoptosis or NFkappaB-mediated survival functions. We examined dental tissues for the presence of trkA-like immunoreactivity (trkA-IR), to determine which nonneuronal cell types express it in normal compared with inflamed teeth and how the trkA-positive cells relate to those expressing the p75 receptor and/or NGF. Normal and injured rat molars (dentin cavity for 4 h, <em>16</em>-24 h, 3 days, <em>16</em> days, or 5 weeks) were immunoreacted using the ABC detection system for two anti-trkA antibodies (sTA, Santa Cruz Biotechnology; rTA, L. Reichardt) and antibodies against p75 and NGF, all of which also stained pulpal nerve fibers. We report that, when using the sTA antibody (recognizing the intracellular carboxy terminal), nonneuronal trkA-IR was found in odontoblasts of normal teeth and also in invading polymorphonuclear leukocytes (PMNs) and reparative odontoblasts after injury. When using rTA (recognizing the extracellular domain of the receptor), nonneuronal trkA-IR was only found in odontoblasts. Odontoblasts also had NGF-IR but did not label for NGF mRNA. The lack of odontoblast NGF mRNA suggests that NGF is passed from <em>fibroblasts</em> to the adjacent odontoblasts, where it is picked up by receptor-mediated mechanisms for regulation of odontoblast function. Tooth injury disrupts this system such that trkA-IR decreases in injured odontoblasts, p75 decreases in <em>fibroblasts</em>, and NGF is upregulated by <em>fibroblasts</em> and accumulates in the injured pulp and surviving odontoblasts. Pulpal NGF may contribute to chemoattraction for the invading leukocytes or their sTA-IR may have been induced in response to pulpal NGF. Thus, tooth pulp has a different distribution of nonneuronal NGF and its paracrine receptors during inflammation compared with normal conditions.

OBJECTIVE

To investigate intraperitoneal transplantation of microencapsulated hepatic-like cells from human umbilical cord blood for treatment of hepatic failure in rats.

METHODS

CD34+ cells in umbilical cord blood cells were isolated by magnetic cell sorting. In the in vitro experiment, sorted CD34+ cells were amplified and induced into hepatic-like cells by culturing with a combination of fibroblast growth factor 4 and hepatocyte growth factor. Cultures without growth factor addition served as controls. mRNA and protein levels for hepatic-like cells were analyzed by reverse transcription-polymerase chain reaction, immunohistochemistry and immunofluorescence. In the in vivo experiment, the hepatic-like cells were encapsulated and transplanted into the abdominal cavity of acute hepatic failure (AHF) rats at 48 h after D-galactosamine induction of acute hepatic failure. Transplantation with PBS and unencapsulated hepatic-like cells served as controls. The mortality rate, hepatic pathological changes and serum biochemical indexes were determined. The morphology and structure of microcapsules in the greater omentum were observed.

RESULTS

Human albumin, alpha-fetoprotein and GATA-4 mRNA and albumin protein positive cells were found among cultured cells after 16 d. Albumin level in culture medium was significantly increased after culturing with growth factors in comparison with culturing without growth factor addition (P < 0.01). Compared with the unencapsulated group, the mortality rate of the encapsulated hepatic-like cell-transplanted group was significantly lower (P < 0.05). Serum biochemical parameters, alanine aminotransferase, aspartate aminotransferase and total bilirubin in the encapsulated group were significantly improvement compared with the PBS control group (P < 0.01). Pathological staining further supported these findings. At 1-2 wk post-transplantation, free microcapsules with a round clear structure and a smooth surface were observed in peritoneal lavage fluid, surviving cells inside microcapsules were found by trypan blue staining, but some fibrous tissue around microcapsules was also detected in the greater omentum of encapsulated group by hematoxylin and eosin staining.

CONCLUSIONS

Transplantation of microencapsulated hepatic-like cells derived from umbilical cord blood cells could preliminarily alleviate the symptoms of AHF rats.

BACKGROUND

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is the most common orofacial birth defect with a wide range prevalence among different populations. Previous association studies with populations from Europe and Asia have identified putative susceptibility markers for NSCL/P in fibroblast growth factor 12 (FGF12), vinculin (VCL), connexin 43 (CX43) and in a region close to the ventral anterior homeobox 1 (VAX1) gene. However, there have thus far been no studies of these markers in NSCL/P Brazilian patients, and as the genetic ancestry of the Brazilian population is highly varied, the predisposition to those disease markers can be different.

METHODS

Herein we conducted a structured association study conditioned on the individual ancestry proportions to determine the role of 16 polymorphic markers within those genes in 300 patients with NSCL/P and 385 unaffected controls.

RESULTS

None of the alleles and genotypes showed association with NSCL/P, though there was a significant association of the haplotype formed by VAX1 rs10787760, rs6585429 and rs1871345 polymorphisms with NSCL/P that did not persist Bonferroni correction for multiple tests.

CONCLUSIONS

Our results are consistent with a lack of involvement of FGF12, VCL and CX43 variants with NSCL/P pathogenesis in Brazilian patients. Furthermore, the higher frequency of a haplotype of VAX1 with NSCL/P patients suggests a low penetrant gene for oral cleft, and warrants further studies.

We investigated long-term effects of low carbohydrate diets on wild type mice, streptozotocin-injected and KKAy obese diabetic mice. These mice were pair-fed three different types of diets, standard chow (SC, C∶P∶F = 63∶15∶22), a low carbohydrate (LC, C∶P∶F = 38∶25∶37) diet and a severely carbohydrate restricted (SR, C∶P∶F = 18∶45∶37) diet for <em>16</em> weeks. Despite comparable body weights and serum lipid profiles, wild type and diabetic mice fed the low carbohydrate diets exhibited lower insulin sensitivity and this reduction was dependent on the amount of carbohydrate in the diet. When serum fatty acid compositions were investigated, monounsaturation capacity, i.e. C<em>16</em>:1/C<em>16</em>:0 and C18:1/C18:0, was impaired in all murine models fed the low carbohydrate diets, consistent with the decreased expression of hepatic stearoyl-CoA desaturase-1 (SCD1). Interestingly, both the hepatic expressions and serum levels of <em>fibroblast</em> <em>growth</em> <em>factor</em> 21 (FGF21), which might be related to longevity, were markedly decreased in both wild type and KKAy mice fed the SR diet. Taking into consideration that fat compositions did not differ between the LC and SR diets, we conclude that low carbohydrate diets have deleterious metabolic effects in both wild type and diabetic mice, which may explain the association between diets relatively low in carbohydrate and the elevated risk of cardiovascular events observed in clinical studies.

The influences of specific <em>growth</em> <em>factors</em> upon binding, internalization and degradation of low density lipoproteins (LDL) were investigated in cultured vascular smooth muscle cells and skin <em>fibroblasts</em>. Platelet-derived <em>growth</em> <em>factor</em> (PDGF) and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) significantly stimulated the binding of LDL to high affinity receptors of smooth muscle cells and <em>fibroblasts</em>. The effects of enhanced binding were reflected in elevated rates in internalization and degradation of LDL. FGF and PDGF elicited a mitogenic response as measured by [3H]thymidine autoradiography, indicating that altered LDL metabolism was associated with entry into the cell cycle. When <em>fibroblasts</em> were exposed to mitogen for periods long enough to commit the cells to the <em>growth</em> cycle, after which <em>growth</em> <em>factor</em> was removed before addition of LDL however, enhanced LDL binding to cycling <em>fibroblasts</em> appeared to be dependent upon the length of the period of exposure to <em>growth</em> <em>factors</em> in the early part of the cell cycle. LDL binding was stimulated in the presence of PDGF or FGF but not after their removal within 8 h of entry into the cell cycle. Exposure to the <em>growth</em> <em>factors</em> for <em>16</em> h or longer resulted in stimulation of LDL metabolism whether or not mitogens were present at the cell surface. PDGF and FGF, therefore, appear to exert a direct influence upon LDL receptor expression in addition to that mediated via the cell cycle.

Imatinib mesylate is a specific tyrosine kinase inhibitor that may block the platelet-derived <em>growth</em> <em>factor</em> and transforming <em>growth</em> <em>factor</em> pathways. These pathways are known to provoke <em>fibroblast</em> activation. We evaluated whether imatinib, by inhibiting these pathways, prevents diastolic dysfunction and attenuates myocardial remodeling using spontaneously hypertensive rats (SHRs). Eight-week-old male SHRs were randomly assigned to either imatinib treatment group (30 mg/kg per day; n=10; SHR-I) or hypertensive control group (distilled water, n=10; SHR-C). Wistar-Kyoto rats were used as normal controls (n=10). At <em>16</em> weeks, all rats underwent hemodynamic studies and Doppler echocardiography and then were euthanized. Their hearts were extracted for histopathologic, immunoblotting, and quantitative reverse transcriptase polymerase chain reaction analyses. Although imatinib did not affect blood pressure, it markedly reduced perivascular and interstitial fibrosis in the hearts of SHR. Echocardiogram showed that imatinib significantly reduced the left ventricular wall thickness (septal/posterior wall; SHR-C versus SHR-I, 18±1/19±2 versus 15±1/15±1 mm; P<0.001) and increased the E/A ratio (SHR-C versus SHR-I, 1.59±0.11 versus 1.84±0.<em>16</em>; P=0.001). Also, imatinib significantly reduced the mRNA expression of collagen type I, III, and platelet-derived <em>growth</em> <em>factor</em> receptor-β phosphorylation in the hearts of SHR. In addition, imatinib reduced collagen production by inhibiting the phosphorylation of c-abl and platelet-derived <em>growth</em> <em>factor</em> receptor-β in rat cardiac <em>fibroblasts</em>. In conclusion, these results suggest that imatinib could attenuate myocardial remodeling and improve left ventricular diastolic dysfunction in a hypertensive rat model by affecting platelet-derived <em>growth</em> <em>factor</em> and transforming <em>growth</em> <em>factor</em>-β1 pathway without the blood pressure-lowering effect.

Although <em>growth</em> hormone (GH) receptor (GHR) mRNA and protein are present in fetal tissues such as the lung, there is little evidence that GH mediates <em>growth</em> in the fetus. We have identified functional responses to GH in fetal rat lung epithelia and suggest a possible role for GHR in the developing lung. GHR mRNA in lung extracts was high before birth at day <em>16</em> of gestation (<em>16</em>f), decreased to low levels at day 22f but increased again after birth. At day 20f GHR mRNA levels were higher in lung than in liver, whereas <em>growth</em> hormone binding protein mRNA levels were approximately equal in lung and liver. Stimulation of primary cell cultures of day 19f lung epithelia with GH caused increased tyrosine phosphorylation in specific proteins, demonstrating functional GHR. Lung <em>fibroblasts</em> isolated at the same time did not respond to GH. Ligand and Northern blot analysis of the epithelial cultures revealed that GH stimulation increased insulin-like <em>growth</em> <em>factor</em> binding protein-2 (IGFBP-2) activity and mRNA. These experiments demonstrate the functional activity of GHR, specifically in fetal lung epithelium. We suggest that one role for GH in vivo may be indirectly to modify insulin-like <em>growth</em> <em>factor</em> activity in the developing fetal lung by increasing IGFBP-2.

Prostatic cancer typically produces osteoblastic metastases which are not attended by marrow fibrosis (i.e., osteoblast but not stromal <em>fibroblast</em> proliferation). In the present study we sought to test the hypothesis that prostatic cancer cells produce <em>factor</em>(s) which act selectively on human osteoblasts. Such a paracrine mechanism would explain the observed increase in osteoblasts, unaccompanied by an increase in marrow <em>fibroblasts</em>. To test this hypothesis we investigated the mitogenic activity released by the human prostatic tumor cell line, PC3. PC3 cells have been reported previously to produce mitogenic activity for cells that was relatively specific for rat osteoblasts compared to rat <em>fibroblasts</em>. However, the effects of this activity on human cells has not been examined previously. PC3-conditioned medium (CM) (5-50 micrograms CM protein/ml) stimulated human osteoblast proliferation by 200-950% yet did not stimulate human <em>fibroblast</em> proliferation [( 3H]thymidine incorporation). PC3 CM also increased cell numbers in human osteoblast but not <em>fibroblast</em> cell cultures. To determine whether the osteoblast-specific mitogenic activity could be attributed to known bone <em>growth</em> <em>factors</em>, specific assays for these <em>growth</em> <em>factors</em> were performed. PC3 CM contained 10 pg insulin-like <em>growth</em> <em>factor</em> (IGF) I, less than 2 pg IGF II, 54 pg basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and <em>16</em> pg transforming <em>growth</em> <em>factor</em> beta/microgram CM protein. None of these <em>growth</em> <em>factors</em> alone or in combination could account for the observed osteoblast-specific PC3 cell-derived mitogenic activity. Furthermore, when 5 micrograms/ml PC3 CM was tested in combination with maximally effective concentrations of either basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, IGF I, IGF II, or transforming <em>growth</em> <em>factor</em> beta, it produced an additive effect suggesting that PC3 CM stimulates osteoblast proliferation by a mechanism independent of these bone mitogens. Biochemical characterization supported the hypothesis that the PC3 cell <em>growth</em> <em>factor</em> was unique from other <em>growth</em> <em>factors</em>. The PC3 <em>growth</em> <em>factor</em> did not bind to heparin and was resistant to acid as well as the reducing agent, dithiothreitol. Sephadex G-75 and fast protein liquid chromatography Mono S cation-exchange chromatography revealed the PC3-derived mitogen to be an Mr 26,000-30,000 basic protein. Therefore, we conclude that PC3 cells release a mitogen which exhibits higher specificity for human osteoblasts than human <em>fibroblasts</em> and is unique from other <em>growth</em> <em>factors</em> tested. Production of this mitogen by human prostatic carcinoma cells could play an etiological role in the intense osteoblast-specific stimulation that occurs at sites of bone metastases.

The extracellular adherence protein (Eap), a broad-spectrum adhesin secreted by Staphylococcus aureus, was previously shown to curb acute inflammatory responses, presumably through its binding to endothelial cell (EC) ICAM-1. Examining the effect of Eap on endothelial function in more detail, we here show that, in addition, Eap functions as a potent angiostatic agent. Concomitant treatment of EC with purified Eap resulted in the complete blockage of the mitogenic and sprouting responses elicited by vascular endothelial <em>growth</em> <em>factor</em> (VEGF)<em>16</em>5 or basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). Moreover, the induction of tissue <em>factor</em> and decay-accelerating <em>factor</em> were repressed by Eap, as determined by qRT-polymerase chain reaction (qRT-PCR), with a corresponding reduction in Egr-1 protein up-regulation seen. This angiostatic activity was accompanied by a corresponding inhibition in ERK1/2 phosphorylation, while activation of p38 was not affected. Inhibition occurred downstream of tyrosine kinase receptor activation, as comparable effects were seen on TPA-induced ERK1/2 phosphorylation. Similar to previously described angiostatic agents like angiopoietin-1 or the <em>16</em>-kDa prolactin fragment, Eap blockage of the Ras/Raf/MEK/ERK cascade was localized by pull-down assay at the level of Ras activation. Eap's combined anti-inflammatory and antiangiogenic properties render this bacterial protein not only an important virulence <em>factor</em> during S. aureus infection but open new perspectives for therapeutic applications in pathological neovascularization.

Mesangial cells and RAW 264.7 macrophages respond to different nitric oxide (NO) donors within <em>16</em> to 24 h or 6 to 8 h, respectively, with apoptotic cell death. RAW 264.7 macrophages also die in response to endogenous NO production. In contrast, endogenous NO production fails to significantly induce cell death in mesangial cells. It was hypothesized that differences in the expression of antiapoptotic proteins, in particular the inhibitor of apoptosis (IAP) protein family, might be responsible for this cell type-specific behavior. Therefore, IAP expression was investigated in relation to apoptosis induction in response to NO and cytokines in both cell types. In mesangial cells, interleukin-1beta (IL-1beta) and tumor necrosis <em>factor</em>-alpha induced cellular inhibitor of apoptosis 1 (cIAP1) mRNA expression within 3 h. In contrast, X chromosome-linked inhibitor of apoptosis (XIAP) mRNA levels remained unaffected by cytokines. Although coincubation of cells with IL-1beta and tumor necrosis <em>factor</em>-alpha or IL-1beta and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> resulted in synergistic induction of inducible NO synthase, comparable potentiating effects on cIAP1 induction were absent. Exogenously released NO from NO donors promoted cIAP1 mRNA upregulation in mesangial cells, whereas XIAP mRNA was downregulated. However, the changes observed on the mRNA level were not adequately translated to the protein level, and corresponding values for cIAP1 and XIAP were only slightly affected. In contrast, in lipopolysaccharide/interferon-gamma-stimulated RAW 264.7 macrophages, massive NO-dependent downregulation of cIAP1 and XIAP protein levels, which correlated temporally with the induction of apoptosis, was observed. This effect was at least partially reversed by N(G)-monomethyl-L-arginine, an inhibitor of NO synthase activity. In summary, a direct correlation between the downregulation of IAP protein levels and the induction of apoptosis by endogenous NO was observed in macrophages. In contrast, a stable level of IAP protein in mesangial cells might represent a mechanism for the resistance of the cells to endogenously produced NO.

The binding of 125I-labelled epidermal <em>growth</em> <em>factor</em> (EGF) was utilized to monitor possible cell surface effects of polycyclic aromatic hydrocarbon carcinogens. Exposure of confluent C3H 10T1/2 mouse <em>fibroblasts</em> to 1 muM benzo[a]pyrene led to a time-dependent decrease of EGF binding. By 24 h, EGF binding was only 5% that of control cultures. In contrast, benzo[a]pyrene-7,8-diol-9,10-oxide did not significantly alter EGF binding, indicating that the inhibition by benzo[a]pyrene was not simply due to DNA damage. A curvilinear Scatchard plot in the control cells was consistent with the presence of two classes of EGF receptors having differing affinities. Our results suggest that the major effect of benzo[a]pyrene was a reduction in receptor number rather than affinity, although other interpretations have not been excluded. Progesterone, 17 beta-estradiol, benzo[e]pyrene, cholesterol, phenobarbital, 1,1-bis-(p-chlorophenyl)-2,2,2-trichloroethane, hexachlorobenzene or pregnenolone-<em>16</em> alpha-carbonitrile, did not inhibit EGF binding. On the other hand, several known inducers of P1-450 were very effective inhibitors of EGF binding. These included: dimethylbenz[a]anthracene, 3-methylcholanthrene, benzo[a]pyrene, benz[a]anthracene, beta-naphthoflavone and alpha-naphthoflavone. We postulate that the binding of certain polycyclic aromatic hydrocarbons to the Ah receptor may induce not only specific drug metabolizing enzymes but also inhibition of EGF binding, and possible other cell effects. Further studies are required to verify this hypothesis.

In a fraction of patients surgically treated for cleft lip/palate, excessive scarring disturbs maxillary <em>growth</em> and dento-alveolar development. Since certain genes are involved in craniofacial morphogenesis as well as tissue repair, a primary defect causing cleft lip/palate could lead to altered wound healing. We performed in vitro wound healing assays with primary lip <em>fibroblasts</em> from <em>16</em> cleft lip/palate patients. Nine foreskin <em>fibroblast</em> strains were included for comparison. Cells were grown to confluency and scratch wounds were applied; wound closure was monitored morphometrically over time. Wound closure rate showed highly significant differences between <em>fibroblast</em> strains. Statistically, <em>fibroblast</em> strains from the 25 individuals could be divided into three migratory groups, namely "fast", "intermediate", and "slow". Most cleft lip/palate <em>fibroblasts</em> were distributed between the "fast" (5 strains) and the "intermediate" group (10 strains). These phenotypes were stable over different cell passages from the same individual. Expression of genes involved in cleft lip/palate and wound repair was determined by quantitative PCR. Transforming <em>growth</em> <em>factor</em>-α mRNA was significantly up-regulated in the "fast" group. 5 ng/ml transforming <em>growth</em> <em>factor</em>-α added to the culture medium increased the wound closure rate of cleft lip/palate strains from the "intermediate" migratory group to the level of the "fast", but had no effect on the latter group. Conversely, antibody to transforming <em>growth</em> <em>factor</em>-α or a specific inhibitor of its receptor most effectively reduced the wound closure rate of "fast" cleft lip/palate strains. Thus, <em>fibroblasts</em> from a distinct subgroup of cleft lip/palate patients exhibit an increased migration rate into wounds in vitro, which is linked to higher transforming <em>growth</em> <em>factor</em>-α expression and attenuated by interfering with its signaling.

Although the pituitary is known to produce several <em>growth</em> <em>factors</em>, their effects on pituitary cell <em>growth</em> and differentiation are still unclear, particularly in normal tissue. Using primary cultures of aged ewe pituitaries cultured in serum-free conditions, we studied the effects of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), epidermal <em>growth</em> <em>factor</em> (EGF), transforming <em>growth</em> <em>factor</em>-beta (TGF beta 1), insulin, and 17 beta-estradiol (E2) on the <em>growth</em>, differentiation, and expression of gonadotropin subunit genes. After 72-h incubation of the monolayer (day 5) with optimal concentrations of each <em>factor</em>, [3H]thymidine incorporation was increased significantly (P < 0.01) over the control values by 33 +/- 8% (mean +/- SEM; n = 3; 10 nM E2), 36 +/- 10% (1 ng/ml TGF beta 1), 83 +/- 12% (10 ng/ml bFGF), and 118 +/- 12% (1 nM EGF). Insulin showed a two-phase dose-response curve, increasing [3H]thymidine uptake by 34 +/- 9% at 10 ng/ml and by 63 +/- 13% at 10 micrograms/ml. Cell counting using a Coulter counter confirmed these results. Characterization of cell types by immunocytochemistry (avidin-biotin-peroxidase complex technique) revealed that the cell cultures were predominantly gonadotrophs. However, the cultures contained cells that did not stain with any specific ovine or human antiserum against LH beta, FSH, TSH beta, PRL, GH, ACTH, or glial fibrillary acidic protein, but were of epithelial cell lineage, as shown by positive keratin staining. Treatment with EGF and bFGF increased the proportion of these undifferentiated pituitary cells and induced changes in their morphology to large cuboidal cells containing large nuclei. After treatment with E2, insulin, and TGF beta 1, pituitary cells remained differentiated, although with E2, staining for gonadotrophs was much reduced. Northern blot analysis revealed that E2 treatment for 0-48 h progressively reduced the mRNA for FSH beta (<em>16</em> +/- 4.5% of control values) and LH beta (12.4 +/- 2.5%), but had little effect on the common alpha-subunit (88.4 +/- 4.6%). TGF beta 1, however, stimulated the expression of FSH beta subunit gene by 142 +/- 4.6% (P < 0.01) of the control value, but had no significant effect on LH beta and common alpha-subunit genes. Insulin, EGF, and bFGF showed no significant effect on the expression of these three subunit genes. The data define the direct effects of <em>growth</em> <em>factors</em> and E2 on the <em>growth</em> and differentiation of normal sheep pituitary cells and gonadotrophs in particular, which may be of relevance to the pathophysiology of the pituitary and in the multistage process of pituitary tumorigenesis.

Background: A wealth of research has reported on the anti-obesity effects of green tea extract (GTE). Although browning of white adipose tissue (WAT) has been reported to attenuate obesity, no study has disclosed the effects of GTE on browning in Sprague Dawley rats. Objectives: The aims of the study were to investigate the effects of GTE on anti-obesity and browning, and their underlying mechanisms. Methods: Four groups of rats (n=10/group) were used including a normal diet with vehicle treatment, and a high-energy diet (HED) with vehicle or GTE by oral gavage at 77.5 or 155 mg/kg/day for 8 weeks. Body weight, fat accumulation, and serum biochemical parameters were used to evaluate obesity. The gene expressions were analyzed using RT-qPCR and western blotting. Results: GTE modulated HED-induced body weight, fat accumulation, and serum levels of triacylglycerol, total cholesterol, low-density lipoprotein, free fatty acids, aspartate aminotransferase, and alanine aminotransferase. Moreover, GTE enhanced the serum high-density lipoprotein. Most importantly, the biomarkers of beige adipose tissue were up-regulated in WAT in GTE-given groups. GTE induced genes involved in different pathways of browning, and reduced transducin-like enhancer protein-3 in WAT. Conclusion: Our results suggest that GTE may improve obesity through inducing browning in HED-fed rats. Abbreviations: ALT: Alanine transaminase; AST: Aspartate transaminase; BAT: Brown adipose tissue; BMP-7: Bone morphogenetic protein-7; BW: Body weight; CIDEA: Cell death activator; CPT-1: Carnitine palmitoyltransferase-1; EFP: Epididymal fat pad; FFA: Free fatty acid; FGF-21: <em>Fibroblast</em> <em>growth</em> <em>factor</em>-21; GTE: Green tea extract; HDL: High-density lipoprotein; HED: high-energy diet; LDL: Low-density lipoprotein; MFP: Mesenteric fat pad; PGC-1α: Activates PPAR-γ coactivator-1; PPAR-γ: Peroxisome proliferator-activated receptor-γ; PRDM-<em>16</em>: PR domain containing <em>16</em>; RFP: Renal fat pad; SD: Sprague Dawley; TC: Total cholesterol; TG: Triacylglycerol; TLE-3: Transducin-like enhancer protein-3: UCP-1: Uncoupling protein-1; WAT: White adipose tissue.