The events linking rhinovirus (RV) infection to airway symptoms are poorly understood. The virus initially infects airway epithelium followed by a vigorous inflammatory response that may entail spread of RV from epithelium to other cells in the airway wall. However, RV has fastidious <em>growth</em> characteristics and to date reproductive infection of primary cells other than human airway epithelium has not been confirmed. Airway <em>fibroblasts</em> are adjacent to and in contact with epithelial cells, play a key role in innate immune responses, and may participate in the evolution of inflammation. To investigate <em>fibroblast</em> actions, we first determined whether RV could infect and replicate in primary culture human lung <em>fibroblasts</em>. RV serotype <em>16</em> (RV<em>16</em>) was used to infect <em>fibroblasts</em> grown from lung tissue, and virus infection with replication was demonstrated by a combination of techniques. RT-PCR was used to show an increase in RV transcription; confocal microscopy demonstrated colocalization of the replicative form of RV genome (double-stranded RNA) and RV<em>16</em> proteins; infectious virus was also recovered from the culture supernatant of infected <em>fibroblasts</em>. Functional consequences of RV infection were next examined. RV infection of <em>fibroblasts</em> was followed by an increase in epithelial neutrophil-activating peptide-78 (ENA-78) mRNA and protein. The permeability <em>factor</em> vascular endothelial <em>growth</em> <em>factor</em> (VEGF) was also induced over a similar time course. These data suggest that interactions between RV and human <em>fibroblasts</em> are feasible, may coordinate neutrophil chemoattraction with enhanced vascular permeability and that <em>fibroblasts</em> may contribute to inflammatory responses following RV infections.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) induces a protein kinase C (PKC)-dependent mitogenic response in transformed fetal bovine aortic endothelial GM 7373 cells. A long-lasting interaction of bFGF with the cell is required to induce cell proliferation. bFGF-treated cells are in fact committed to proliferate only after they have entered the phase S of the cell cycle, 12-14 h after the beginning of bFGF treatment. Before that time, the mitogenic response to bFGF is abolished by 1) removal of extracellular bFGF by suramin, 2) addition of neutralizing anti-bFGF antibodies to the culture medium, 3) inhibition of PKC activity by the protein kinase inhibitor H-7, and 4) down-regulation of PKC by cotreatment with phorbol ester. Thus the requirement for a prolonged interaction of bFGF with the cell reflects the requirement for a prolonged activation of PKC. Similar conclusions can be drawn for the PKC activators 12-O-tetradecanoyl phorbol 13-acetate and 1,2-dioctanoyl-sn-glycerol. The two molecules require <em>16</em> and 6 h, respectively, of activation of PKC to induce 50% of maximal cell proliferation. The requirement for a long-lasting activation of PKC appears to be a mechanism for the control of cell proliferation capable of discriminating among transient nonmitogenic stimuli and long-lasting mitogenic stimuli.

BACKGROUND

Flavopiridol is a flavonoid with antiproliferative effects mediated, in part, by inhibition of cyclin-dependent kinases. Clinical manifestations in a previous Phase I trial in patients with refractory malignancies treated with a 72-h flavopiridol infusion included a proinflammatory syndrome consisting of fever, fatigue, and "local" tumor pain with concomitant alterations in plasma acute-phase reactant proteins.

OBJECTIVE

The aim of this study was to determine whether the proinflammatory syndrome observed in this trial was associated with modulation of plasma cytokines.

METHODS

Patients receiving flavopiridol (n = 76) had serial plasma samples drawn preinfusion and during the infusion for evaluation of interleukin (IL)-6, IL-10, IL-12, granulocyte macrophage colony-stimulating factor, basic-fibroblast growth factor, transforming growth factor-beta, and tumor necrosis factor-alpha levels by standard ELISA assays. The Wilcoxon signed rank test was used to test the significance of the difference between the baseline (time 0) plasma cytokine levels compared with the values of each subsequent data collection time points (8, 24, 48, and 72 h).

RESULTS

There was a significant and sustained increase in plasma IL-6 levels at all time points when compared with baseline values. Paired values were used in the statistical analysis. Median plasma (interquartile range) values of IL-6 were elevated from 15.5 (9-52) pg/ml at baseline to 23 (4-48) pg/ml (P < 0.01) at 8 h; from 15 (2-48) pg/ml at baseline to 46 (21-105) pg/ml (P < 0.001) at 24 h; from 16 (9-52) pg/ml at baseline to 61 (32-170) pg/ml (P < 0.001) at 48 h; and from 15.5 (6-48) pg/ml to 68 (40-200) pg/ml (P < 0.001) at 72 h. Significance was maintained even when adjusted for multiple comparisons. The relative increase in IL-6 concentration was dose-dependent. Moreover, IL-6 elevation had a direct correlation with flavopiridol peak plasma concentration, flavopiridol area under the curve, and plasma C-Reactive protein levels. A significant decrease in plasma granulocyte macrophage colony-stimulating factor occurred at the 8-h sampling point: 50 pg/ml (interquartile range 10-205 pg/ml, P < 0.01) when compared with baseline plasma levels and 71 pg/ml (interquartile range 5-152 pg/ml, P < 0.01). No changes in the other pro or anti-inflammatory cytokines were observed. Immunohistochemistry studies in bone marrow aspirates from a prospective group of patients in this trial demonstrated approximately 4-fold induction of IL-6 (compared with baseline), mostly in non-T cells.

CONCLUSIONS

Biochemical analysis of plasma in patients undergoing infusional flavopiridol found a significant dose-dependent induction of IL-6. IL-6 elevation could be a marker for the process leading to the appearance of the proinflammatory syndrome observed in patients treated with infusional flavopiridol. The mechanism(s) underlying IL-6 induction and its significance are still unknown but may influence strategies to modulate flavopiridol's clinical effects.

This study examined the influence of the following <em>growth</em> <em>factors</em> and cytokines on early embryonic development: insulin-like <em>growth</em> <em>factors</em> I and II (IGF-I, IGF-II), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), transforming <em>growth</em> <em>factor</em> (TGF-beta), granulocyte-macrophage colony-stimulating <em>factor</em> (GM-CSF), and leukemia inhibitory <em>factor</em> (LIF). Synthetic oviduct fluid (SOF) was used as the culture medium. We studied the development of bovine embryos produced in vitro and cultured until Day 9 after fertilization. TGF-beta1, bFGF, GM-CSF, and LIF used on their own significantly improved the yield of hatched blastocysts. IGF-I, bFGF, TGF-beta1, GM-CSF, and LIF significantly accelerated embryonic development, especially the change from the expanded blastocyst to hatched blastocyst stages. Use of a combination of these <em>growth</em> <em>factors</em> and cytokines (GF-CYK) in SOF medium produced higher percentages of blastocysts and hatched blastocysts than did use of SOF alone (45% and 22% vs. 24% and 12%; P<0.05) on Day 8 after in vitro fertilization and similar results to use of SOF+10% fetal calf serum (38% and <em>16</em>%, at the same stages, respectively). The averages of total cells, inner cell mass cells, and trophectoderm cells of exclusively in vitro Day-8 blastocysts for pooled GF-CYK treatments were higher than those for SOF and similar to those for fetal calf serum. The presence of these <em>growth</em> <em>factors</em> and cytokines in the embryo culture medium therefore has a combined stimulatory action on embryonic development; in particular through an increase in hatching rate and in the number of cells of both the inner cell mass and trophoblast. These results are the first to demonstrate that use of a combination of recombinant <em>growth</em> <em>factors</em> and cytokine, as IGF-I, IGF-II, bFGF, TGF-beta1, LIF, and GM-CSF, produces similar results to 10% fetal calf serum for the development of in vitro-produced bovine embryos. This entirely synthetic method of embryo culture has undeniable advantages for the biosecurity of embryo transfer.

Acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) is a potent mitogenic and chemotactic agent for vascular endothelial cells, dermal <em>fibroblasts</em>, and epidermal keratinocytes, the principal cellular constituents of skin. To explore its potential to heal chronic dermal wounds, we applied pure recombinant human aFGF topically to full-thickness excisional injuries in healing-impaired genetically diabetic mice. Transformation of the nonlinear percent initial wound areas as a function of time to linear rates of tissue in<em>growth</em> from the original wound edges showed that aFGF increased wound closure in a dose-dependent manner. Optimal 3-micrograms/cm2 doses of aFGF nearly tripled the linear rate of healing. The median time to complete closure decreased from 46 d in vehicle-treated wounds to only <em>16</em> d in those treated with aFGF. Histomorphometric analyses established that aFGF increased granulation tissue formation and reepithelialization throughout healing. Vehicle- and aFGF-treated wounds appeared to be histologically equivalent by the time of closure. Therefore, aFGF has potential therapeutic applications for promoting healing of dermal ulcers, especially in healing-impaired individuals.

<AbstractText>Children with X-linked hypophosphataemia have high concentrations of circulating phosphatonin <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23), which causes renal phosphate wasting and hypophosphataemia, rickets, skeletal deformities, and <em>growth</em> impairment. Burosumab, a human monoclonal antibody against FGF23, improves phosphate homoeostasis and rickets in children aged 5-12 years with X-linked hypophosphataemia. We aimed to assess the safety and efficacy of burosumab in younger children with X-linked hypophosphataemia.</AbstractText><AbstractText>In this open-label, phase 2 trial at three hospitals in the USA, children (aged 1-4 years) with X-linked hypophosphataemia received burosumab (0·8 mg/kg) via subcutaneous injection every 2 weeks for 64 weeks. The dose was increased to 1·2 mg/kg if two consecutive pre-dose serum phosphorus concentrations were below 1·03 mmol/L (3·2 mg/dL), serum phosphorus had increased by less than 0·<em>16</em> mmol/L (<0·5 mg/dL) from baseline, and a dose of burosumab had not been missed. Participants could continue to receive burosumab for up to an additional 96 weeks during the extension period. Key inclusion criteria were age 1-4 years at the time of informed consent; fasting serum phosphorus concentration of less than 0·97 mmol/L (3·0 mg/dL); serum creatinine 8·8-35·4 μmol/L (0·1-0·4 mg/dL); radiographic evidence of rickets (at least five participants were required to have a Thacher Rickets Severity Score of ≥1·5 at the knee); and a confirmed PHEX mutation or a variant of unknown significance in the patient or direct relative also affected with X-linked hypophosphataemia. Conventional therapy was stopped upon enrolment. The coprimary endpoints were safety and change from baseline to week 40 in fasting serum phosphorus concentrations. Changes in rickets severity from baseline to weeks 40 and 64 (assessed radiographically using Thacher Rickets Severity Score and an adaptation of the Radiographic Global Impression of Change), and recumbent length or standing height, were key secondary outcomes. This trial is registered with ClinicalTrials.gov, number NCT02750618, and is ongoing.</AbstractText><AbstractText>Between May <em>16</em>, 20<em>16</em>, and June 10, 20<em>16</em>, we enrolled 13 children with X-linked hypophosphataemia. All 13 children completed 64 weeks of treatment and were included in the efficacy and safety analysis; none exceeded 70 weeks of treatment at the time of analysis. Serum phosphorus least squares mean increase from baseline to week 40 of treatment was 0·31 mmol/L (SE 0·04; 95% CI 0·24-0·39; 0·96 mg/dL [SE 0·12]; p<0·0001). All patients had at least one adverse event. 14 treatment-related adverse events, mostly injection site reactions, occurred in five children. One serious adverse event considered unrelated to treatment (tooth abscess) occurred in a child with a history of tooth abscess. All other adverse events were mild to moderate, except a severe food allergy considered unrelated to treatment. No instances of nephrocalcinosis or noteworthy changes in the results of a standard safety chemistry panel emerged. Total Thacher Rickets Severity Score decreased by a least squares mean of -1·7 (SE 0·1; p<0·0001) from baseline to week 40 and by -2·0 (SE 0·1; p<0·0001) by week 64. The Radiographic Global Impression of Change score also indicated significant improvement, with a least squares mean score of +2·3 (SE 0·1) at week 40 and +2·2 (0·1) at week 64 (both p<0·0001). Mean length or standing height Z score was maintained from baseline to week 64.</AbstractText><AbstractText>Burosumab had a favourable safety profile, increased serum phosphorus, and improved rickets and prevented early declines in <em>growth</em> in children aged 1-4 years with X-linked hypophosphataemia. These findings could substantially alter the treatment of young children with X-linked hypophosphataemia.</AbstractText><AbstractText>Ultragenyx Pharmaceutical and Kyowa Kirin International.</AbstractText>

We have previously established an ovariectomized (OVX) ewe model to study how steroid removal and replacement affects uterine blood vessel and tissue <em>growth</em>. Using this model, endometrial expression of mRNA for 14 angiogenic <em>factors</em> (7 genes and their respective receptors) in caruncular (CAR) and intercaruncular (ICAR) endometrium were evaluated by quantitative real time RT-PCR at 0 (control), 2, 4, 8, <em>16</em>, or 24 h after treating OVX ewes with an estradiol-17beta (E2) implant. In CAR and ICAR, compared to 0 h, the mRNA expression of vascular endothelial <em>growth</em> <em>factor</em> (VEGF), VEGF receptor (R)1, soluble guanylate cyclase (GUCY1B3; the R for nitric oxide [NO]), hypoxia inducible <em>factor</em> (HIF)1alpha, and placental <em>growth</em> <em>factor</em> (PlGF) increased by 4 h after E2-treatment, but basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF2), endothelial NO synthase (NOS3), angiopoietin (ANGPT)1, ANGPT2, ANGPT receptor Tie2 by 2 h after E2. Expression of mRNA for FGFR2 IIIc was increased at 2 h by E2-treatment in ICAR, but not in CAR. By contrast, expression of neuropilin (NP)1 mRNA was increased at 2 h in CAR, but not ICAR. The mRNA expression of VEGF, FGF2, HIF1 alpha, and PlGF was positively correlated with mRNA expression of NOS3, VEGFR1, and Tie2 suggesting some E2-stimulated interactions between these <em>factors</em> in promoting blood vessel <em>growth</em>. Thus, several major angiogenic <em>factors</em> and their receptors are increased within hours after E2-treatment, which indicates that E2 plays a role in regulation of angiogenesis in the uterus. By using the OVX ewe model, we may begin to understand the molecular basis of E2 effects on angiogenesis in the endometrium and, eventually, how angiogenesis is regulated in normal versus pathological conditions.

Alzheimer's disease is characterized mainly by loss of neurons from the septal nucleus. In this study, neurons from the septal nucleus of the embryonic day <em>16</em> (E<em>16</em>) rat were grown in culture with a plane of astrocytes from the embryonic rat and in a defined medium in the absence of serum. Neurons were treated with beta-amyloid (Abeta: 0.1, 1 and 10 microM) on day in vitro (DIV) 1 and DIV 4 and fluorescent microscopy was used to measure survival and apoptosis following exposure of the treated cells on DIV 7. Reversal of neurotoxicity was studied using the potentially neuroprotective agents nerve <em>growth</em> <em>factor</em> (NGF, 100 ng/ml), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF, 5 ng/ml), insulin-like <em>growth</em> <em>factors</em> (IGF1 and IGF2, 10 ng/ml) and estrogen (10 nM), administered on DIV 4 and DIV 5, that is, subsequent to the Abeta (10 microM)-induced neurotoxicity. Abeta caused a significant decrease in survival at 10 microM, and a significant increase in apoptosis at 0.1 and 10 microM. IGF1, IGF2 and bFGF all caused a reversal of the Abeta-induced neurotoxic effect on survival while NGF and estrogen did not under these experimental conditions.

Disruption of the X-chromosome <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>16</em> (Fgf-<em>16</em>) gene, a member of the FGF-9 subfamily with FGF-20, was linked with an effect on cardiac development in two independent studies. However, poor trabeculation with lethality by embryonic day (E) 11.5 was associated with only one, involving maintenance in Black Swiss (Bsw) versus C57BL/6 mice. The aim of this study was to examine the potential influence of genetic background through breeding the null mutation onto an alternate (C57BL/6) background. After three generations, 25% of Fgf-<em>16</em>(-/Y) mice survived to adulthood, which could be reversed by reducing the contribution of the C57BL/6 genetic background by back crossing to another strain. There was no significant difference between FGF-9 and FGF-20 RNA levels in Fgf-<em>16</em> null versus wild-type mice regardless of strain. However, FGF-8 RNA levels were reduced significantly in Bsw but not C57BL/6 mice. FGF-8 is linked to anterior heart development and like the FGF-9 subfamily is reportedly expressed at E10.5. Like FGF-<em>16</em>, neuregulin as well as signaling via ErbB2 and ErbB4 receptors have been linked to trabeculae formation and cardiac development around E10.5. Basal neuregulin, ErbB2, and ErbB4 as well as FGF-8, FGF-9, and FGF-<em>16</em> RNA levels varied in Bsw versus C57BL/6 mice. These data are consistent with the ability of genetic background to modify the phenotype and affect embryonic survival in Fgf-<em>16</em> null mice.

Developmentally competent bovine blastocysts were produced by adding transforming <em>growth</em> <em>factor</em> beta (TGF beta) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) to serum-free cultures of in vitro produced, 2-cell bovine embryos. The effects of TGF beta were evaluated because this <em>growth</em> <em>factor</em> signals synthesis and secretion of the extracellular matrix component fibronectin and its receptor. Previous investigations have demonstrated that fibronectin promotes early bovine embryo development in vitro. The effects of TGF beta can be potentiated by bFGF; bFGF itself is an effector of protein synthesis and a potent mitogen. A positive interaction between the 2 <em>growth</em> <em>factors</em> resulted in 38.8% of fertilized oocytes maturing beyond the <em>16</em>-cell stage; of these, 24.6% formed blastocysts. Transfer of early blastocysts produced using serum-free medium supplemented with <em>growth</em> <em>factors</em> resulted in pregnancy in 3 of 9 recipients. These results support the hypothesis that TGF beta and bFGF act synergistically to promote development of bovine embryos beyond the "8-cell block" observed in vitro.

We characterized a gene of the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) homologous to the mammalian <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family. We termed it vfgf, and examined its transcription and the properties of the gene product (vFGF). RT-PCR analysis showed that vfgf is one of the baculovirus early genes, although there are no consensus sequences of the baculovirus early gene promoters. 5'-RACE analysis revealed that its transcription started at 10 nucleotides upstream of the translation start codon. vFGF has a hydrophobic amino terminus (approximately <em>16</em> amino acids), which is a typical signal sequence. As expected, vFGF was efficiently secreted from BmNPV-infected BmN cells. Because possible glycosylation sites are found at positions 44 (Asn) and 171 (Asn), we examined whether BmNPV vFGF is glycosylated or not. Cleavage of recombinant vFGF with PNGase F revealed that BmNPV vFGF was glycosylated. We also found that secretion of vFGF is completely blocked by the treatment of Tunicamycin, which blocks N-linked glycosylation. This is the first report to characterize a virus-encoded FGF.

The structural gene for human keratinocyte <em>growth</em> <em>factor</em> (KGF), a member of the <em>fibroblast</em> <em>growth</em> <em>factor</em> family, consists of three coding exons and two introns typical of other <em>fibroblast</em> <em>growth</em> <em>factor</em> loci. A portion of the KGF gene, located on chromosome 15, is amplified to approximately <em>16</em> copies in the human genome, and these highly related copies (which consist of exon 2, exon 3, the intron between them, and a 3' noncoding segment of the KGF transcript) are dispersed to multiple human chromosomes. The KGF-like sequences are transcriptionally active, differentially regulated in various tissues, and composed of three distinct classes of coding sequences that are 5% divergent from each other and from the authentic KGF sequence. Multiple copies of KGF-like genes were also discovered in the genomic DNAs of chimpanzee and gorilla but were not found in lesser apes (gibbon), Old World monkeys (African green monkey and macaques), mice, or chickens. The pattern of evolutionary occurrence suggests that a primordial KGF gene was amplified and chromosomally dispersed subsequent to the divergence of orangutan from African apes but before the trichotomous divergence of human, chimpanzee, and gorilla 5-8 million years ago. The appearance of a transcriptionally active and chromosomally dispersed multigene KGF family may have implications in the evolution of the great apes and humans.

We tested the hypothesis that aged animals are as responsive as the young adult animals in expanding collateral vasculature under a similar treatment of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). Two age groups of male Fischer 344 rats (11 mo old; n = 32, 23 mo old; n = 43) weighing approximately 385 g were subdivided into normal, acute ligation [femoral artery (FA) ligated 3 days before blood flow (BF) measurement] or ligated groups for <em>16</em> days and received recombinant human bFGF intra-arterial infusion at doses of 0, 0.5, 5, and 50 microg x kg(-1) x day(-1). BF was determined with (85)Sr- and (141)Ce-labeled microspheres during treadmill running at 15 and 20 m/min at 15% grade. Blood pressure (BP) values were approximately 149 and approximately <em>16</em>3 mmHg (p < 0.05); heart rates were approximately 496 and approximately 512 beats/min in the aged and young adult groups during running, respectively. Maximal collateral BF values were confirmed by no additional BF increase in the calf muscle at the higher speed. Ligation of the FA for 3 days reduced the BF reserve to the calf muscle by approximately 90%. Calf muscle BF was modestly greater (10 ml x min(-1) x 100 g(-1)) by <em>16</em> days in the carrier group. bFGF infusion expanded collateral BF in a dose-dependent manner with an increase of 33 and 42 ml x min(-1) x 100 g(-1) (P < 0.001) in the 5 and 50 microg x kg(-1) x day(-1) bFGF groups, respectively. Aged animals showed similar BF improvements as observed with the adult groups in response to ligation surgery and bFGF treatment. Our data indicate that the aged rats (approximately 23 mo old) remain responsive to exogenous bFGF induced in developing collateral-dependent BF as the young adult (approximately 11 mo old) controls. This suggests that the influence of bFGF in expanding collateral BF should not be preempted in the aged group, the population most affected by peripheral arterial insufficiency.

A cascade of signaling events triggers myogenesis in vertebrates. Although studies of zebrafish indicate that <em>fibroblast</em> <em>growth</em> <em>factor</em> (Fgf), Hedgehog (Hh), and the T-box transcription <em>factors</em>, No tail (Ntl) and T-box gene <em>16</em> (Tbx<em>16</em>), regulate myogenesis, the hierarchy of these <em>factors</em> has not been determined. Recently, another transcriptional co<em>factor</em>, Smarcd3, a subunit of the SWI/SNF chromatin-remodeling complex, has been shown to be required for heart muscle formation in mouse. In zebrafish, fgf8 and ntl expression commences during blastula stages, whereas myogenesis, as indicated by myod expression, does not begin until much later during mid-gastrula stages. smarcd3b expression, on the other hand, becomes enriched in the marginal zone just prior to the beginning of myod expression. Overexpression of smarcd3 shifts the onset of myod and myf5 expression earlier, and myod and myf5 expression in adaxial cells, the earliest muscle precursors, requires Smarcd3, indicating that Smarcd3 is the limiting <em>factor</em> that regulates the onset of myogenesis. Smarcd3 physically interacts with Ntl, and Smarcd3 overexpression fails to rescue myod expression in ntl mutants, demonstrating that function of Smarcd3 depends on Ntl activity. We propose a model in which cooperative activity of Fgf, Ntl, and Smarcd3 is required for the onset of myogenesis, with Smarcd3b serving as the primary regulator of the timing of myogenesis onset.

The relationship of production of interleukin 1 (IL-1)-like <em>factor</em> to accessory function of Epstein-Barr virus (EBV)-transformed B lymphocytes was examined. Six of eight human EBV-B cell lines spontaneously produced and released detectable levels of thymocyte comitogenic <em>factor</em> in vitro, but no interleukin 2 (IL-2) activity. Eight of eight produced <em>fibroblast</em> proliferation activity. Culture supernatants from the two apparent nonproducers of thymocyte comitogenic activity induced the proliferation of the IL-1-dependent murine helper-T-cell clone D10G4.1 in the presence of concanavalin A (Con A). One of the EBV-B cell lines produced a potent inhibitory <em>factor</em> in addition to IL-1-like thymocyte comitogenic and <em>fibroblast</em> proliferation <em>factors</em>. The inhibitory <em>factor</em> inhibited mouse thymocyte proliferative response to Con A, and the proliferation of the IL-2-dependent CT6 cell line, but not human <em>fibroblast</em> <em>growth</em>. All but one of the eight EBV-B cell lines tested, the exception being the line that produced an inhibitory <em>factor</em>, were able to serve as antigen-presenting cells that enabled purified human T lymphocytes to proliferate in one-way mixed lymphocyte reactions (MLR) and in response to Con A. The supernatants of 14 of <em>16</em> clones derived from two of the EBV-B cell line cells contained thymocyte comitogenic activity and all <em>16</em> stimulated <em>fibroblast</em> proliferation. The phenotypic characteristics of the EBV-B cell lines were heterogeneous, but there was no clear-cut relationship between the cell surface phenotypes of either the cloned or uncloned EBV-B cells and their ability to produce these <em>factors</em>. These studies show that all of the EBV-B cell lines that can function as accessory cells have the capacity to produce an IL-1-like <em>factor</em>.

Although insulin-like <em>growth</em> <em>factor</em>-I (IGF-I) is known as an important stimulator of collagen biosynthesis in collagen-producing cells, the mechanism and endpoints by which it regulate the process remain largely unknown. Serum of acutely fasted rats contained reduced amount of IGF-I (72+/-<em>16</em> ng/ml) and showed about 75% reduced ability to stimulate collagen and DNA synthesis in confluent human skin <em>fibroblasts</em> in comparison to the effect of control rat serum (IGF-I, <em>16</em>8+/-19 ng/ml). An addition of IGF-I (at least 40 ng/ml) to fasted rat serum restored its mitogenic activity but could not restore its ability to stimulate collagen biosynthesis to control values during 24 h of incubation. However, when the cells were incubated in fasted rat serum supplemented with 40 ng/ml of IGF-I for 48 h, collagen biosynthesis was restored to control values. It suggests that the stimulatory role of IGF-I in collagen biosynthesis undergo indirectly. We considered pyrroline-5-carboxylate (P5C) as a candidate to play a direct role in this process. Since IGF-I and P5C are known to be decreased in serum of fasted rats it seems that the action of IGF-I on collagen biosynthesis may involve participation of P5C. We have found that serum of fasted rats (showing low level of P5C) supplemented with 1 mmol/l P5C induced collagen biosynthesis in confluent human skin <em>fibroblasts</em> during 24 h to control values. Supporting evidence comes from the experiment showing stimulatory action of P5C on collagen biosynthesis in <em>fibroblasts</em> cultured in serum-free medium. Our results postulate potential role of P5C in regulation of collagen biosynthesis and indicate participation of this molecule in the pathway of IGF-I action in this process.

Camelpox virus (CMLV) gene 176R encodes a protein with sequence similarity to murine schlafen (m-slfn) proteins. In vivo, short and long members of the m-slfn family inhibited T-cell development, whereas in vitro, only short m-slfns caused arrest of <em>fibroblast</em> <em>growth</em>. CMLV 176 protein (v-slfn) is most closely related to short m-slfns; however, when expressed stably in mammalian cells, v-slfn did not inhibit cell <em>growth</em>. v-slfn is a predominantly cytoplasmic 57 kDa protein that is expressed throughout infection. Several other orthopoxviruses encode v-slfn proteins, but the v-slfn gene is fragmented in all sequenced variola virus and vaccinia virus (VACV) strains. Consistent with this, all <em>16</em> VACV strains tested do not express a v-slfn detected by polyclonal serum raised against the CMLV protein. In the absence of a small animal model to study CMLV pathogenesis, the contribution of CMLV v-slfn to orthopoxvirus virulence was studied via its expression in an attenuated strain of VACV. Recombinant viruses expressing wild-type v-slfn or v-slfn tagged at its C terminus with a haemagglutinin (HA) epitope were less virulent than control viruses. However, a virus expressing v-slfn tagged with the HA epitope at its N terminus had similar virulence to controls, implying that the N terminus has an important function. A greater recruitment of lymphocytes into infected lung tissue was observed in the presence of wild-type v-slfn but, interestingly, these cells were less activated. Thus, v-slfn is an orthopoxvirus virulence <em>factor</em> that affects the host immune response to infection.

De novo point mutations arise predominantly in the male germline and increase in frequency with age, but it has not previously been possible to locate specific, identifiable mutations directly within the seminiferous tubules of human testes. Using microdissection of tubules exhibiting altered expression of the spermatogonial markers MAGEA4, FGFR3, and phospho-AKT, whole genome amplification, and DNA sequencing, we establish an in situ strategy for discovery and analysis of pathogenic de novo mutations. In 14 testes from men aged 39-90 y, we identified 11 distinct gain-of-function mutations in five genes (<em>fibroblast</em> <em>growth</em> <em>factor</em> receptors FGFR2 and FGFR3, tyrosine phosphatase PTPN11, and RAS oncogene homologs HRAS and KRAS) from <em>16</em> of 22 tubules analyzed; all mutations have known associations with severe diseases, ranging from congenital or perinatal lethal disorders to somatically acquired cancers. These results support proposed selfish selection of spermatogonial mutations affecting <em>growth</em> <em>factor</em> receptor-RAS signaling, highlight its prevalence in older men, and enable direct visualization of the microscopic anatomy of elongated mutant clones.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> receptors (FGFRs) play an important role in development and tumorigenesis. Mutations in FGFR2 cause more than five craniosynostosis syndromes. The FGFR2 genomic structure is the largest of the FGFR family. We have refined and extended the genomic organization of the FGFR2 gene by sequencing more than 119 kb of PACs, cosmids, and PCR products and assembling a region of approximately 175 kb. Although the gene structure has been reported to include only 20 exons, we have verified the presence of at least 22 exons, some of which are alternatively spliced. The sizes of six exons differed from those reported previously. Comparison of our sequence and those in the NCBI database detected more than 300 potential single nucleotide polymorphisms (SNPs). However, sequencing regions containing 52 of these potential SNPs verified only 14 in PCR products generated from <em>16</em> CEPH alleles. In contrast, direct sequencing of the CEPH DNAs revealed 21 other polymorphisms. Only one SNP was found in the 2,926 bp of coding sequence. Twenty-seven SNPs, two insertion polymorphisms and five microsatellite polymorphisms are contained in approximately <em>16</em>.6 kb of non-coding sequence. These data yield an average of one polymorphism for approximately 488 bp of non-coding sequence examined. This collection of SNP, insertion, and repeat polymorphisms will aid future association studies between the FGFR2 gene and human disease and will enhance mutation detection.

Angiogenesis is essential in tissue <em>growth</em> and regeneration. There are several <em>factors</em> that are able to stimulate vascular endothelial cell <em>growth</em>, including platelet-derived <em>growth</em> <em>factor</em> (PDGF) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF). Disc herniation tissue (DHT) contains vascular in<em>growth</em>, which promotes granulation tissue formation. In this study we observed 50 disc herniations for PDGF and VEGF immunoreactivity. PDGF immunopositivity was detected in 38 samples (78%). In 28 samples (56%) there were PDGF immunopositive capillaries, PDGF immunopositive disc cells were detected in 19 samples (38%) and PDGF immunopositive <em>fibroblasts</em> in 6 DHT samples (12%). VEGF immunopositive capillaries were identified in 44 DHT samples (88%). For neither <em>growth</em> <em>factor</em> was immunopositivity dependent on preoperative radicular pain duration. In extrusions (n = 25) VEGF immunopositive capillaries were detected in 23 samples (92%) and PDGF immunopositivity in 21 samples (84%). PDGF immunopositivity was more commonly associated with capillaries than with nuclei of disc cells. In sequesters (n = 20) VEGF immunopositive capillaries were identified in all samples and PDGF immunopositivity in <em>16</em> (80%). As in extrusions, PDGF immunoreaction was more prevalent in capillaries than in disc cells. Patient age did not relate to VEGF expression. In all age groups it was higher than 80%. Thus capillaries in disc herniation tissue are evidently newly formed and our results demonstrate that PDGF and VEGF participate in the neovascularization process. The presence of PDGF in <em>fibroblasts</em> and in disc cells suggests that this <em>growth</em> <em>factor</em> regulates the function of these cells, possibly the proliferation of the cells and the production of extracellular matrix components.

The Saccharomyces cerevisiae cwh43-2 mutant, originally isolated for its Calcofluor white hypersensitivity, displays several cell wall defects similar to mutants in the PKC1-MPK1 pathway, including a <em>growth</em> defect and increased release of beta-1,6-glucan and beta-glucosylated proteins into the <em>growth</em> medium at increased temperatures. The cloning of CWH43 showed that it corresponds to YCR017c and encodes a protein with 14-<em>16</em> transmembrane segments containing several putative phosphorylation and glycosylation sites. The N-terminal part of the amino acid sequence of Cwh43p shows 40% similarity with the mammalian FRAG1, a membrane protein that activates the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor of rat osteosarcoma (FGFR2-ROS) and with protein sequences of four uncharacterized ORFs from Caenorhabditis elegans and one from Drosophila melanogaster. The C-terminus of Cwh43p shows low similarities with a xylose permease of Bacillus megaterium and with putative sugar transporter from D. melanogaster, and has 52% similarity with a protein sequence from a Schizosaccharomyces pombe cDNA. A Cwh43-GFP fusion protein suggested a plasma membrane localization, although localization to the internal structure of the cells could not be excluded, and it concentrates to the bud tip of small budded cells and to the neck of dividing cells. Deletion of CWH43 resulted in cell wall defects less pronounced than those of the cwh43-2 mutant. This allele-specific phenotype appears to be due to a G-R substitution at position 57 in a highly conserved region of the protein. Genetic analysis places CWH43 upstream of the BCK2 branch of the PKC1 signalling pathway, since cwh43 mutations were synthetic lethal with pkc1 deletion, whereas the cwh43 defects could be rescued by overexpression of BCK2 and not by high-copy-number expression of genes encoding downstream proteins of the PKC1 pathway However, unlike BCK2, whose disruption in a cln3 mutant resulted in <em>growth</em> arrest in G(1), no <em>growth</em> defect was observed in a double cwh43 cln3 mutants. Taken together, it is proposed that CWH43 encodes a protein with putative sensor and transporter domains acting in parallel to the main PKC1-dependent cell wall integrity pathway, and that this gene has evolved into two distinct genes in higher eukaryotes.

Levels of multiplication-stimulating activity (MSA) in fetal rat serum are high (2-4 micrograms/ml), suggesting that MSA may have a role in fetal <em>growth</em>. We now demonstrate that <em>fibroblasts</em> derived from rat embryos (REFs) have specific MSA receptors and respond to MSA with increased DNA synthesis. Two types of insulin-like <em>growth</em> <em>factor</em> (IGF) receptors were demonstrated by competitive binding of radioiodinated MSA, IGF-I, and IGF-II and by chemical cross-linking of [125I]iodo-MSA and [125I]iodo-IGF-I to REFs. One type of receptor (mol wt, 260,000 under reducing conditions) did not interact with insulin, and another type of receptor (mol wt, 130,000, under reducing conditions) was recognized by insulin. Scatchard analysis of [125I]iodo-MSA binding data was consistent with one class of noninteracting binding sites. A biological response of MSA, increased DNA synthesis, was demonstrated with autoradiography in REFs. During a <em>16</em>-hr incubation, DNA synthesis was stimulated by normal rat serum, and platelet-poor plasma plus platelet-derived <em>growth</em> <em>factor</em> (PDGF), but not by serum from hypophysectomized (hypox) rats or hypophysectomized (hypox) platelet-poor plasma plus PDGF. However, when MSA was added to hypox serum or to hypox platelet-poor plasma plus PDGF, DNA synthesis was stimulated to the level achieved by normal rat serum. By contrast, during a longer cell multiplication experiment, REFs grew equally well in normal or hypox rat serum, raising the possibility that REFs may produce a MSA-like <em>factor</em>.

Hepatitis C virus (HCV) often causes persistent infection in humans. This could be due in part to the effect of viral proteins on cellular gene expression. Earlier observations suggest that the HCV core protein expressed from genotype 1a modulates important cellular genes at the transcriptional level, affects programmed cell death (apoptosis) and promotes cell <em>growth</em>. Recently, different groups of investigators have reported the translation of an approximately <em>16</em> kDa protein (named F/ARFP/core+1 ORF) from an alternate open reading frame of the HCV core-encoding genomic region. The functional significance of this F protein is presently unknown. Thus, whether the F and core proteins have both shared and distinct functions was investigated here. The experimental observations suggested that the F protein does not significantly modulate c-myc, hTERT and p53 promoter activities, unlike the HCV core protein. Interestingly, the F protein repressed p21 expression. Further studies indicated that the F protein does not inhibit tumour necrosis <em>factor</em> alpha-mediated apoptosis of HepG2 cells or promote rat embryo <em>fibroblast</em> <em>growth</em>. Taken together, these results suggest that the F protein does not share major properties identified previously for the HCV core protein, other than regulating p21 expression.

Surgical biopsies from ten head and neck squamous cell carcinomas were labeled in vitro with bromodeoxyuridine. In histological sections, bromodeoxyuridine-positive nuclei and beta-<em>fibroblast</em> <em>growth</em> <em>factor</em> (beta-FGF) were stained using immunohistochemistry. In clearly discernible clusters of tumor cells, the cytoplasm shows strong positive beta-FGF staining, whereas other tumor regions are completely beta-FGF negative. Within positively stained areas, the tumor cell bromodeoxyuridine labeling index is higher in comparison to beta-FGF-negative areas by a <em>factor</em> of 5 +/- 0.8. This is reflected in a positive correlation of the tumor cell labeling index and the relative extent of beta-FGF-positive tumor areas. Viable tumor areas bordering on necrosis, which are known to be hypoxic, are beta-FGF negative. The average tumor endothelial cell labeling index was 1.8 +/- 0.6%, as compared to 0.<em>16</em>% in adjacent normal mucosa. Since endothelial cell pulse labeling indices are too low for a further quantitative analysis, the relationship of beta-FGF expression and endothelial cell turnover was studied in more detail in two fairly well-differentiated murine squamous cell carcinoma lines (AT 84 and AT 478). Labeling indices were higher and endothelial cell doubling times were significantly shorter in beta-FGF-positive as compared to beta-FGF-negative tumor areas (AT 84, 9.3 h versus 25.4 h; AT 478/25, 6.8 h versus <em>16</em> h). Thus, the discrete expression of beta-FGF is associated with regional differences in endothelial cell kinetics. In two generations of the tumor line AT 478, characterized by different volume doubling times of 18 days (AT 478/25) and 36 days (AT 478/4), beta-FGF-positive areas represent 75.5 +/- 6% and 19.7 +/- 7% of the viable tumor tissue, respectively. This indicates a correlation between beta-FGF production of tumor cells and <em>growth</em> rate.