No abstract

Activation of fibroblast growth factor receptor 3 (FGFR3) leads to attenuation of cartilage growth. The members of the STAT family of transcription factors are believed to participate in FGFR3 signaling in cartilage, however the molecular mechanism of this action is poorly understood. Here, we demonstrate that a chronic FGF stimulus leads to accumulation of STAT1, 3, 5 and 6, evident in both in vitro chondrocyte model and murine limb explant cultures. Despite the accumulation, both endogenous and cytokine-induced activation of STAT1 and STAT3 is impaired by FGF, as demonstrated by imaging of active STAT nuclear translocation and analyses of STAT activatory phosphorylation and transcriptional activation. Further, we demonstrate that FGF induces expression of CIS, SOCS1 and SOCS3 inhibitors of gp130, a common receptor for the IL6-family of cytokines. Since cytokine-gp130 signaling represents an important positive regulator of cartilage, its inhibition may contribute to the growth-inhibitory effect of FGFR3 in cartilage.

The apoptosis inhibitor-5 [Api5; antiapoptosis clone 11 (AAC-11); fibroblast growth factor-2 (FGF2)-interacting factor (FIF)] is a 504-aa nuclear protein frequently up-regulated in tumor cells. Several studies have shown potent antiapoptotic action of Api5, which appears to be, at least in part, mediated via suppression of apoptosis depending on the E2F family of transcription factors [1][2][3][4].Human FGF2 is a prototypic member of the FGF family of growth factors and exists in several variants generated by alternative translation of a single transcript. The smallest variant, 18 kDa FGF2, is released from cells and acts through binding and activation of cell-surface FGF-receptor tyrosine kinases. In contrast, the high molecular weight (HMW) FGF2 variants (22, 22.5, and 24 kDa) localize predominantly to the nucleus and have an intracrine, FGF receptor-independent mode of action [5]. It is interesting that HMW but not 18 kDa FGF2 interacts with Api5 in mammalian cells [3]. Similar to Api5, HMW FGF2 has a prosurvival action in various experimental models [6], although it is unknown to what extent HMW FGF2 and Api5 depend functionally on each other.In B cell chronic lymphoid leukemia (B-CLL), FGF2 is up-regulated with an 18-kDa FGF2 peripheral blood concentration significantly exceeding control levels in the majority of patients with advanced disease [7]. This deregulation also involves HMW FGF2, which is overexpressed in B-CLL cells [8]. As Api5 represents the binding partner of HMW FGF2 [3], we asked whether Api5 is also up-regulated in B-CLL. We compared the expression of Api5 and HMW FGF2 in PB-MCs obtained from eight B-CLL patients and five control individuals. Figure 1A shows up-regulation of 55 kDa (full-length) Api5 and HMW FGF2 in the majority of B-CLL patients when compared with control samples. In addition to the full-length Api5, a 25-kDa truncated variant of Api5, generated by alternative translation [1], was found in some samples.The up-regulation of full-length Api5 in B-CLL (Fig. 1A) might be specific to the leukemic clones or simply represent B cell enrichment in the B-CLL PBMCs compared with control PBMCs. We therefore determined the levels of Api5 and FGF2 in PBMCs enriched for B cells via negative selection, i.e., removal of T cells, NK cells, and monocytes using magnetic bead separation. Compared with controls, there was a clear up-regulation of full-length Api5 and HMW FGF2 in the B cell-enriched PBMCs of all five B-CLL individuals (Fig. 1B). Similar results were obtained with CD19ϩ B cells purified from PBMCs via magnetic bead separation, thus confirming that B-CLL leukemic clones have up-regulated full-length Api5 and HMW FGF2 (Fig. 1C). Considering the nature of the defect leading to B-CLL, i.e., decreased apoptosis of mature B cells, Api5 and HMW FGF2 may contribute to the pathology of B-CLL.

The Erk MAP kinase pathway contributes to tumor development and thus represents an important therapeutic target. Several inhibitors of the Erk pathway are presently being evaluated in clinical trials for cancer, but show limited efficiency thus warranting discovery of more potent inhibitors. We have developed a novel mammalian cell-based assay that should facilitate the identification of such compounds by screening molecular libraries. In rat chondrosarcoma (RCS) cells, treatment with fibroblast growth factor 2 (FGF2) leads to sustained activation of the Erk pathway, resulting in growth arrest with more than an 80% cell count difference between control and FGF2-treated cells after 72 h of treatment. The extent of both Erk activation and the growth arrest can be precisely modulated by the FGF2 dose. We also demonstrate that FGF2-mediated activation of the Erk pathway is robust and has only a limited sensitivity to the available MEK inhibitors. The assay is rapid, sensitive and easily adapted to high throughput screening. A major advantage of this system is exclusion of toxic compounds as false-positive hits, given the nature of the RCS response to inhibition of the Erk pathway, i.e. growth.

From the epidemiological point of view, dogs are very important since they are considered a suitable indicator of the spread of human borreliosis. Serum samples obtained from healthy, asymptomatic military dogs from 12 different areas in the Czech Republic were examined for IgG antibodies to Borrelia burgdorferi sensu lato (s.l.). The total of 399 serum samples were tested by a whole-cell ELISA. Specific antibodies to Borrelia burgdorferi s.l. were detected in 26 cases (6.5%). In different localities, the seroprevalence varied from 0.0% to 28.6%. Two local isolated strains Br-75 (Borrelia afzelii) and Br-97 (Borrelia garinii) were used as antigens. A total of 22 (5.5%) were positive for antibodies to Borrelia afzelii and 19 (4.8%) were positive for antibodies to Borrelia garinii. Fifteen cases were positive for both antibodies. A significantly higher seroprevalence was found in younger dogs (1-3 years) than in older ones (p < 0.05). An analysis of seroprevalence by months of sampling showed no significant difference (p > 0.05).