P.P. Parnigotto

In vitro biological activity of bovine milk ribonuclease-4

Several members of the ribonuclease superfamily possess a variety of interesting biological properties, including ribonucleolytic, angiogenic, antiproliferative, cytotoxic, embryotoxic, aspermatogenic and antitumoral activity. In this study, we report the purification from bovine milk of a protein with structural and enzymatic properties very similar to those of ribonuclease-4 (RNase-4), which is normally present in the liver and lungs, and examined its functional properties, biological activity and cytotoxic effects. RNase-4, at physiological concentrations, had a positive effect on the vitality and proliferation of human umbilical vein endothelial cells. Moreover, it induced an increase in cellular migration and the formation of in vitro capillary-like structures. We also evaluated the effect of RNase-4 in vitro on human breast, colorectal and cervical carcinoma cell lines. The protein was revealed to have a cytotoxic effect similar to that of RNase-A. We suggest that the positive effects of RNase-4 on normal cells were due to its particularly close interaction with RNase inhibitor, while good conformational stability and resistance to proteolytic degradation potentially favour ribonuclease cytotoxicity.

Recombinant human TAT-OP1 to enhance NGF neurogenic potential: preliminary studies on PC12 cells

Osteogenic protein 1 (OP1), also known as bone morphogenic protein-7 (BMP7), is a multifunctional cytokine with demonstrated neurogenic potential. As the recombinant OP1 (rhOP1) was shown to provide axonal guidance cues and to prevent the reduction of dendritic growth in the injury-induced cortical cultures, it was suggested that an in vivo efficient rhOP1 delivery could enhance neurite growth and functional reconnectivity in the damaged brain. In the present work, we engineered a chimeric molecule in which rhBMP7 was fused to a protein transduction domain derived from HIV-1 TAT protein to deliver the denatured recombinant BMP7 into cells and obtain its chaperone-mediated folding, circumventing the expensive and not much efficient in vitro refolding procedures. When tested on rat PC12 cells, a widely used in vitro neurogenic differentiation model, the resulting fusion protein (rhTAT-OP1) demonstrated to enter fastly into the cells, lose HIV-TAT sequence and interact with membrane receptors activating BMP pathway by SMAD 1/5/8 phosphorylation. In comparison with nerve growth factor (NGF) and BMP7, it proved itself effective to induce the formation of more organized H and M neurofilaments. Moreover, if used in combination with NGF, it stimulated a significant (P < 0.05) and more precocious dendritic outgrowth with respect to NGF alone. These results indicate that rhTAT-OP1 fused with TAT transduction domain shows neurogenic activity and may be a promising enhancer factor in NGF-based therapies.

Sex-dependent differences in inflammatory responses during liver regeneration in a murine model of acute liver injury

A sexual dimorphism in liver inflammation and repair was previously demonstrated. Its cellular dissection in the course of acute liver injury (ALI) was explored. BALB/c mice were treated with carbon tetrachloride (CCl4) by intraperitoneal injection and killed after 3, 5, and 8 days. Histological and hepatic cell population analyses were performed. The correlation between androgen receptor (AR) expression and liver recruited inflammatory cells was investigated by treatment with the AR antagonist flutamide. Additionally, patients with a diagnosis of drug induced liver injury (DILI) were included in the study, with a particular focus on gender dimorphism in circulating monocytes. A delayed resolution of necrotic damage and a higher expression of proinflammatory cytokines were apparent in male mice along with a slower recruitment of inflammatory monocytes. F4/80+CD11b+ macrophages and CD11bhighGr-1high monocytes expressed AR and were recruited later in male compared with female livers after CCl4 treatment. Moreover, CD11bhighAR+Gr-1high recruitment was negatively modulated by flutamide in males. Analysis of DILI patients showed overall a significant reduction in circulating mature monocytes compared with healthy subjects. More interestingly, male patients had higher numbers of immature monocytes compared with female patients.A stronger cytotoxic tissue response was correlated with an impaired recruitment of CD11bhighAR+Gr-1high cells and F4/80+CD11b+ macrophages in the early inflammatory phase under AR signaling. During DILI, a dimorphic immune response was apparent, characterized by a massive recruitment of monocytes to the liver both in males and females, but only in males was this recruitment sustained by a turnover of immature monocytes.

5-Azacytidine Makes Human Preadipocytes Able to Differentiate into Mesoderm-Derived Cell Lineages

In the present study we have evaluated whether (i) 5-azacytidine (AZA), a well-known demethylating agent, could be able to modify the phenotype of human preadipocytes and (ii) the modified cells could possess multilineage differentiation potential. Human preadipocytes at the 3rd passage were treated for 48 or 96 h with 10 μM AZA and then expanded up to passage 5. Stem cell markers, such as OCT-4, Nanog, and Sox2, were upregulated after 96 h of treatment with the demethylating treatment. Further, decreases in the expression of genes, such as adipose differentiation-related protein, characterizing the preadipocytes were noted. Our data showed that AZA-treated preadipocytes differentiated into cell lineages derived from mesoderm. Indeed, after incubation with inductive media for 3 weeks, osteblast-, chondrocyte-, and myoblast-like cells were detected in the cultures. Interestingly, both upregulation of stem cell markers and differentiation potential were maintained by the treated cultures expanded until the 5th passage. Taken together, our results suggest that AZA, without the use of transduction methods, convert preadipocytes to a less differentiated state that can be induced, under suitable stimuli, to the formation of mesoderm-derived cell lineages.

In vitro evaluation of TAT-OP1 osteogenic properties and prospects for in vivo applications.

So far, osteogenic protein 1 (OP1) is biotechnologically produced and approved for the treatment of human lumbar spine fusion and long bone non‐union fractures. When combined with the TAT sequence, it has been demonstrated in vitro to be easily taken up by PC12 neuronal cells and to acquire its biological activity after intracellular refolding. In this study, TAT‐OP1 was shown to be a useful strategy to efficiently drive denatured OP1 into mouse MC3T3E1 pre‐osteoblasts. The correct in vitro protein refolding was verified by the activation of the BMP cascade, while the osteogenic potential of OP1 was demonstrated by increased expression of alkaline phosphatase, osteonectin and osteocalcin. Copyright

Inflammatory profile of neurotrophins, IL-6, IL1-β, TNF-α, VEGF, ICAM-1 and TGF-β in the Human Waldeyer’s ring

The palatine tonsils, nasopharyngeal tonsil (adenoid) and lingual tonsil constitute the major part of Waldeyer’s ring, with the tubal tonsils and lateral pharyngeal bands as less prominent components. The lymphoid tissue of Waldeyer’s ring is located at the gateway of the respiratory and alimentary tract and belongs to the mucosa-associated lymphoid tissue (MALT). The lymphatic tissue is known to interact with the nervous system and several organs implicated in the host response to a wide range of stressors (Otten et al., 1995; Kaneko et al., 2012; Ogasawara et al., 2011). This study focusses on the expression of some neurotrophins (NTs), their high- and low-affinity receptors in human adenoid tissues, lingual and palatine tonsils via immunohistochemical analysis, as well as on the expression of some inflammatory cytokines and other tissue growth factors (IL-6, IL1-β, TNF-α, VEGF, ICAM-1 and TGF-β)). Light microscopy immunohistochemistry performed on human samples showed to be generally positive for all the NTs investigated (NGF, BDNF, NT-3) and their receptors (TrKA, TrKB and TrKC) as well as the other cytokines and growth factors studied with some different expression levels. Real time PCR analysis is in progress to quantitate these data. Our data corroborate previous studies, suggesting that neurotrophins and inflammatory cytokines may mediate functional signals in lymphoid aggregates (Yusuf-Makagiansar et al., 2002; Ruoco et al., 1990).

Inflammatory profile of neurotrophins, IL-6, IL1-β, TNF-α, VEGF, ICAM-1 and TGF-β in the human Waldeyer's ring

The palatine tonsils, nasopharyngeal tonsil (adenoid) and lingual tonsil constitute the major part of Waldeyers ring, with the tubal tonsils and lateral pharyngeal bands as less prominent components. The lymphoid tissue of Waldeyers ring is located at the gateway of the respiratory and alimentary tract and belongs to the mucosa-associated lymphoid tissue (MALT). Mucosae-associated lymphoid tissues are richly innervated and the mucosae contain peptidergic nerve endings associated with different types of cells and macrophages. The lymphatic tissue is known to interact with the nervous system and several organs implicated in the host response to a wide range of stressors. This study focuses on the expression of some neurotrophins (NTs), their high- and low-affinity receptors in human adenoid tissues, lingual and palatine tonsils via immunohistochemical analysis, as well as on the expression of some inflammatory cytokines and other tissue growth factors. Light microscopy immunohistochemistry showed human samples to be generally positive for all the NTs investigated (NGF, BDNF, NT-3) and their receptors (TrKA, TrKB and TrKC) with some different expression levels. IL-6, IL1-b, TNF-α, VEGF, ICAM-1 and TGF-b were also investigated by immunohistochemistry. These results suggest the presence of a pattern of neurotrophic innervation in the human lymphatic tissues which may play a role in sustaining inflammatory conditions and in modulating a close interaction between the nervous system and the different immune cellular subtypes. Our data also corroborate previous studies, suggesting that neurotrophins and inflammatory cytokines may mediate functional signals in lymphoid aggregates. In this context, owing to their widespread expression in immune organs and immunocompetent cells, NTs and inflammatory cytokines are potential candidates for a prominent role in the regulation of immune and neuroimmune interactions.

1070 IMMUNE RESPONSE AND GENDER DISPARITY INFLUENCE LIVER REGENERATION AFTER ACUTE HEPATIC INJURY

and therefore regulate development and regression of liver fibrosis in sortilin −/− mice. Methods: Expression of neurotrophin receptors and ligands was measured by qRT-PCR in quiescent, activated and fully activated rat HSC, then the effect of NGF, proNGF and BDNF on proliferation, activation and apoptosis of HSC was assessed in 7d and 14d cultures. Thioacetamide-induced fibrosis was performed in wild type (WT) and sortilin −/− mice. Results: Fully activated rat HSC express increased mRNA levels for NGF, BDNF, trkB, p75NTR and sortilin compared to quiescent and activated HSC, while trkA is unchanged. Interestingly, neurotrophins strongly induce mRNA expression of a-smooth muscle actin (a-SMA), collagen I and TGF-b1 in activated HSC, but not in fully activated HSC. On the other hand, fully activated HSC display a dramatic increase in proinflammatory cytokines TNFa, SDF-1 and MCP-1 after neurotrophin treatment. Surprisingly, we observed that NGF and proNGF induce a modest statistically significant, and BDNF a strong mitogenic effect both in activated and fully activated HSC, but no apoptosis. In vivo results with sortilin −/− mice demonstrate that these mice have increased fibrosis, accompanied by enhanced aSMA and collagen I compared to WT mice, in a model of thioacetamide-induced fibrosis. Conclusions: The neurotrophin system plays different roles during HSC activation stages. Whereas, at earlier stages of HSC activation, neurotrophins increase activation, at later stages of activation, they induce a proinflammatory phenotype. Sortilin may participate in the response of HSC to neurotrophins, as sortilin −/− mice have increased liver fibrosis.