OBJECTIVE

To investigate the expression of fibroblast growth factor receptor-4 (FGFR4) in fetal kidneys and pathological kidneys of children in order to show the roles of fibroblast growth factor (FGF) and FGFR4 in the development of fetal kidneys and renal diseases.

METHODS

The expression of FGFR4 was detected by immumohistochemistry in the normal fetal kidney at 8 to 34 weeks of gestation age (n=18) and 82 children with renal disease, including 28 cases of primary nephrotic syndrome (PNS), 12 acute glomerulonephritis (AGN), 20 Henoch-Schoenlein purpura nephritis (HSPN), and 22 isolated hematuria (IHU). A correlation analysis between renal pathological scores and FGFR4 expression was performed.

RESULTS

1) FGFR4 expression was weakly in renal vesicle and primitive tubules of S-shaped body, irrecognizable in urteric bud and podocytes of C-stage, and negative in mesenchyme and condensing mesenchyme. The immunostaining of FGFR4 was intense in distal tubules and collecting ducts, but was negative in mature glomeruli and proximal tubules. 2) FGFR4 was expressed in all pathological sections of various renal diseases. FGFR4 expression was intense in tubules but weak in glomeruli. It was principally expressed in distal tubules and partially in proximal tubules. The tubules with very strong expressions of FGFR4 presented abnormal structures including dilation and atrophy, especially in proximal tubules. 3) There were no significant differences in the FGFR4 expression in various parts of the kidney among various renal diseases. There were also no significant differences in the FGFR4 expression in renal tubules among the four different pathological types of renal diseases: focal segmental glomerularsclerosis (FSGS), membranoproliferative glomerulonephritis (MPGN), mesangial proliferative glomerulonephritis (MsPGN), and minimal change disease (MCD). The FGFR4 expression in podocytes in the MPGN group was noticeably higher than that of the other pathological type group (P < 0.05). 4) The FGFR4 expression in proximal tubules positively correlated with the pathological score of tubules (r=0.463682, P < 0.05) but negatively correlated with the pathological score of glomeruli (r=- 0.0277, P < 0.05). The FGFR4 expression in both distal tubules and podocytes negatively correlated with the pathological score of tubules or glomeruli (P < 0.05).

CONCLUSIONS

The development of fetal kidneys in the early period could not be regulated by FGF-FGFR4 signal which takes part in the development of renal tubules and collecting duct in the mature period. The FGFR4 expression is related with renal pathology in children with PNS, AGN, HSPN or IHU. A proper increase of FGFR4 expression is beneficial to the recovery of renal tissues but an over-expression relates to a severe renal damage.

OBJECTIVE

To explore the expression of bFGF gene in normal and wounded rat skeletal muscles.

METHODS

In situ hybridization and quantitative PCR methods were used to evaluate the location of basic fibroblast growth factor (bFGF) mRNA and the amount of bFGF mRNA expression in normal, ischemic or ischemic and reperfused rat skeletal muscles.

RESULTS

According to the intensity of the stain in situ hybridization, four main classes of fibers could be identified: strong, moderate, weak and negative stained fibers. bFGF mRNA was found in the cytoplasm in both normal and wounded muscles; however, the signal intensity was much stronger in normal muscles. In the normal muscles, bFGF mRNA was distributed irregularly and sparsely, and 82% of fibers had positive bFGF mRNA staining, and only 52% and 22% of fibers in ischemic or ischemic and reperfused muscles were positive for bFGF mRNA staining in situ hybridization. The results in quantitative PCR study supported the concept of the reduced bFGF mRNA expression in the wounded muscles.

CONCLUSIONS

The loss of stored bFGF in wounded skeletal muscles as we showed previously is also caused by its increased damage and reduced secretion.

In squamous cell carcinoma (SCC) of the lung, mutations within the genes of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptors (FGFR) such as K660N/K660E in FGFR2 and R248C/S249C in FGFR3 and FGFR1 gene amplification have been described, but their prognostic relevance still remains unclear. In order to detect the mutation frequencies and to define their prognostic value for associated clinicopathologic features and survival of patients, resected ΔNp63/p40-positive SCC of the lung (n = 101) were screened for FGFR1 gene amplification by fluorescence in situ hybridization performed on formalin-fixed paraffin embedded tissues and for the presumed driver mutations in genes of FGFR2 and FGFR3 by PCR and Sanger sequencing. Twenty-two of 101 SCCs (<em>22</em>%) were positive for amplification based on a FGFR1/centromere (chromosome 8) ratio>> 2.0 or higher. In advanced tumor stages (III-IV), the overall survival of patients carrying FGFR1 gene amplification was significantly higher (p = 0.006). Among women, FGFR1 gene amplification was significantly associated with longer overall survival (p = 0.023). The presence of FGFR1 gene amplification was associated with patient age (65 versus 69 years, p = 0.046), but not with gender, tumor stage, histologic subtype, tumor grade, or ΔNp63/p40 immunoreactivity. The S249C mutation in the FGFR3 gene was identified in one out of 101 SCCs (1%); the K600N, K660E, or R248C mutations were not identified. These results suggest that FGFR1 gene amplification is a frequent alteration in SCC of the lung and appears not to be a negative but rather a favorable prognostic marker for women and particularly for patients with advanced SCC of the lung (stage III-IV).

<AbstractText>The behavior of pleural fluid cytokine (PFCs) levels and their association with pleurodesis after indwelling pleural catheter (IPC) placement is unknown.</AbstractText><AbstractText>A prospective exploratory study was conducted to obtain preliminary data on PFC levels after IPC placement.</AbstractText><AbstractText>The PFC panel consisted of 4 cytokines [interleukin -8 (IL-8), vascular endothelial <em>growth</em> <em>factor</em>, total (but not activated) transforming <em>growth</em> <em>factor</em> betas, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>], measured across 5 time points (T0: insertion; T1: 24 to 48 h; T2: 72 to 96 h; T3: 1 wk; and T4: 2 wk). Profile plots were used to identify patterns of change of PFC levels. Correlation matrices for each PFC over time were computed, and area under the curve (AUC) categories were used to compare the cumulative incidence of IPC removal. Auto pleurodesis was defined as elective catheter removal because of decreased drainage within 90 days of insertion.</AbstractText><AbstractText>A total of <em>22</em> patients provided complete data. Except for IL-8, the majority of PFCs demonstrated strong positive correlations across measurement time points. Patients with high AUCs for IL-8, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and vascular endothelial <em>growth</em> <em>factor</em> had a higher cumulative incidence of IPC removal by 90 days than did patients with low AUCs.</AbstractText><AbstractText>This is the first study to evaluate longitudinal changes of pleural cytokine levels with respect to the likelihood of IPC removal and provide early evidence that the cytokine profile may be associated with the outcome of pleurodesis induced by IPCs. However, this is an exploratory study and further studies are needed to assess if these findings can be validated in further studies.</AbstractText>

We recently generated transgenic mice expressing the RET proto-oncogene with a multiple endocrine neoplasia type 2A mutation (RET-MEN2A). Mammary tumors with frequent lung metastasis were developed in <em>22</em>% of female transgenic mice in a stochastic fashion. In the current study, we established two cell lines (named MKK-f and MKK-s) from mammary tumors developed in RET-MEN2A transgenic mice. MKK-f and MKK-s were derived from well-differentiated ductal carcinoma and sarcomatous spindle cell carcinoma, respectively. MKK-f cells show epithelial-like morphology with a doubling time of 19 h, and MKK-s cells show spindle-shaped morphology with a doubling time of 15 h. When inoculated in immunodeficient mice, both cell lines were tumorigenic, metastasized to the lung and displayed histological features similar to those of the primary tumors. They maintained a high level of RET expression and activation of signaling molecules downstream of RET. Consistent with the histological phenotype, expression of E-cadherin was almost undetectable in MKK-s cells, whereas its expression was very high in MKK-f cells. When the difference of gene expression between the two cell lines was analyzed using cDNA microarrays including approximately 900 genes/ESTs, a total of 21 up- or down-regulated >> 2.0-fold) genes were identified. Differentially regulated genes included thymosin beta-10, <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 4, aldo-keto reductase and caspase 6 genes, which are known to be associated with tumor development and progression. These results may reflect the profiles of the transcriptional changes associated with dedifferentiation or progression of mammary carcinomas developed in genetically engineered mice.

Breast cancer is a malignancy with a strong heritable component. Genetic counseling has been principally focused on families carrying high-penetrance breast cancer 1/2, early onset genes. Current modeling suggests that the majority of the unexplained fraction of familial risk is likely to be explained by a polygenic model. The aim of the present study was to estimate the heritability (h2) of breast cancer susceptibility through the analysis of 6 single nucleotide polymorphisms (SNPs), nuclear mitotic apparatus protein 1, cyclin D1, cytochrome C oxidase copper chaperone, <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2, TOX high mobility group box family member 3 and solute carrier family 4 member 7. These 6 SNPs, previously identified by genome-wide association studies, were considered to evaluate the additive and common environmental components that contribute to the development of breast cancer in nuclear (pedigrees including only first degree relationships) and in extended families (with at most third degree relationships). A total of <em>22</em> extended pedigrees, subsequently split into 52 nuclear pedigrees were analyzed. An example of splitting process from extended to nuclear pedigree is shown in Fig. 1. Firstly, an underline latent continuous trait (Y*) using breast cancer status and information of 6 breast cancer-associated SNPs was calculated. This novel trait summarized the susceptibility of breast cancer in each individual. Secondly, the h2 of Y* was estimated using an additive polygenic-common environment-unique error model. h2 was evaluated in extended and immediate pedigrees, obtaining comparable results. h2 accounts for ~40% of the total phenotypic variance, indicating a fairly strong additive genetic effect of breast cancer susceptibility. The present study indicated the importance of the evaluation and consideration of these six SNPs, which can be used as instrumental variables in order to obtain improved genetic models that are useful for h2 analysis.

Background: Experimental studies have shown fibroblast growth factor 23 (FGF23)-mediated upregulation of the distal tubule sodium/chloride (Na⁺Cl^-) co-transporter leading to increased Na reabsorption, volume expansion and hypertension. However, data on the associations of FGF23 with renal Na regulation and blood pressure (BP) are lacking in young CKD patients.

Methods: FGF23 and other determinants of mineral metabolism, plasma renin activity (PRA), fractional excretion of Na (FENa) and BP, were analyzed at a single center in 60 patients aged 5-22 years with CKD Stages 1 (n = 33) and Stages 2-3 (n = 27) defined by cystatin C- and creatinine-based estimating equations (estimated glomerular filtration rate, eGFR). Associations between FGF23 and renal Na handling were explored by regression analysis.

Results: Median FGF23 levels were higher in CKD Stages 2-3 versus CKD 1 (119 versus 79 RU/mL; P < 0.05), with hyperparathyroidism [parathyroid hormone (PTH) >69 pg/mL] in only few subjects with CKD Stages 2-3. Median FENa was comparable in both subgroups, but with proportionally more values above the reference mean (0.55%) in CKD Stages 2-3 and 3-fold higher (1.6%) in CKD Stage 3. PRA was higher in CKD Stages 2-3 (P < 0.05). Meanwhile in CKD Stage 1, FGF23 did not associate with FENa, and in CKD Stages 2-3 FGF23 associated positively with FENa (r = 0.4; P < 0.05) and PTH (r = 0.45; P < 0.05), and FENa associated with FE of phosphate (r = 0.6; P < 0.005). Neither FGF23 nor FENa was associated with systolic or diastolic BP in either subgroup. The negative association of eGFR by cystatin with FENa remained the strongest predictor of FENa by multivariable linear regression in CKD Stages 2-3.

Conclusions: The elevated FGF23, FENa and PRA and the positive association of FGF23 with FENa do not suggest FGF23-mediated increased tubular Na reabsorption and volume expansion as causing hypertension in young patients with incipient CKD.

Keywords: CKD; FGF23; blood pressure; fractional sodium excretion.

We used high-resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) to identify hormonally regulated proteins in cultured human fetal lung. Proteins labeled with [35S]methionine were separated by 2-D PAGE, and fluorograms were analyzed by computer-assisted analysis of densitometric scans. Dexamethasone (10 nM) and gamma-interferon (10 ng/ml) induced (2- to <em>22</em>-fold vs. control) distinct sets of proteins (comprising approximately 2% of approximately 1,000 resolved proteins). Treatment with forskolin (10 microM) plus 3-isobutyl-1-methylxanthine (IBMX, 100 microM), which increases intracellular adenosine 3',5'-cyclic monophosphate (cAMP), induced both unique proteins and several proteins induced by dexamethasone. One protein (Mr 40,000, pI 4.4) was induced only with combined dexamethasone and cAMP treatment. Dexamethasone repressed four proteins, but inhibition was not observed with other hormones. Some of the regulated proteins were enriched in either <em>fibroblasts</em> or type II cells isolated from lung explants. We found no proteins that were consistently regulated by triiodothyronine (T3) (2 nM) or transforming <em>growth</em> <em>factor</em>-beta (10 ng/ml). Additionally, none of the hormonal treatments substantially altered the rate of methionine incorporation into total protein. Thus we have identified separate subsets of proteins that are regulated by glucocorticoids, gamma-interferon, and cAMP; these proteins may be important mediators of hormonal effects in the developing fetal lung.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2) is a multifunctional cell <em>growth</em> <em>factor</em> that regulates cell proliferation, differentiation, adhesion, migration, and apoptosis. FGF2 has multiple isoforms, including an 18-kDa low molecular weight isoform (lo-FGF2) and <em>22</em>-, 23-, 24-, and 34-kDa high molecular weight isoforms (hi-FGF2). Hi-FGF2 overexpression induces chromatin compaction, which requires the mitochondria and leads to apoptosis. Complement component 1 Q subcomponent-binding protein (C1QBP) plays an important role in mitochondria-dependent apoptosis by regulating the opening of the mitochondrial permeability transition pore. However, the interaction between C1QBP and hi-FGF2 and its role in hi-FGF2-mediated apoptosis remain unclear. Here, we found that hi-FGF2 overexpression induced depolarization of the mitochondrial membrane, cytochrome c release into the cytosol, and a considerable increase in C1QBP messenger RNA and protein expression. Furthermore, coimmunoprecipitation results showed that the mitochondrial protein, C1QBP, interacts with hi-FGF2. C1QBP knockdown using small interfering RNA significantly decreased the localization of hi-FGF2 to the mitochondria and increased the rate of apoptosis. Our results highlight a novel mechanism underlying hi-FGF2-induced, mitochondria-driven cell death involving the direct interaction between hi-FGF2 and C1QBP and the upregulation of C1QBP expression.

<AbstractText>Children with Hutchinson-Gilford progeria syndrome (HGPS), a rare premature aging disease, exhibit extraskeletal calcifications detected by radiographic analysis and on physical examination. The aim of this study was to describe the natural history and pathophysiology of these abnormal calcifications in HGPS, and to determine whether medications and/or supplements tested in clinical trials alter their development.</AbstractText><AbstractText>Children from two successive clinical trials administering 1) lonafarnib (n = 26) and 2) lonafarnib + pravastatin + zoledronic acid (n = 37) were studied at baseline (pre-therapy), one year on therapy, and at end-of-therapy (3.3-4.3 years after the baseline visit). Calcium supplementation (oral calcium carbonate) was administered during the first year of the second trial and was subsequently discontinued. Information on calcifications was obtained from physical examinations, radiographs, and serum and urinary biochemical measures. The mineral content of two skin-derived calcifications was determined by x-ray diffraction.</AbstractText><AbstractText>Extraskeletal calcifications were detected radiographically in 12/39 (31%) patients at baseline. The odds of exhibiting calcifications increased with age (p = 0.045). The odds were unaffected by receipt of lonafarnib, pravastatin, and zoledronate therapies. However, administration of calcium carbonate supplementation, in conjunction with all three therapeutic agents, significantly increased the odds of developing calcifications (p = 0.009), with the odds plateauing after the supplement's discontinuation. Composition analysis of calcinosis cutis showed hydroxyapatite similar to bone. Although serum calcium, phosphorus, and parathyroid hormone (PTH) were within normal limits at baseline and on-therapy, PTH increased significantly after lonafarnib initiation (p < 0.001). Both the urinary calcium/creatinine ratio and tubular reabsorption of phosphate (TRP) were elevated at baseline in <em>22</em>/39 (56%) and 31/37 (84%) evaluable patients, respectively, with no significant changes while on-therapy. The mean calcium × phosphorus product (Ca × Pi) was within normal limits, but plasma magnesium decreased over both clinical trials. <em>Fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23) was lower compared to age-matched controls (p = 0.03).</AbstractText><AbstractText>Extraskeletal calcifications increased with age in children with HGPS and were composed of hydroxyapatite. The urinary calcium/creatinine ratio and TRP were elevated for age while FGF23 was decreased. Magnesium decreased and PTH increased after lonafarnib therapy which may alter the ability to mobilize calcium. These findings demonstrate that children with HGPS with normal renal function and an unremarkable Ca × Pi develop extraskeletal calcifications by an unidentified mechanism that may involve decreased plasma magnesium and FGF23. Calcium carbonate accelerated their development and is, therefore, not recommended for routine supplementation in these children.</AbstractText>

In order to design new potent inhibitors of the epidermal <em>growth</em> <em>factor</em> receptor (EGF-R) associated protein tyrosine kinase (PTK) activity as antitumor agents, several families of phenylhydroquinone derivatives were synthesized. Some of these compounds were shown to block PTK activity in vitro, but also efficiently to inhibit EGF-stimulated DNA synthesis in ER <em>22</em> cells (CCL 39 hamster <em>fibroblasts</em> transfected with EGF-R) with IC50 values in the range 1-10 microM. In some cases, a correlation between the two sets of data was observed, allowing structure-activity relationships to be established. However, inhibitors which had in vitro specificity with regard to other kinases were not specific in the cellular test. Similar effects on DNA synthesis were observed after stimulation by various activating agents, suggesting that our compounds may also act against other cellular targets involved in the EGF-dependent pathways leading to cell proliferation.

Erythromycin (0.2-20 micrograms/ml) induced the proliferation of macrophages of mouse peritoneal exudate cells (PEC) in a liquid medium without exogenous <em>growth</em> <em>factors</em>. The proliferating macrophages formed giant colonies between days <em>22</em> and 26 of culture; these colonies continued to proliferate even after subculture. The erythromycin-induced cell proliferation was independent of <em>fibroblasts</em>, T cells, B cells, or endotoxins. This activity seemed to be specific to erythromycin since other antibiotics such as tetracycline, streptomycin, gentamicin, penicillin G, and josamycin did not induce the proliferation of macrophages. Any known cytokines, including IL-2, IL-3, IL-4, IL-6, and GM-CSF, were not detectable by ELISA tests in any of the culture supernatants sampled from day 7 through day 28. The culture supernatants, however, had the capability of inducing the <em>growth</em> of macrophages, only in the presence of bioactive erythromycin at concentrations higher than 1.6 micrograms/l. Moreover, the culture supernatants, sampled after giant colonies had been formed, were capable of inducing giant colonies in the culture of adherent PEC. Thus, the erythromycin-induced macrophage proliferation might be due to the direct effect of this antibiotic, whereas the formation of giant colonies might be due to the production of some unidentified soluble <em>factor</em> produced by the proliferating macrophages. These data indicate that mouse PEC contain a subset of peritoneal macrophages capable of responding to erythromycin by forming proliferating colonies without exogenous <em>growth</em> <em>factors</em>.

Human granulocyte-macrophage colony stimulating <em>factor</em> (GM-CSF) is a <em>22</em> kD glycoprotein derived from activated T cells, endothelial cells, <em>fibroblasts</em> and macrophages. It stimulates the proliferation and differentiation of haematopoietic cells and certain nonhaematopoietic cells. This study investigated the effect of GM-CSF on cultured human keratinocytes and determined whether these cells express GM-CSF receptors. Measurement of keratinocyte <em>growth</em> by image analysis showed that GM-CSF mildly stimulated the <em>growth</em> of keratinocytes. With 4 ng/ml GM-CSF the <em>growth</em> of primary keratinocytes and subcultured keratinocytes was only stimulated up to 25% and 18% of the control cell level, respectively. Human keratinocytes were incubated with GM-CSF at 4 ng/ml for 4 days to induce receptor expression. These cells showed only a weak positive reaction on affinity labelling using digoxigenated GM-CSF. We conclude that keratinocyte <em>growth</em> may be stimulated by GM-CSF but that the presence of GM-CSF receptors on these cells is equivocal.

The Tientsin Albino 2 (TA2) mouse has a high incidence of spontaneous breast cancer (SBC) in the absence of external inducers or carcinogens. The initiation of SBC is related to mouse mammary tumor virus (MMTV) infection and pregnancy. Pathologic analysis showed that breast cancer cells in TA2 mice are triple negative. Our previous study confirmed that <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 (FGFR2) expression increased in SBC tissue compared to that in their corresponding normal breast tissues of TA2 mice. The present study focused on the function of the FGFR2/STAT3 signaling pathway in the initiation of SBC. In this study, the expression of FGF3, FGFR2, STAT3, p-STAT3^Tyr705, and p-STAT3^Ser727 was detected in serum and normal mammary gland tissues of TA2 mice with different number of pregnancies and SBC. The proliferation, invasiveness, and migration abilities of MA-891 cells from TA2 SBC were compared before and after cryptotanshinone and Stattic treatment. Transient siRNA transfection was used to detect the invasiveness, and migration abilities to avoid the off-targets effects. Downstream protein expression of STAT3 was also detected in MA-891 cells and TA2 xenografts from MA-891 inoculation. In addition, STAT3 expression was analyzed in 139 cases of human breast cancer including 117 cases of non-triple negative breast cancer (non-TNBC) (group I) and <em>22</em> cases of triple-negative breast cancer (TNBC) (group II). Results of our study confirmed that MMTV-LTR amplification, and FGFR2, p-STAT3^Tyr705, p-STAT3^Ser727 expression increased with the number of pregnancies in the breast tissue of TA2 mice and were the highest in SBC. Serum FGF3 expression of SBC was higher than it of TA2 mice with different number of pregnancies. After STAT3 was inhibited, the abilities of proliferation, invasiveness, and migration in MA-891 decreased and the expression levels of STAT3, p-STAT3^Ser727, p-STAT3^Tyr705, Bcl2, cyclin D1, and c-myc in MA-891 and animal xenografts were also down-regulated. In human breast cancer, STAT3 expression was significantly higher in TNBC than that in non-TNBC. Our results showed that the FGFR2/STAT3 signaling pathway may be related to SBC initiation in TA2 mice. Inhibition of STAT3 can decrease proliferation, invasiveness, and migration in MA-891 cells and the <em>growth</em> of TA2 xenografts.

Keywords: FGFR2/STAT3; MMTV; spontaneous breast cancer; tientsin albino 2; triple-negative breast cancer.

Disruption of epidermal barrier is an important trigger in abnormal cutaneous inflammation. Phospholipase C epsilon (PLCε), a Ras/Rap1 effector, is essential for regulating cytokines production in different types of skin inflammation. Our previous studies have demonstrated that elevated expression of PLCε participates in the psoriasis-like inflammation in PLCε overexpressing transgenic mice model, while the reduction in PLCε expression attenuates inflammatory responses in either TPA- or DNFB-induced cutaneous inflammation. Here, we determined the role of PLCε in cutaneous inflammation induced by acute abrogation of epidermal permeability barrier. In comparison to wild type controls, PLCε KO mice exhibited reduced ear swelling and infiltration of granulocytes after tape-stripping. Moreover, expression levels of pro-inflammatory cytokines (IL-1α, IL-1β), chemokines (CXCL-1, CXCL-2, CCL20), and antimicrobial peptides (S100 proteins, MBD3) were lower in PLCε-deficient versus wild type mice. Likewise, expression levels of cytokines and chemokines were also lower in PLCε deficient keratinocytes and <em>fibroblasts</em> following IL-<em>22</em> stimulation <i>in vitro</i>. Furthermore, knockdown of PLCε with its siRNA decreased expression of IL-1α, CCL20, and S100 proteins, and MBD3 in HEK cultures. Collectively, these results suggested that PLCε mediated cytokine cascade induced by acute barrier disruption. IL-<em>22</em> is likely the upstream of PLCε-mediated cytokine cascade following acute barrier disruption.

<strong class="sub-title"> Keywords: </strong> BMP4, bone morphogenetic protein 4; Barrier function; CCL20, chemokine (C–C motif) ligand 20; CXCL, chemokine (C-X-C motif) ligand; FLG, filaggrin; HEK, human epidermal keratinocytes; IL-<em>22</em>; IVL, involucrin; K1, keratin 1; K15, keratin 15; LHX2, LIM homeobox 2; LOR, loricrin; MBD, murine beta defensin; PLCε; PLCε, phospholipase C epsilon; PMA, Phorbol-12-myristate-13-acetate; Psoriasis; SHH, sonic hedgehog; SOX9, SRY-box 9; SPT1, serine palmitoyltransferase 1; STAT3, transducer and activator of transcription 3; Skin inflammation; TGF, transforming growth factor; TLR2, toll like receptor 2; TNF, tumor necrosis factor.

<b>Background:</b> Bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stem cells (MSCs) are used increasingly for autologous cell therapy in equine practice to treat musculoskeletal and other injuries. Current recommendations often call for 10-100 million MSCs per treatment, necessitating the expansion of primary cells in culture prior to therapeutic use. Of concern, human and rodent studies have shown a decline of both MSC recovery from sampled tissue and <i>in vitro</i> proliferative capacity with increasing donor age. This may be problematic for applications of autologous cell-based therapies in the important equine demographic of older patients. <b>Objectives:</b> To investigate the effect of donor age on the cellular proliferation of equine BM- and AT-MSCs. <b>Study Design:</b> <i>In vitro</i> study. <b>Methods:</b> BM- and AT-MSCs and dermal <em>fibroblasts</em> (biological control) were harvested from horses in five different age groups (<i>n</i> = 4, <i>N</i> = 60); newborn (0 days), yearling (15-17 months), adult (5-8 years), middle-aged (12-18 years), and geriatric (≥<em>22</em> years). Proliferation of the cells was tested using an EdU incorporation assay and steady state mRNA levels measured for targeted proliferation, aging, and senescence biomarkers. <b>Results:</b> The cellular proliferation of equine BM- and AT-MSCs declined significantly in the geriatric cohort relative to the younger age groups. Proliferation levels in the two MSC types were equally affected by donor age. Analysis of steady state mRNA levels showed an up-regulation in tumor suppressors, apoptotic genes, and multiple <em>growth</em> <em>factors</em> in MSCs from old horses, and a down-regulation of some pro-cycling genes with a few differences between cell types. <b>Main Limitations:</b> Potential age-dependent differences in cell function parameters relevant to cell-therapy application were not investigated. <b>Conclusions:</b> The cellular proliferation of equine BM- and AT-MSCs declined at advanced donor ages. High levels of <i>in vitro</i> proliferation were observed in both MSC types from horses in the age groups below 18 years of age.

Keywords: aging; donor age; horse; mesenchymal stem cells; proliferation; tumor suppressors.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF-2), ubiquitously expressed in humans and mice, is functionally involved in cell <em>growth</em>, migration and maturation in vitro and in vivo. Based on the same mRNA, an 18-kilo Dalton (kDa) FGF-2 isoform named FGF-2 low molecular weight (FGF-2LMW) isoform is translated in humans and rodents. Additionally, two larger isoforms weighing 21 and <em>22</em> kDa also exist, summarized as the FGF-2 high molecular weight (FGF-2HMW) isoform. Meanwhile, the human FGF-2HMW comprises a <em>22</em>, 23, 24 and 34 kDa protein. Independent studies verified a specific intracellular localization, mode of action and tissue-specific spatiotemporal expression of the FGF-2 isoforms, increasing the complexity of their physiological and pathophysiological roles. In order to analyze their spectrum of effects, FGF-2LMW knock out (ko) and FGF-2HMWko mice have been generated, as well as mice specifically overexpressing either FGF-2LMW or FGF-2HMW. So far, the development and functionality of the cardiovascular system, bone formation and regeneration as well as their impact on the central nervous system including disease models of neurodegeneration, have been examined. This review provides a summary of the studies characterizing the in vivo effects modulated by the FGF-2 isoforms and, thus, offers a comprehensive overview of its actions in the aforementioned organ systems.

Keywords: FGF-2HMWko; FGF-2HMWtg; FGF-2LMWko; FGF-2LMWtg; FGF-2ko; bone physiology; cardiovascular system; central nervous system; mu-tant mice.

Background: Given the important emerging field of fibroblast growth factor 23 (FGF23) biology, there is a growing need for reliable data on FGF23 assay characteristics. We therefore evaluated the effects of different processing and storage conditions on FGF23 stability, as well as the assay precision of FGF23 measurements using 2 commercially available FGF23 ELISA kits.

Methods: We measured plasma concentrations of intact FGF23 (iFGF23) and C-terminal FGF23 (cFGF23) in duplicate in 12 patients with a wide range of kidney function. We used blinded replicate samples to calculate the interassay CV for both assays. We processed the samples immediately after collection, after 6 h at 22 °C, or after 24 h at 4 °C. We also exposed samples to 0, 1, 2, or 3 freeze-thaw cycles.

Results: The interassay CVs for iFGF23 and cFGF23 were 5.2% and 7.2%, respectively. Delayed processing for either 6 h at 22 °C or 24 h at 4 °C had no significant effect on either iFGF23 or cFGF23, although a nonsignificant trend toward decreased iFGF23 concentrations was observed compared with immediate processing (23% relative decline in concentrations under both delayed processing conditions). Three freeze-thaw cycles had no effect on either iFGF23 or cFGF23 concentrations.

Conclusions: FGF23 measurements in human plasma are stable with delayed processing or after undergoing multiple freeze-thaw cycles.

The cytotoxic and mutagenic effects of a series of carcinogenic mutagens in human peripheral blood T cells exposed in culture were determined and compared with results obtained previously with diploid human skin <em>fibroblasts</em>. The mutagens studied were: ethylnitrosourea (ENU), (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and UV (254 nm) radiation. Resistance to 6-thioguanine (TG) served as the genetic marker. The T lymphocytes, cultured in the presence of 10% fetal bovine serum, T cell <em>growth</em> <em>factor</em> and lethally-irradiated B lymphoblastoid cells (allogeneic stimulators), exhibited population doubling times of <em>22</em> h for as long as tested, i.e. greater than 40 days, and cloning efficiencies between 30 and 50%. The T cells were exposed to the mutagens in exponential <em>growth</em>, 3 days after being isolated and primed to begin blast formation, using doses that reduced cell survival to between 80 and 25% of the untreated control. The background frequency of TG-resistant cells in the 14 independent populations assayed ranged from 1.2 x 10(-6) to 10.6 x 10(-6), with an average of 5.1 x 10(-6) +/- 2.8 x 10(-6). Each agent induced a dose-dependent increase in TG-resistant cells, reaching 85 x 10(-6) for UV, 260 x 10(-6) for ENU and 70 x 10(-6) for BPDE. When compared on the basis of fluence or applied concentration, T cells were 1.5-times more sensitive than <em>fibroblasts</em> to the cytotoxic effects of ENU, but equally as sensitive as <em>fibroblasts</em> to its mutagenic effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Lactic acid bacteria, either alive or dead, can improve villous atrophy caused by weaning in both piglets and mice. In this experiment, we tried to detect the molecules involved in this phenomenon with a real-time RT-PCR array approach. Weaning pups of mice were administered either a suspension of an Enterococcus faecalis EC-12 dried cell preparation (EC-12) or saline for 11 consecutive days after weaning. The jejunal and ileal villous heights were measured histologically, and the expression levels of 86 genes were analyzed for the jejunal and ileal epithelial cells and the lamina propria (LP). EC-12 induced significantly higher villous height in the jejunum and the ileum. Interleukin (IL)-6, <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-7, -10, and -<em>22</em>, and the platelet-derived <em>growth</em> <em>factor</em> (PDGF) beta in the jejunal and the ileal LP were the most enhanced genes by EC-12. The possible role of these molecules in the improvement of villous atrophy is discussed.

<AbstractText>Evaluation of importance of serum levels of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) in patients with ovarian cancer, patients with border-line ovarian tumor, patients with benign ovarian cyst and women with normal ovarian tissue.</AbstractText><AbstractText>Prospective clinical study.</AbstractText><AbstractText>Department of Gynecology and Obstetrics, Charles University, Faculty of Medicine in Hradec Kralove and University Hospital Hradec Kralove.</AbstractText><AbstractText>Measurement of serum levels of bFGF by ELISA using reagents of company R&D Systems prior to treatment in a total of 74 consecutive coming women.</AbstractText><AbstractText>Serum level of bFGF from peripheral blood before treatment was significantly higher (p &lt; 0.05) in patients with newly diagnosed ovarian cancer (n = <em>22</em>), Med = 10.35 pg/ml (1.2-46.2 pg/ml) compared to patients with a border-line ovarian tumor (n = 9), Med = 5.4 pg/ml (1.6-6.8 pg/ml), patients with benign ovarian cyst (n = 24), Med = 5.2 pg/ml (0.1-67.2 pg/ml), and to women with normal ovarian tissue (n = 19) Med = 4.3 pg/ml (0.9-13.4 pg/ml). There isnt strong linear correlation (Spearmans rank correlation coefficient = 0.208791) between the serum level of bFGF and CA125 collected from peripheral blood before primary surgery or neoadjuvant chemotherapy in a group of patients with ovarian cancer (n = 14). We have not found significance correlation between age and serum levels of bFGF in patients with ovarian cancer, with border-line ovarian tumor, with benign ovarian cyst and in women with normal ovarian tissue.</AbstractText><AbstractText>Serum levels of bFGF in patients with ovarian cancer are significantly higher than in patients with a border-line ovarian tumor, with benign ovarian cyst and in women with normal ovarian tissue regardless of age of patients.</AbstractText>

<b>Background:</b> Our previous work demonstrated that application of transforming <em>growth</em> <em>factor</em> beta 1 (TGF-β1) and forskolin to the repair site after chronic denervation and axotomy has a mitogenic effect, reactivates Schwann cells (SCs), and supports axonal regeneration. We found decreased expression of <em>fibroblast</em> <em>growth</em> <em>factor</em> 7 (FGF-7), a <em>factor</em> involved in synaptic organization and maintenance. Using an in vitro system, we examined the molecular mechanism of TGF-β1 and forskolin on the regulation of FGF-7 expression in SCs. <b>Methods:</b> SCs were prepared from the sciatic nerve and stimulated with forskolin (0.5 μM), TGF-β1 (1 ng/mL), or TGF-β1 + forskolin for 6 or 24 hours. SCs were also pretreated with LY2109761 (0.5 μM), a TGF-β receptor inhibitor, prior to stimulation with TGF-β1 + forskolin for 6 hours. Real-time TaqMan quantitative polymerase chain reaction analyses for FGF-7, myelin basic protein, and peripheral myelin protein <em>22</em> expression were performed. Cycle threshold (Ct) data were normalized to a reference gene, and fold changes relative to untreated SCs were determined using the 2^-ΔΔCt method. Statistical analysis was done using <i>t</i> test (<i>P</i><0.05). <b>Results:</b> TGF-β1 alone or in combination with forskolin for 24 hours resulted in a 3.3- and 2.8-fold decrease in FGF-7 expression in SCs, respectively. No change in FGF-7 expression was found with forskolin alone. TGF-β1 + forskolin treatment for 6 hours resulted in a 4.0-fold decrease in FGF-7 expression, while the addition of LY2109761 resulted in a 2.7-fold decrease in FGF-7 expression. <b>Conclusion:</b> We showed that SC expression of FGF-7 is regulated by TGF-β1. The positive effect of TGF-β1 and forskolin on SC reactivation and axonal regeneration may involve modulation of FGF-7 expression and activity in SCs.

Conditioned media collected from arterial endothelial cells contain protein <em>factor</em>(s) that promotes the contraction of collagen lattices made with skin <em>fibroblasts</em>. Based on the lattice contraction-promoting activity, a protein with an apparent molecular weight of <em>22</em> kDa was identified. This <em>22</em>-kDa protein stimulated lattice contraction in both serum-containing and serum-free media. When assayed at a 30% equivalent of the conditioned medium, the contraction-promoting activity of the purified <em>factor</em> was about 50 to 60% of that elicited by the unfractionated conditioned medium. Some contraction-promoting activity was also present in certain subfractions of the conditioned medium generated during the separation of the <em>22</em>-kDa protein. Taken together, the results indicate that the lattice contraction-promoting activity in the endothelial cell-conditioned medium is probably aided by multiple active principles. The biochemical and biological characteristics indicated that the <em>22</em>-kDa protein is not a transforming <em>growth</em> <em>factor</em>-beta-related <em>factor</em> nor a <em>fibroblast</em> <em>growth</em> promoter.

Information on the heterogeneity of phosphaturic mesenchymal tumor, a rare entity associated with tumor-induced osteomalacia, is limited. In this retrospective analysis of <em>22</em>2 phosphaturic mesenchymal tumors, <em>22</em> cases exhibited mixed mesenchymal and epithelial elements, which we propose to term "phosphaturic mesenchymal tumor, mixed epithelial, and connective tissue type." Phosphaturic mesenchymal tumor of the mixed epithelial and connective tissue type showed a distinctive and significant male predominance (male:female = 2.67:1), with most patients diagnosed at <40 years old. Moreover, all tumors were mainly located in the alveolar bone with focal invasion into surrounding soft tissue and oral mucosa, which could be detected preoperatively by oral examination. The mesenchymal component, composed of spindled cells resembling <em>fibroblasts</em> or myo<em>fibroblasts</em> arranged in a storiform or fascicular pattern, exhibited a less prominent vasculature and lower cellularity than the typical phosphaturic mesenchymal tumor (mixed connective tissue type). The epithelial component was typically haphazardly and diffusely distributed throughout the tumor, forming small, irregular nests resembling odontogenic epithelial nests. All cases were immunoreactive for <em>fibroblast</em> <em>growth</em> <em>factor</em>-23, somatostatin receptor 2A, and NSE in both components. Mostly also demonstrated positive staining for CD99 (21/<em>22</em>, 96%), CD56 (16/<em>22</em>, 73%), Bcl-2 (21/<em>22</em>, 96%), and D2-40 (19/<em>22</em>, 86%) in one or both components. S100 was positive in both components in one of seven cases. Interestingly, immunoreactivity was typically stronger and more diffuse in the epithelial than in the paired mesenchymal components. The mesenchymal component was also diffusely positive for CD68 (17/17, 100%) and showed variable focal staining for SMA (15/<em>22</em>, 68%) and CD34 (9/19, 47 %). These results indicate that phosphaturic mesenchymal tumor of the mixed epithelial and connective tissue type has distinctive clinicopathological characteristics and a polyimmunophenotypic profile.