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Background Lactoferrin (LF) is an 80 kDa glycoprotein which is known for its effects against bacteria, viruses and other pathogens. It also has a high potential in nutrition therapy and welfare of people and a variety of animals, including piglets. The ability to bind lipopolysaccharide (LPS) is one of the described anti-inflammatory mechanisms of LF. Previous studies suggested that cells can be stimulated even by LPS-free LF. Therefore, the aim of our study was to bring additional information about this possibility. Porcine monocyte derived macrophages (MDMF) and human embryonic kidney (HEK) cells were stimulated with unpurified LF in complex with LPS and with purified LF without bound LPS. Results Both cell types were stimulated with unpurified as well as purified LF. On the other hand, neither HEK0 cells not expressing any TLR nor HEK4a cells transfected with TLR4 produced any pro-inflammatory cytokine transcripts after stimulation with purified LF. This suggests that purified LF without LPS stimulates cells via another receptor than TLR4. An alternative, TLR4-independent, pathway was further confirmed by analyses of the NF-kappa-B-inducing kinase (NIK) activation. Western blot analyses showed NIK which activates different NFκB subunits compared to LF-LPS signaling via TLR4. Though, this confirmed an alternative pathway which is used by the purified LF free of LPS. This stimulation of MDMF led to low, but significant amounts of pro-inflammatory cytokines, which can be considered as a positive stimulation of the immune system. Conclusion Our results suggest that LF’s ability is not only to bind LPS, but LF itself may be a stimulant of pro-inflammatory pathways.

In the veterinary field, only limited information is available about interleukin-17A (IL-17), despite the fact that this cytokine plays an important role during pro-inflammatory immune responses and induces the production of chemotactic factors for neutrophils. The aim of this study was to characterize porcine IL-17-producing cells. We tested the cross-reactivity of five anti-human IL-17 monoclonal antibodies because such antibodies against porcine IL-17 are currently unavailable. Whole blood cells (WBCs) were stimulated with phorbol-myristate-acetate (PMA) and ionomycin and subsequently analyzed by flow cytometry. The antibody clone SCPL1362 was found to cross-react with porcine IL-17, whereas the other four antibodies tested did not recognize this cytokine. Using this antibody, we characterized porcine WBC-secreting IL-17 after PMA and ionomycin stimulation. All IL-17-producing WBCs were positive for the T lymphocyte marker CD3. Myeloid cells (CD172α(+)) and B lymphocytes (CD79α(+)) were IL-17 negative. The major subset of IL-17 positive T lymphocytes was the CD4(+) lymphocytes (about 60% of all IL-17 positive WBCs). The remaining IL-17 positive WBCs were γδTCR(+) lymphocytes. CD8 positive and CD8 negative cells were found within both CD4(+) and γδTCR(+) cells producing the cytokine. Moreover, IL-17 positive cells were mostly CD45RA negative, therefore activated cells or memory cells. Flow cytometry data were confirmed using sorted cells. Both sorted CD4(+) and γδTCR(+) cells produced IL-17 at mRNA level after PMA and ionomycin stimulation while double negative CD4(-)γδTCR(-) cells were negative for IL-17. We can conclude that only two subpopulations of porcine WBCs are sources of IL-17 after non-specific stimulation: CD3(+)CD4(+) and CD3(+)γδTCR(+).

The FGFR3 receptor tyrosine kinase represents an attractive target for therapy due to its role in several human disorders including skeletal dysplasias, multiple myeloma, and cervical and bladder carcinomas. By using molecular library screening, we identified a compound named NF449 with inhibitory activity towards FGFR3 signaling. In cultured chondrocytes and murine limb organ culture, NF449 rescued FGFR3-mediated extracellular matrix loss and growth inhibition, which represent two major cellular phenotypes of aberrant FGFR3 signaling in cartilage. Similarly, NF449 antagonized FGFR3 action in the multiple myeloma cell lines OPM2 and KMS11, as evidenced by NF449-mediated reversal of ERK MAP kinase activation and transcript accumulation of CCL3 and CCL4 chemokines, both of which are induced by FGFR3 activation. In cell-free kinase assays, NF449 inhibited the kinase activity of both wild-type and a disease-associated FGFR3 mutant (K650E) in a fashion that appeared noncompetitive with ATP. Our data identify NF449 as a novel antagonist of FGFR3 signaling, useful for FGFR3 inhibition alone or in combination with inhibitors that target the ATP binding site

Fibroblast growth factor 2 (FGF2) is one of the most studied growth factors to date. Most attention has been dedicated to the smallest, 18 kDa FGF2 variant that is released by cells and acts through activation of cell-surface FGF-receptor tyrosine kinases. There are, however, several higher molecular weight (HMW) variants of FGF2 that rarely leave their producing cells, are retained in the nucleus and act independently of FGF-receptors (FGFR). Despite significant evidence documenting the expression and intracellular trafficking of HMW FGF2, many important questions remain about the physiological roles and mechanisms of action of HMW FGF2. In this review, we summarize the current knowledge about the biology of HMW FGF2, its role in disease and areas for future investigation.

Oncogenic activation of the RAS-ERK MAP kinase signaling pathway can lead to uncontrolled proliferation but can also result in apoptosis or premature cellular senescence, both regarded as natural protective barriers to cell immortalization and transformation. In FGFR3-related skeletal dyplasias, oncogenic mutations in the FGFR3 receptor tyrosine kinase cause profound inhibition of cartilage growth resulting in severe dwarfism, although many of the precise mechanisms of FGFR3 action remain unclear. Mutated FGFR3 induces constitutive activation of the ERK pathway in chondrocytes and, remarkably, can also cause both increased proliferation and apoptosis in growing cartilage, depending on the gestational age. Here, we demonstrate that FGFR3 signaling is also capable of inducing premature senescence in chondrocytes, manifested as reversible, ERK-dependent growth arrest accompanied by alteration of cellular shape, loss of the extracellular matrix, upregulation of senescence markers (alpha-GLUCOSIDASE, FIBRONECTIN, CAVEOLIN 1, LAMIN A, SM22alpha and TIMP 1), and induction of senescence-associated beta-GALACTOSIDASE activity. Our data support a model whereby FGFR3 signaling inhibits cartilage growth via exploiting cellular responses originally designed to eliminate cells harboring activated oncogenes.

Activating mutations in FGFR3 tyrosine kinase cause several forms of human skeletal dysplasia. Although the mechanisms of FGFR3 action in cartilage are not completely understood, it is believed that the STAT1 transcription factor plays a central role in pathogenic FGFR3 signaling. Here, we analyzed STAT1 activation by the N540K, G380R, R248C, Y373C, K650M and K650E-FGFR3 mutants associated with skeletal dysplasias. In a cell-free kinase assay, only K650M and K650E-FGFR3 caused activatory STAT1(Y701) phosphorylation. Similarly, in RCS chondrocytes, HeLa, and 293T cellular environments, only K650M and K650E-FGFR3 caused strong STAT1 activation. Other FGFR3 mutants caused weak (HeLa) or no activation (293T and RCS). This contrasted with ERK MAP kinase activation, which was strongly induced by all six mutants and correlated with the inhibition of proliferation in RCS chondrocytes. Thus the ability to activate STAT1 appears restricted to the K650M and K650E-FGFR3 mutants, which however account for only a small minority of the FGFR3-related skeletal dysplasia cases. Other pathways such as ERK should therefore be considered as central to pathological FGFR3 signaling in cartilage.