This pilot study investigated the potential use of three circulating angiogenesis-related cytokines, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), angiogenin (ANG) and endostatin, as tumour markers and prognostic <em>factors</em> in patients with head and neck squamous cell carcinoma (HNSCC). A total of 30 patients with HNSCC treated with curative intent and <em>15</em> healthy controls were studied. Serum (bFGF and ANG) and plasma (endostatin) was assayed by enzyme-linked immunoabsorbance assay (ELISA). None of the cytokines was raised in HNSCC patients when compared with controls. Serum bFGF was not associated with any clinico-pathological or outcome parameters, although there was a trend towards higher levels in more advanced and aggressive tumours. Lower serum angiogenin (sANG) levels were associated with loco-regional disease recurrence (P = 0.036). Using a cut-off level of 400 pg/mL, a low level of sANG predicted tumour recurrence with a relative risk of 4.0 (95% CI: 0.7-24.0). Plasma endostatin was associated with higher histological grade (P = 0.01) and with both disease recurrence (P = 0.045) and death from disease (P = 0.021). Plasma endostatin above a cut-off point of 70 ng/mL could predict tumour recurrence with a relative risk of 4.7 (95% CI: 1.1-19.7). These data suggest that plasma endostatin and sANG have potential roles as prognostic <em>factors</em> and require further investigation.

A useful system to evaluate the angiogenic activity of hormones and <em>growth</em> <em>factors</em> is the chorioallantoic membrane (CAM) of chick embryos. The present studies examined the angiogenic activity of chicken anterior pituitary glands and both <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) and <em>growth</em> hormone (GH). Grafts of anterior pituitary gland evoked an angiogenic response on the CAM which was lost if the adenohypophyseal tissue was first boiled. The magnitude of the angiogenic response to anterior pituitary glands increased with the age of the donor (from a minimum <em>15</em> days of embryonic development to a maximum between 2 and 6 weeks old). In view of the similarity of the profile of the angiogenic response and the reported changes in GH secretion, the angiogenic activity of GH was then examined. Considerable angiogenic responses were observed with GH; there being increases (P < 0.05) in number of new blood vessels on the CAM of chick embryos on which native chicken GH or native bovine GH or recombinant bovine GH were added. These data support GH having an angiogenic action.

Granulocyte-macrophage colony-stimulating <em>factor</em> (GM-CSF) is a hemopoietic <em>growth</em> <em>factor</em> that is expressed in activated T cells, <em>fibroblasts</em>, macrophages, and endothelial cells. Although GM-CSF does not appear to be essential for normal hemopoiesis, overexpression of GM-CSF has been implicated in the pathogenesis of some diseases such as myeloid leukemia and chronic inflammation. An NF-kappaB/Rel binding site within the GM-CSF promoter, termed the kappaB element appears to be important for controlling expression in reporter gene assays in response to a number of stimuli in T cells. We investigated oligonucleotide-directed triple helix formation across this regulatory sequence as a potential tool to inhibit GM-CSF gene transcription. A <em>15</em>-base oligonucleotide, GM3, was targeted to a purine-rich region in the GM-CSF proximal promoter, which overlaps the kappaB element. Gel mobility shift assays and DNase I footprinting demonstrated that GM3 formed a sequence-specific collinear triplex with its double-stranded DNA target. Triplex formation by GM3 blocked recombinant and nuclear NF-kappaB proteins binding to the GM-CSF element. GM3 also caused selective inhibition of the human T-cell lymphotrophic virus-1 Tax transactivator-induced luciferase activity from a reporter construct driven by the GM-CSF promoter in Jurkat T cells. Finally, GM3 greatly reduced the concentration of endogenous GM-CSF mRNA induced by different stimuli in Jurkat T cells but did not affect interleukin 3 mRNA levels in the same cells. We conclude that the kappaB element in the GM-CSF promoter plays a central role in the transcriptional activation of the endogenous GM-CSF gene. Colinear triplex formation acts as a selective transcriptional repressor of the GM-CSF gene and may have potential therapeutic application in cases of undesirable overexpression of this protein.

BACKGROUND

The fibroblast growth factor 23 (FGF23) is the most important hormonal regulator of circulating phosphate levels. Apart from this essential role, it may also act as a 'hormone-like' factor involved in glucose and lipid metabolism. It is believed to have a potential role in the development of insulin resistance.

OBJECTIVE

The aim of the study was to compare FGF23 levels between two groups of obese adolescents: insulin resistant and non-insulin resistant.

METHODS

The study included 36 obese, insulin-resistant adolescents (21 boys and 15 girls) of pubertal age (mean age, 13.95 years; Tanner stage IV or V). The control group consisted of 21 obese peers with normal HOMA-IR values.

METHODS

FGF23 levels were measured in a fasting blood sample by Human Intact FGF-23 ELISA Kit (Immunotopics Inc., San Clemente, CA, USA). A standard oral glucose tolerance test was performed, which assessed fasting and 120 min postload plasma glucose and serum insulin levels; the insulin resistance index HOMA-IR was calculated. The definition of insulin resistance was based on a HOMA-IR threshold set for adolescents >> or = 3.16).

RESULTS

There was a significant inverse correlation between FGF23 levels and HOMA-IR (R = -0.26, p < 0.05) in the study group. FGF23 levels were also significantly lower in the study group (9.8 vs. 11.9 pg/mL, p = 0.026).

CONCLUSIONS

In adolescents with simple obesity and insulin resistance, FGF23 levels are lower compared with obese adolescents with normal HOMA-IR.

A turbot, Scophthalmus maximus, fin (TF) cell line was established and susceptibility to turbot reddish body iridovirus (TRBIV) was determined in this study. Primary culture of TF cells was initiated from fin tissue pieces partially digested with trypsin, collagenase II and hyaluronidase. Digested tissue pieces were cultured at 24 degrees C in Leibovitz-<em>15</em> medium (pH 7.2), supplemented with 20% fetal bovine serum, carboxymethyl chitosan, N-acetylglucosamine hydrochloride, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and epidermal <em>growth</em> <em>factor</em>. The cultured TF cells, in <em>fibroblast</em> shape, proliferated to 100% confluency 50 days later. A TF cell line, with a population doubling time of 45.6 h at passage 80, has been established and subcultured to passage 133. Chromosome analyses indicated that the TF cells exhibited chromosomal aneuploidy with a modal chromosome number of 44 which displayed the normal diploid karyotype of S. maximus at least up to passage 80. TRBIV susceptibility testing demonstrated that cytopathic effect and propagated viral particles were observed in TF cells after TRBIV infection. In conclusion, a continuous TRBIV susceptible TF cell line has been established successfully, and the cell line may serve as a valuable tool for studies of cell-virus interactions and has applications for different kinds of cytotechnological studies as well.

We reported induction of broad-spectrum chemoresistance by acidic and basic <em>fibroblast</em> <em>growth</em> <em>factors</em> and chemosensitization by their nonspecific inhibitor suramin at nontoxic and subtherapeutic doses. This study evaluated whether low-dose suramin enhances paclitaxel activity in chemotherapy-naïve and paclitaxel-pretreated human MCF7 breast xenograft tumors in mice. Suramin, 10 mg/kg, and/or paclitaxel, <em>15</em> mg/kg, were administered intravenously, twice weekly for 2 to 3 weeks. In addition to conventional end points [tumor size change, median survival time (MST)], we also used clinically relevant end points [partial (PR) and complete response rates (CR); progressive disease (PD); stable disease (SD); time to tumor progression (TTP)]. In chemotherapy-naïve mice, the control and suramin groups showed identical TTP (3 days) and MST (21 days). Single-agent paclitaxel produced 47% PR and 24% CR, and prolonged both TTP and MST to 73 days. The addition of suramin further improved the total response rate to 100% with a dramatically greater 63% CR, shortened the time to attain PR and CR, and prolonged TTP and MST to>> or =136 days. In the paclitaxel-pretreated group, single-agent paclitaxel resulted in 67% SD and 33% PD, whereas the combination produced 50% PR and 50% SD. Suramin also significantly enhanced the apoptotic effect of paclitaxel in tumors. In conclusion, suramin improved the activity of paclitaxel in both chemotherapy-naïve and paclitaxel-pretreated animals, without enhancing host toxicity (< or =10% body weight loss in all groups). These data have led to the initiation of phase I/II trials of paclitaxel and low-dose suramin combination in advanced metastatic breast cancer patients.

Precursors of osteoclasts seeded on top of a confluent layer of osteoblasts/bone lining cells induced retraction of the latter cells. The (pre)osteoclasts then migrated in the formed cell-free areas and fused to form osteoclast-like cells. Retraction of the osteoblasts/bone lining cells proved to depend on activity of matrix metalloproteinases, and TGF-beta1 prevented the retraction.

BACKGROUND

It is well known that osteoblasts have a profound effect on (pre)osteoclasts in inducing the formation of bone-resorbing osteoclasts. Whether, on the other hand, (pre)osteoclasts also modulate osteoblast activity is largely unknown. Because osteoblasts/bone lining cells have to retract from the surface before resorption of bone by osteoclasts, we addressed the question of whether (pre)osteoclasts have the capacity to induce such an activity.

METHODS

Rabbit calvarial osteoblasts/bone lining cells or periosteal <em>fibroblasts</em> were cultured until confluency, after which rabbit peripheral blood mononuclear cells (PBMCs) were seeded on top of them. The co-cultures were maintained for up to <em>15</em> days in the presence or absence of the cytokines transforming <em>growth</em> <em>factor</em> (TGF)-beta1 and TNF-alpha and selective inhibitors of matrix metalloproteinases and serine proteinases. The formation of cell-free areas and the number of TRACP+ multinucleated osteoclast-like cells were analyzed. In addition, formation of cell-free areas was analyzed in co-cultures of osteoblasts with mature osteoclasts.

RESULTS

The seeding of PBMCs on a confluent layer of osteoblasts/bone lining cells resulted in the following sequence of events. (1) A low number of PBMCs strongly attached to osteoblasts. 2) At these sites of contact, the osteoblasts retracted, thus forming cell-free areas. (3) The PBMCs invaded these areas and attached to the surface of the well, after which they fused and formed multinucleated TRACP+ osteoclast-like cells. Retraction was only seen if the cells were in direct contact; conditioned media from cultured PBMCs added to osteoblasts had no effect. Mature osteoclasts seeded on osteoblasts similarly induced retraction, but this retraction occurred at a much faster rate (within 2 days) than the retraction effectuated by the osteoclast precursors (after 8 days in co-culture). Inhibition of matrix metalloproteinase activity, but not of serine proteinases, strongly reduced retraction of the osteoblasts, thus indicating that this type of cell movement depends on the activity of matrix metalloproteinases. A similar inhibitory effect was found with TGF-beta1. TNF-alpha had no effect on osteoblast retraction but enhanced the formation of multinucleated osteoclast-like cells. Addition of PBMCs to confluent layers of periosteal fibroblasts resulted in similar phenomena as observed in co-cultures with osteoblasts. However, the cell-free areas proved to be significantly smaller, and the number of multinucleated cells formed within cell-free areas was three to four times lower.

CONCLUSIONS

Our results indicate that osteoclast precursors and mature osteoclasts have the capacity to modulate the activity of osteoblasts and that, yet unknown, membrane-bound signaling molecules are essential in inducing retraction of osteoblasts and the subsequent formation of cell-free areas.

Neurosphere cultures derived from fetal brain regions can proliferate in response to exogenous <em>growth</em> <em>factors</em> such as basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and give rise to undifferentiated precursor cells that form a floating neurosphere. In this study, neurospheres generated from the ganglionic eminence region of embryonic day <em>15</em> (E<em>15</em>) rat embryos were treated in the presence or absence of ethanol. We found that such neurospheres respond to environmental toxins such as alcohol and still retain the multi-potential capability of differentiation into neuronal and glial cell types. Ethanol at high concentration (50 mM) affected proliferation, gliogenesis and neurogenesis, although the most profound effect was observed on glial phenotype. Our findings suggest that extrinsic agents, such as alcohol can alter intrinsic cellular mechanisms of stem cell fate choices contributing to altered neurogenesis and gliogenesis during central nervous system (CNS) maturation, which might in part be responsible for defective astroglial and neuronal functions in fetal alcohol syndrome (FAS).

The controlled accumulation of <em>fibroblasts</em> to sites of inflammation is crucial to effective tissue repair after injury. Either inadequate or excessive accumulation of <em>fibroblasts</em> could result in abnormal tissue function. Prostacyclin (PGI(2)) is a potent mediator in the coagulation and inflammatory processes. The aim of this study was to investigate the effect of PGI(2) on chemotaxis of human fetal lung <em>fibroblasts</em> (HFL-1). Using the blind well chamber technique, we found that the PGI(2) analog carbaprostacyclin (10(-6) M) inhibited HFL-1 chemotaxis to human plasma fibronectin (20 microg/ml) 58.0 +/- 13.2% (P < 0.05) and to platelet-derived <em>growth</em> <em>factor</em> (PDGF)-BB (10 ng/ml) 48.7 +/- 4.6% (P < 0.05). Checkerboard analysis demonstrated that carbaprostacyclin inhibits both directed and undirected migration. The inhibitory effect of the carbaprostacyclin was concentration dependent and blocked by the cAMP-dependent protein kinase (PKA) inhibitor KT-5720, suggesting that a cAMP-PKA pathway may be involved in the process. Two other PGI(2) analogs, ciprostene and dehydro-<em>15</em>-cyclohexyl carbaprostacyclin (both 10(-6) M), significantly inhibited <em>fibroblast</em> migration to fibronectin. In summary, PGI(2) appears to inhibit <em>fibroblast</em> chemotaxis to fibronectin and PDGF-BB. Such an effect may contribute to the regulation of <em>fibroblasts</em> in wound healing and could contribute to the pathogenesis of diseases characterized by abnormal tissue repair remodeling.

BACKGROUND

The use of the erbium-doped:yttrium, aluminum, and garnet (Er:YAG) laser in periodontal therapy has been the focus of much research. Recombinant human platelet-derived growth factor-BB (rhPDGF-BB) is suggested as a potent stimulator and strong mitogen for human periodontal ligament cells. The present study tested the direct effects of Er:YAG laser irradiation, alone or with rhPDGF-BB application, on the biocompatibility of periodontally diseased roots through fibroblast attachment and proliferation.

METHODS

The study examined five healthy and 15 periodontally involved teeth, prepared from proximal surfaces, which were divided randomly into four groups (10 specimens each): group 1: healthy; group 2: untreated diseased; group 3: Er:YAG laser irradiation (60 mJ/pulse, 10 Hz); and group 4: Er:YAG laser irradiation (60 mJ/pulse, 10 Hz) plus rhPDGF-BB application (50 ng/ml). Three subgroups per group (three specimens each) were incubated for three periods (1, 3, or 7 days). The remaining specimen was used to determine surface topography. Fibroblasts were pooled on root specimens and incubated. Results were evaluated using scanning electron microscopy. Repeated cell counts were performed within a representative standard area.

RESULTS

Using paired t tests, all experimental groups (except group 2 diseased) showed statistically significant differences between 1- and 3-day and between 1- and 7-day incubation periods, but not between 3- and 7-day incubation periods. Using analysis of variance, the intergroup comparison showed significant differences favoring group 1 over groups 2 and 3 and group 4 over group 2 at the 1-day incubation period; group 1 was favored over groups 2, 3, and 4 and groups 3 and 4 were favored over group 2 at the 3- and 7-day incubation periods. Comparable effects were shown between groups 3 and 4 for all incubation periods and between groups 2 and 3 and groups 1 and 4 for the 1-day incubation.

CONCLUSIONS

Er:YAG laser used alone or in combination with rhPDGF-BB application may offer a promising periodontal therapy for conditioning root surfaces, although the combined application seemed to be slightly more effective. However, testing laser use in intervals and with parameters <60 mJ/pulse and 10 Hz is required to verify the minimum threshold values necessary to obtain complete root debridement and clarify optimal conditions for fibroblast cell attachment and growth. Further studies are needed to determine ideal parameters for creating the best environment for successful periodontal treatment.

Primary neuroendocrine carcinoma of the breast is a rare variant, accounting for only 2% to 5% of diagnosed breast cancers, and may have relatively aggressive behavior. Mutational profiling of invasive ductal breast cancers has yielded potential targets for directed cancer therapy, yet most studies have not included neuroendocrine carcinomas. In a tissue microarray screen, we found a 2.4% prevalence (9/372) of neuroendocrine breast carcinoma, including several with lobular morphology. We then screened primary or metastatic neuroendocrine breast carcinomas (excluding papillary and mucinous) for mutations in common cancer genes using polymerase chain reaction-mass spectroscopy (643 hotspot mutations across 53 genes), or semiconductor-based next-generation sequencing analysis (37 genes). Mutations were identified in 5 of <em>15</em> tumors, including 3 with PIK3CA exon 9 E542K mutations, 2 of which also harbored point mutations in FGFR family members (FGFR1 P126S, FGFR4 V550M). Single mutations were found in each of KDR (A1065T) and HRAS (G12A). PIK3CA mutations are common in other types of breast carcinoma. However, FGFR and RAS family mutations are exceedingly rare in the breast cancer literature. Likewise, activating mutations in the receptor tyrosine kinase KDR (VEGFR2) have been reported in angiosarcomas and non-small cell lung cancers; the KDR A1065T mutation is reported to be sensitive to VEGFR kinase inhibitors, and <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor inhibitors are in trials. Our findings demonstrate the utility of broad-based genotyping in the study of rare tumors such as neuroendocrine breast cancer.

In this study, nerve guides composed of poly(D,L-lactide) (PDLLA) were fabricated and used in the repair of transected sciatic nerves (<em>15</em>-mm gaps) of rats. Nerve guides with a two-ply structure (inner layer dense, outer layer microporous) were prepared by controlling the solvent evaporation rate. Then basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) was embedded in the inner layer of the nerve guides. Thus the inner dense layer not only could prevent the in<em>growth</em> of <em>fibroblast</em> and avoid the outgrowing nerve cable, but it also could retain the released bFGF in the guide lumen. The outer porous layer allowed vascular in<em>growth</em> and the diffusion of essential nutrients into the guide lumen. The data show that by using this nerve guide, the transected <em>15</em>-mm sciatic nerve was regenerated successfully within 4 months. The recovery of function of the regenerated nerves was significantly accelerated by bFGF, as indicated by an electrostimulation test and histologic assays. In addition, the bFGF retained its bioactivity during embedding and continuously was released from the matrix, as confirmed by the results of both the dorsal root ganglia (DRG) and the Schwann cell culture in the presence of PDLLA matrix containing bFGF. The released bFGF enhanced the ability of the nerve fibers to sprout from dorsal root ganglia, and it accelerated the proliferation of Schwann cells.

<em>Fibroblasts</em> from young patients exhibiting clinical features of progeroidal syndromes showed decreased biosynthesis of the small proteoglycan decorin. Cells in culture were metabolically labeled, and proteoglycans secreted into the medium were analyzed electrophoretically after immunoprecipitation with antibodies raised against decorin and biglycan. Fluorograms showed regularly a reduction to <em>15</em>-30% of the normal amount of mature decorin and its core protein after chondroitin ABC lyase treatment. The size of the glycosaminoglycan chains was increased, but there was no obvious anomaly in the secretion kinetics of the mature proteoglycan. In addition, the patients' <em>fibroblasts</em> synthesized an increased amount of biglycan compared to control cells from healthy donors. Northern blot analysis clearly demonstrated a reduction by 85-94% in decorin mRNA, but biglycan mRNA was concomitantly increased, indicating that these alterations occur at the transcriptional level of protein expression. Transcription of decorin in <em>fibroblasts</em> from one of the patients was stimulated up to 3-fold by treatment with interleukin-1 beta. No response to interleukin-1 beta and transforming <em>growth</em> <em>factor</em>-beta was observed in the cells from another patient. In situ hybridization of cultured cells with an antisense decorin probe showed that decorin levels were reduced throughout the cell population. Surprisingly, subsequent examination of cells from one of the patients, now in mid-teenage, revealed a return to normal levels of decorin expression compared to age-matched controls. These studies suggest that, as in Marfan's syndrome where the primary defect concerns the fibrillin gene, reduced decorin expression contributes to the formation of an abnormal matrix and the pathogenesis of these disorders. They also indicate that this abnormality is likely to represent a secondary phenomenon which leads to a fault in the regulation of decorin gene transcription.

Fibulin-1 and fibulin-2 are two novel rod-like proteins which occur either in basement membranes or in interstitial fibrils in close association with fibronectin. They were examined for their sensitivity to proteolysis by matrix metalloproteinases (stromelysin, matrilysin), circulating proteases (thrombin, plasmin, kallikrein), leucocyte elastase and mast cell chymase. Fibulin-1 (95 kDa) was readily cleaved by leucocyte elastase, weakly by matrilysin and not by the other proteases. Cleavage occurred in a domain-connecting link region close to the N-terminus, giving rise to fragments of 70 kDa and 26 kDa. A much more extensive cleavage by all seven proteases was observed for fibulin-2 (195 kDa), giving rise to many fragments in the range <em>15</em>-<em>15</em>0 kDa. Vulnerable sites included two central link regions, the cysteine-free part of the large N-terminal globular domain but also several regions of epidermal-<em>growth</em>-<em>factor</em>(EGF)-like repeats which are a major part of the rod-like domain. The latter domain became much more sensitive to proteolysis in the presence of EDTA, demonstrating that calcium is required for stabilization. Edman degradation demonstrated cleavage of peptide bonds corresponding to the known specificities of these proteases. A similar proteolysis was also observed for fibulin-2 deposited by cultured <em>fibroblasts</em> into a dense fibrillar network. Since fibulin-2 is an abundant component of small and large blood vessels it could be a major target for proteolysis during vascular injuries.

OBJECTIVE: To explore the safety, efficacy, and biomarkers of bevacizumab with gemcitabine and oxaliplatin in women with recurrent platinum-sensitive ovarian carcinoma. METHODS: The patients received bevacizumab (10 mg/kg), gemcitabine (1000 mg/m(2)), and oxaliplatin (65 mg/m(2)) on days 1 and <em>15</em> in 28-day cycles. The patients with safely accessible tumor underwent intratumoral fluid pressure (IFP) measurements and positron-emission tomographies immediately and 2 weeks after treatment. Blood biomarkers were evaluated at 5 time points. RESULTS: The trial was closed after enrolling 19 of the 53 projected patient accrual. Thirteen (68.5%) of 19 patients showed a response (1 complete response, 12 partial responses), and 6 patients showed stable disease (31.6%). Median progressive-free survival was 36.9 weeks (258.3 days), and the median overall survival was 112.3 weeks (633 days, not reached). Toxicity was acceptable, and there were no arterial thromboses, serious bleeding, gastrointestinal perforations, or complications from the invasive procedures. Bevacizumab with chemotherapy induced a substantial drop in tumor IFP after treatment. The regimen induced sustained elevation in circulating plasma vascular endothelial <em>growth</em> <em>factor</em> (VEGF), placental <em>growth</em> <em>factor</em> (PlGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), soluable vascular endothelial <em>growth</em> <em>factor</em> receptor 2 (sVEGFR2), and circulating progenitor cells. Plasma PlGF, VEGFR2(+) monocytes, and urinary matrix metalloproteinase 2 activity showed differential associations with treatment outcome when evaluated at baseline and after 14 days of treatment. CONCLUSIONS: Despite early termination of the study, the results indicate that the regimen was well tolerated and demonstrated activity in platinum-sensitive ovarian cancer. Biomarker evaluations showed that bevacizumab with chemotherapy significantly changed the levels of several circulating cellular and molecular biomarkers. The increases in plasma PlGF and VEGFR2(+) monocytes showed correlations with outcome. These exploratory data should be further evaluated in future studies of bevacizumab in ovarian cancer.

PD176067 is a reversible and selective inhibitor of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor tyrosine kinase, and was in preclinical development as an angiogenesis inhibitor for the treatment of solid tumors. A 14-day oral toxicity study of PD176067 in young female rats (7 weeks old) was conducted at doses of 2.5, 5, and 10 mg/kg/day (<em>15</em>, 30, and 60 mg/m(2), respectively). Skeletal changes, and vascular and soft tissue mineralization were observed as primary drug-related toxicities. To determine if these changes are specific to young, rapidly <em>growing</em> animals with increased vascular and osseous development, PD176067 was administered to mature (11 months old) rats. Female rats received PD176067 by gavage for 14 days at doses of 2.5, 5, and 10 mg/kg/day and necropsied on day <em>15</em>. Clinical signs of toxicity were seen at>> or =5 mg/kg and one death occurred at 10 mg/kg. Physeal dysplasia (distal femur, proximal tibia, sternum) occurred in all drug-treated animals and was characterized by dose-related increased thickness of the zones of chondrocyte proliferation and hypertrophy, and marked thickening of the zone of ossification. Cartilage hyperplasia was characterized by proliferation of chondrocytes along margins of the synchondrosis and subperiosteum of sternebrae. Serum phosphorus levels increased 47% and 166% at 5 and 10 mg/kg, respectively. Mineralization of cardiac myocytes, aorta, various arteries, renal tubules, and gastric mucosa and muscularis was seen at 10 mg/kg, and consistent with the presence of calcium-phosphorus deposition. Physeal changes occurred at similar plasma PD176067 exposures in young and mature rats (AUC>> or = 4.83 microg.hr/mL). PD176067 produced morphologically similar lesions in young and adult rats.

Hematopoietic prostaglandin D synthase (PGDS) is a key enzyme to produce prostaglandin (PG) D and J series. These PGs are involved in inflammation and immune system. The PGDS complementary DNA (cDNA)-expressing retrovirally transfected <em>fibroblasts</em> were introduced in vivo, and effect of the expression on lung injury induced by bleomycin was investigated in mice. Intravenous injection of PGDS cDNA-expressing <em>fibroblasts</em> significantly reduced lung edema, leukocyte infiltration in bronchoalveolar lavage (BAL) fluid, and pulmonary collagen content at 4 wk after instillation of bleomycin. Survival rate in mice instilled with the PGDS-expressing <em>fibroblasts</em> was higher than that in mice that received the mock transfection. Administration of <em>15</em>-deoxy-Delta 12,14-PGJ2, which is a nonenzymatic metabolite of PGD2, also attenuated the lung injury, suggesting mediation of PGs produced by PGDS for the attenuation. Introduction of PGDS cDNA-expressing <em>fibroblasts</em> suppressed expression of basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, connective tissue <em>growth</em> <em>factor</em>, and collagen messenger RNAs in the lungs, as well as the levels of total proteins and hemoglobin in BAL fluid. These data suggest that the suppressive effect of PGDS on the lung injury could be partly mediated by edema formation and inhibition of genes involved in the fibrotic change.

Germline mutations of the gene encoding human <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) have been shown to be responsible for several related autosomal dominant forms of syndromic craniosynostosis and short limb dwarfism. Somatic activating mutations of FGFR3 were recently reported to occur in three of 12 (25%) uterine cervical carcinomas and nine of 26 (35%) bladder carcinomas, suggesting that constitutive activation of FGFR3 may be an important mechanism underlying the development and/or progression of these common epithelial malignancies. In order to investigate further a possible role for FGFR3 mutations in cervical carcinogenesis, we performed sequence-based mutational analysis of FGFR3 in 51 primary cervical carcinomas and seven cervical carcinoma-derived cell lines. The regions analysed (exons 7, 10, 13, <em>15</em>, and 19) encompassed all previously described FGFR3 mutations. A single nucleotide substitution at codon 249, predicting a serine to cysteine amino acid substitution (S249C) in the FGFR3 extracellular domain, was identified in one primary tumor. Only wild type FGFR3 alleles were identified in the remaining tumors and cell lines. The S249C mutation is the only FGFR3 mutation described to date in cervical carcinomas. These findings suggest that while activating mutations of FGFR3 occur in cervical cancer, they may not be as common as initially reported.

OBJECTIVE

To characterize the growth and maturation of nonimmortalized human lens epithelial (HLE) cells grown in vitro.

METHODS

HLE cells, established from 18-week prenatal lenses, were maintained on bovine corneal endothelial (BCE) extracellular matrix (ECM) in medium supplemented with basic fibroblast growth factor (FGF-2). The identity, growth, and differentiation of the cultures were characterized by karyotyping, cell morphology, and growth kinetics studies, reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, and Western blot analysis.

RESULTS

HLE cells had a male, human diploid (2N = 46) karyotype. The population-doubling time of exponentially growing cells was 24 hours. After 15 days in culture, cell morphology changed, and lentoid formation was evident. Reverse transcription-polymerase chain reaction (RT-PCR) indicated expression of alphaA- and betaB2-crystallin, fibroblast growth factor receptor 1 (FGFR1), and major intrinsic protein (MIP26) in exponential growth. Western analyses of protein extracts show positive expression of three immunologically distinct classes of crystallin proteins (alphaA-, alphaB-, and betaB2-crystallin) with time in culture. By Western blot analysis, expression of p57(KIP2), a known marker of terminally differentiated fiber cells, was detectable in exponential cultures, and levels increased after confluence. MIP26 and gamma-crystallin protein expression was detected in confluent cultures, by using immunofluorescence, but not in exponentially growing cells.

CONCLUSIONS

HLE cells can be maintained for up to 4 months on ECM derived from BCE cells in medium containing FGF-2. With time in culture, the cells demonstrate morphologic characteristics of, and express protein markers for, lens fiber cell differentiation. This in vitro model will be useful for investigations of radiation-induced cataractogenesis and other studies of lens toxicity.

Low oxygen tension has recently been shown to stimulate cell <em>growth</em> and clonal expansion, as well as synthesis and transcription of certain <em>growth</em> <em>factors</em> and extracellular matrix components. These results have been obtained by exposing cell cultures to a hypoxic environment. Using an oxygen probe, we have now studied how experimental conditions affect the oxygen tension detectable at the cell surface. Dissolved oxygen tension was directly related to the height of the medium above the cell surface (r = 0.8793, P = 0.021), but was constant when no cells were present in the flask (r = -0. 9732, P = 0.001). In both human dermal <em>fibroblasts</em> and NIH/3T3 cultures, oxygen tension decreased linearly as cell density increased (r = -0.835, P < 0.0001; r = -0.916, P < 0.0001, respectively). When human dermal <em>fibroblasts</em> were exposed to 2% O(2), maximum hypoxic levels (0 mmHg) were achieved within approximately <em>15</em> min, and the recovery time was within a similar time frame. The addition of rotenone, an inhibitor of cellular respiration, blocked this decrease in oxygen tension at the cell surface, suggesting that cellular consumption of oxygen is responsible for the decline. Finally, we examined the cell-surface oxygen tension in control and acutely wounded human skin equivalents (HSE), consisting of a keratinocyte layer over a type I collagen matrix containing <em>fibroblasts</em>. We found that oxygen tension dropped significantly (P < 0.0001) in acutely wounded areas of HSE as compared to unwounded areas of HSE and that this drop was prevented by the addition of mitomycin C. These results indicate that cell-surface oxygen tension is indirectly related to cell density, and that the amount of detectable oxygen at the cell surface is a function of cell density, the oxygen tension in the incubator, and increased cellular activity, as occurs after injury.

OBJECTIVE

To determine the types and frequency of airway anomalies in patients with Pfeiffer syndrome.

METHODS

Retrospective case series.

METHODS

Academic tertiary care pediatric hospital.

METHODS

Eleven patients with Pfeiffer syndrome, 6 of whom were severely affected, were identified. All were included in the study.

METHODS

Presence of tracheal anomalies, need for tracheotomy, and length of life.

RESULTS

The 6 severely affected patients had mutations in genes that code for <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 (S351C [3 patients]; C342S [2 patients]; and W290C [1 patient]). Five of these patients were diagnosed during bronchoscopy or tracheotomy as having a congenital tracheal cartilaginous sleeve. In 1 patient, supportive care was withdrawn at 2 weeks of life, and the patient died. The remaining 5 patients required tracheotomy because of severe upper airway obstruction. Three of these patients died (at ages 9 months and 7 and <em>15</em> years). Two are still alive at ages 23 and 18 months.

CONCLUSIONS

Patients with Pfeiffer syndrome manifest significant airway pathologic conditions. Upper airway obstruction is related to midface hypoplasia and secondary nasal obstruction. Tracheal anomalies have been infrequently reported.

OBJECTIVE

Platelet concentrates are important for support of patients with malignancies requiring myelotoxic chemotherapy. During storage, 10% to <em>15</em>% of platelets may become activated resulting in the release of alpha-granules, which contain <em>growth</em> <em>factors</em>. We hypothesize that, during storage, <em>growth</em> <em>factors</em> accumulate in the plasma, specifically platelet-derived <em>growth</em> <em>factor</em>, vascular endothelial <em>growth</em> <em>factor</em> (VEGF), transforming <em>growth</em> <em>factor</em>-beta, and <em>fibroblast</em> <em>growth</em> <em>factor</em>-2, which may adversely affect cancer patients.

METHODS

The concentrations of growth factors were measured by ELISA from the plasma of apheresis platelets serially throughout storage (days 1, 3, 5, and 7) and compared with concentrations in fresh plasma from healthy blood donors. Washing was evaluated as a method of growth factor removal, and an in vitro model of platelet transfusion in a patient receiving Bevacizumab (Avastin) using immunoprecipitation was employed to determine if Bevacizumab would be bound by the VEGF in apheresis platelets.

RESULTS

VEGF, platelet-derived growth factor, and transforming growth factor-beta were increased on day 1 versus fresh plasma and throughout storage reaching a relative maximum at outdate (P < 0.01, day 5 or 7). Fibroblast growth factor-2 concentrations were significantly increased on day 7 alone versus day 1 or to fresh plasma (P < 0.01). Washing removed 41 +/- 11% to 56 +/- 2% of the growth factors. Bevacizumab effectively bound the VEGF from apheresis platelets, with significant amounts of VEGF remaining in the supernatant.

CONCLUSIONS

Significant amounts of growth factors are present in apheresis platelets due to the isolation procedures, and these concentrations increase over storage, which may be partially removed by washing. In addition, apheresis platelet transfusion could affect cancer treatment by binding monoclonal antibodies directed against growth factors of tumor origin.

Disease-specific induced pluripotent stem cells (iPS) cells are expected to contribute to exploring useful tools for studying the pathophysiology of inner ear diseases and to drug discovery for treating inner ear diseases. For this purpose, stable induction methods for the differentiation of human iPS cells into inner ear hair cells are required. In the present study, we examined the efficacy of a simple induction method for inducing the differentiation of human iPS cells into hair cells. The induction of inner ear hair cell-like cells was performed using a stepwise method mimicking inner ear development. Human iPS cells were sequentially transformed into the preplacodal ectoderm, otic placode, and hair cell-like cells. As a first step, preplacodal ectoderm induction, human iPS cells were seeded on a Matrigel-coated plate and cultured in a serum free N2/B27 medium for 8 days according to a previous study that demonstrated spontaneous differentiation of human ES cells into the preplacodal ectoderm. As the second step, the cells after preplacodal ectoderm induction were treated with basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) for induction of differentiation into otic-placode-like cells for <em>15</em> days. As the final step, cultured cells were incubated in a serum free medium containing Matrigel for 48 days. After preplacodal ectoderm induction, over 90% of cultured cells expressed the genes that express in preplacodal ectoderm. By culture with bFGF, otic placode marker-positive cells were obtained, although their number was limited. Further 48-day culture in serum free media resulted in the induction of hair cell-like cells, which expressed a hair cell marker and had stereocilia bundle-like constructions on their apical surface. Our results indicate that hair cell-like cells are induced from human iPS cells using a simple stepwise method with only bFGF, without the use of xenogeneic cells.

A form of insulin-like <em>growth</em> <em>factor</em> II (IGF-II) with a mol wt of <em>15</em>,000 has been purified to homogeneity from human Cohn fraction IV1-4. This protein has an amino-terminal sequence through the first 28 residues that is identical to 7.5K IGF-II. The amino acid composition of <em>15</em>K IGF-II, however, indicates that its carboxyl-terminal region may be different from that predicted from the analysis of IGF-II cDNA clones. The affinities of <em>15</em>K IGF-II for receptors on rat placental membranes and for an IGF-binding protein that was isolated from the medium of cultured buffalo rat liver cells were similar to those of the 7.5K form of the <em>growth</em> <em>factor</em>. A best-fit analysis of data from the binding of the two mol wt forms of IGF-II to receptors on rat placental membranes by the LIGAND program was consistent with a model in which 7.5K and <em>15</em>K IGF-II bound to one site with Kd values of 0.27 +/- 0.03 and 0.38 +/- 0.04, respectively. There was an indication that <em>15</em>K IGF-II also bound to a second low affinity site on the membrane. In mitogenesis assays performed on human <em>fibroblasts</em> isolated from the skin of two fetuses of an early gestational age, <em>15</em>K IGF-II stimulated the incorporation of [3H]thymidine into DNA at a half-maximal concentration, i.e. ED50, of 5.7 and 5.0 nM. In these experiments, the ED50 values for 7.5K IGF-II were 8.7 and <em>15</em> nM.