RESUMO
Autologous chondrocyte implantation for cartilage repair represents a challenge because strongly limited by chondrocytes' poor expansion capacity in vitro. Mesenchymal stem cells (MSCs) can differentiate into chondrocytes, while mechanical loading has been proposed as alternative strategy to induce chondrogenesis excluding the use of exogenous factors. Moreover, MSC supporting material selection is fundamental to allow for an active interaction with cells. Here, we tested a novel thermo-reversible hydrogel composed of 8% w/v methylcellulose (MC) in a 0.05 M Na2SO4 solution. MC hydrogel was obtained by dispersion technique and its thermo-reversibility, mechanical properties, degradation and swelling were investigated, demonstrating a solution-gelation transition between 34 and 37 °C and a low bulk degradation (<20%) after 1 month. The lack of any hydrogel-derived immunoreaction was demonstrated in vivo by mice subcutaneous implantation. To induce in vitro chondrogenesis, MSCs were seeded into MC solution retained within a porous polyurethane (PU) matrix. PU-MC composites were subjected to a combination of compression and shear forces for 21 days in a custom made bioreactor. Mechanical stimulation led to a significant increase in chondrogenic gene expression, while histological analysis detected sulphated glycosaminoglycans and collagen II only in loaded specimens, confirming MC hydrogel suitability to support load induced MSCs chondrogenesis.
Assuntos
Materiais Biocompatíveis , Técnicas de Cultura de Células , Diferenciação Celular , Condrogênese , Hidrogéis , Células-Tronco Mesenquimais/citologia , Metilcelulose , Animais , Materiais Biocompatíveis/química , Biomarcadores , Reatores Biológicos , Diferenciação Celular/genética , Condrogênese/genética , Perfilação da Expressão Gênica , Humanos , Teste de Materiais , Células-Tronco Mesenquimais/metabolismo , CamundongosRESUMO
This paper contains original data supporting the antibacterial activities of Gallium (Ga(3+))-doped pro-osteointegrative titanium alloys, obtained via Anodic Spark Deposition (ASD), as described in "The effect of silver or gallium doped titanium against the multidrug resistant Acinetobacter baumannii" (Cochis et al. 2016) [1]. In this article we included an indirect cytocompatibility evaluation towards Saos2 human osteoblasts and extended the microbial evaluation of the Ga(3+) enriched titanium surfaces against the biofilm former Escherichia coli and Staphylococcus epidermidis strains. Cell viability was assayed by the Alamar Blue test, while bacterial viability was evaluated by the metabolic colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Finally biofilm morphology was analyzed by Scanning Electron Microscopy (SEM). Data regarding Ga(3+) activity were compared to Silver.
RESUMO
Implant-related infection of biomaterials is one of the main causes of arthroplasty and osteosynthesis failure. Bacteria, such as the rapidly-emerging Multi Drug Resistant (MDR) pathogen Acinetobacter Baumannii, initiate the infection by adhering to biomaterials and forming a biofilm. Since the implant surface plays a crucial role in early bacterial adhesion phases, titanium was electrochemically modified by an Anodic Spark Deposition (ASD) treatment, developed previously and thought to provide osseo-integrative properties. In this study, the treatment was modified to insert gallium or silver onto the titanium surface, to provide antibacterial properties. The material was characterized morphologically, chemically, and mechanically; biological properties were investigated by direct cytocompatibility assay, Alkaline Phosphatase (ALP) activity, Scanning Electron Microscopy (SEM), and Immunofluorescent (IF) analysis; antibacterial activity was determined by counting Colony Forming Units, and viability assay. The various ASD-treated surfaces showed similar morphology, micrometric pore size, and uniform pore distribution. Of the treatments studied, gallium-doped specimens showed the best ALP synthesis and antibacterial properties. This study demonstrates the possibility of successfully doping the surface of titanium with gallium or silver, using the ASD technique; this approach can provide antibacterial properties and maintain high osseo-integrative potential.
Assuntos
Infecções por Acinetobacter/prevenção & controle , Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Materiais Revestidos Biocompatíveis/farmacologia , Gálio/farmacologia , Prata/farmacologia , Infecções por Acinetobacter/etiologia , Acinetobacter baumannii/fisiologia , Antibacterianos/química , Aderência Bacteriana/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Linhagem Celular , Materiais Revestidos Biocompatíveis/química , Resistência a Múltiplos Medicamentos , Gálio/química , Humanos , Próteses e Implantes/efeitos adversos , Prata/química , Propriedades de Superfície , Titânio/químicaRESUMO
BACKGROUND: Recent reports have revealed the therapeutic potential of cell-mediated immunity in neoplasms such as cutaneous squamous cell carcinoma (SCC). OBJECTIVES: To define the antigenic coexpression of regulatory T cells (Tregs) and plasmacytoid dendritic cells (pDCs) and assess the CD8(+) /Foxp3(+) CD25(+) cell ratio at peritumoral and intratumoral levels in order to investigate a correlation with the aggressiveness of SCC tumours. METHODS: We evaluated the content and distribution of Foxp3(+) CD25(+) Treg and CD123(+) pDC infiltration and assessed CD8(+) /Foxp3(+) CD25(+) cell ratio at peritumoral and intratumoral levels in 40 SCCs (20 well-differentiated, G1; and 20 moderately to poorly differentiated, G2-G3) to investigate a correlation with their aggressiveness. We determined the profiles of Tregs and CD123(+) cells; immunostained for CD4, CD8, CD123, interleukin (IL)-1 and transforming growth factor (TGF)-ß1; and unequivocally double stained for Foxp3CD25. RESULTS: Peritumorally, CD4, CD8 and Foxp3 expression showed no difference between the two groups. CD123(+) cells were fewer in G2-G3 (P = 0·0005), while Foxp3(+) CD25(+) cells were more numerous (P = 0·0005). The Foxp3(+) CD25(+) /Foxp3(+) ratio was higher in G2-G3 cases (P = 0·0005), confirming the trend in this group of activated T lymphocytes towards total Treg Foxp3(+) cells, while the CD8(+) /Foxp3(+) CD25(+) ratio was higher in G1 (P = 0·0005). Intratumorally, CD4(+) and CD8(+) cells infiltrated G2-G3 (P = 0·048) more than G1 (P = 0·004), whereas almost all cells were CD123 negative. Regarding Foxp3CD25, TGF-ß1 and IL-10, they were less expressed in G1, whereas they were positive in G2-G3 (P < 0·05). The CD8(+) /Foxp3(+) CD25(+) ratio was similar to that observed in peritumoral infiltration. CONCLUSIONS: Our data suggest that intratumoral recruitment of Tregs, high expression of TGF-ß1 and IL-10, almost negative CD123+, and a low CD8(+) /Foxp3(+) CD25(+) T-cell ratio may contribute to the aggressiveness of cutaneous SCC, as already evidenced for other solid tumours.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Carcinoma de Células Escamosas/imunologia , Fatores de Transcrição Forkhead/metabolismo , Imunidade Celular/fisiologia , Neoplasias Cutâneas/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/patologia , Células Dendríticas/imunologia , Feminino , Humanos , Interleucina-10/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Neoplasias Cutâneas/patologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
AIMS: To investigate whether the expression of interferon (IFN)-inducible gene IFI16 is inversely related to proliferative activity in vivo, we compared immunohistochemical reactivity of IFI16 in a series of head and neck squamous cell carcinomas (HNSCCs) with their proliferation index and the cell cycle regulator pRb. As human papillomavirus (HPV) infection is manifested by changes in the function or expression level of host genes such as IFN-inducible genes, we also investigated the presence of HPV DNA to determine whether head and neck cancers associated with HPV DNA can be distinguished from tumours that are presumably transformed by other mechanisms. METHODS: Thirty-six HNSCCs were evaluated for IFI16, pRb and Ki67 expression by immunohistochemistry. The presence of HPV was also detected by polymerase chain reaction. Nine tumours were located in the oropharynx (tonsillar area) and 27 in the larynx. RESULTS: HPV DNA was found in 14 of 25 (56%) laryngeal SCCs and in five of nine (56%) tonsillar SCC specimens examined; 17 out of the 19 HPV-DNA-positive cases showed high-grade IFI16 expression. Overall, proliferative activity was significantly related to tumour differentiation and histological grading. IFI16 protein expression was significantly inversely correlated with Ki67 (P = 0.039). Low-proliferating tumours positive for IFI16 staining showed a marked expression of pRb and a better prognosis than those whose tumours had low IFI16, pRb levels and a high proliferation index. CONCLUSIONS: To our knowledge, this is the first expression analysis of the IFN-inducible IFI16 gene in HNSCC. Low-proliferating tumours positive for IFI16 staining showed a marked expression of pRb and a better prognosis than those whose tumours had low IFI16, pRb levels and a high proliferation index.
Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/patologia , Proteínas Nucleares/biossíntese , Fosfoproteínas/biossíntese , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/virologia , DNA Viral/genética , Feminino , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Imuno-Histoquímica , Antígeno Ki-67/análise , Masculino , Pessoa de Meia-Idade , Papillomaviridae/genética , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase , Proteína do Retinoblastoma/análiseRESUMO
We have previously demonstrated that overexpression of p204, a member of the Ifi 200 gene family, inhibits growth, delays G0/G1 progression into S phase, and impairs E2F-mediated transcriptional activity. In this study, we show that p204 directly binds the retinoblastoma protein (pRb) in vivo to exert its activity. Transient p204 overexpression in Rb+/+ mouse embryo fibroblasts (MEF) inhibits cell proliferation, but does not affect cell growth in MEF derived from Rb-/- mice. Two human cell lines, Saos2 and C33A, bearing an inactive pRb, but not primary human embryo fibroblasts, are resistant to the p204 antiproliferative activity. p204 contains two 200 amino acid motifs, designated as type a or b domains, each containing a canonical Rb binding motif (LXCXE). When dominant-negative mutants at the Rb binding motif were transfected in Rb+/+ MEF, p204 lost its ability to inhibit cell growth, delay cell transition from G1 to S phase, and impair DNA synthesis. Moreover p204 overexpression in Rb+/+ MEF led to a significant decrease of both DHFR and PCNA proteins, two S phase markers. By contrast, this effect was not observed when Rb+/+ MEF were transfected with a p204 mutated at both Rb binding sites. Finally, overexpression of the LXCXE p204 mutant rendered Rb+/+ MEF resistant to the IFN-alpha antiproliferative activity, in comparison to the untransfected Rb+/+ MEF. As expected, Rb-/- cells were unsensitive to the IFN-alpha induced growth inhibition. Taken as a whole, these results suggest that (i) p204 contributes to the IFN-alpha antiproliferative activity and (ii) the primary target of p204 leading to efficient G1 arrest as well as to blockade of DNA replication from G1 phase is the pRb regulatory system.
Assuntos
Inibidores do Crescimento/metabolismo , Interferon-alfa/farmacologia , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteína do Retinoblastoma/metabolismo , Células 3T3 , Animais , Sítios de Ligação , Ciclo Celular , Divisão Celular , DNA/biossíntese , Fase G1 , Expressão Gênica , Inibidores do Crescimento/genética , Humanos , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Fosfoproteínas/genética , Coelhos , Proteína do Retinoblastoma/genética , Fase S , Células Tumorais CultivadasRESUMO
To examine whether Ifi 200 genes are involved in antiviral state induction by IFNs we expressed mutant forms capable of inactivating the endogenous p204 and analyzed replication of both RNA and DNA viruses following IFN-alpha treatment. Inactivation of p204 does not impair replication of vesicular stomatitis virus, encephalomyocarditis virus, ectromelia virus, and herpes simplex virus 1 and does not alter an IFN-alpha induced antiviral state. By contrast, in cells lacking functional p204, mouse cytomegalovirus (MCMV) replication is strongly inhibited and is not further modulated by IFN-alpha. These results suggest that p204, a member of the Ifi 200 gene family, is not involved in the IFN-alpha-induced antiviral activity against some RNA or DNA viruses, but is required by MCMV for its replication.
Assuntos
Interferon-alfa/farmacologia , Família Multigênica/genética , Muromegalovirus/fisiologia , Proteínas Nucleares/genética , Fosfoproteínas/genética , Replicação Viral/genética , Células 3T3 , Animais , Células Cultivadas , Técnica Indireta de Fluorescência para Anticorpo , Camundongos , Muromegalovirus/efeitos dos fármacos , Muromegalovirus/genética , Mutação/genética , Mutação/fisiologia , Proteínas Nucleares/análise , Proteínas Nucleares/fisiologia , Fosfoproteínas/análise , Fosfoproteínas/fisiologia , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologiaRESUMO
Recent evidence has shown that human papillomavirus (HPV) is involved in both the development of carcinoma and in premalignant mucosal lesions of the oral cavity. This study examined the relationship of HPV infection to some pathological features in precancerous lesions of the larynx, not examined extensively so far. Fifty formalin-fixed paraffin-embedded tissue sections containing human laryngeal precancerous lesions were screened for the presence of HPV infection by polymerase chain reaction, and for capsid protein expression by immunohistochemistry with polyclonal antibody directed against the L1 protein. The presence of HPV DNA was detected in 28 of 50 specimens (56%), including 9/12 cases with mild dysplasia (75%), 3/6 cases with moderate dysplasia (50%), and 7/11 cases with severe dysplasia (64%). Multiple HPV infections, containing two or three types, were detected in 17 of the 28 HPV-positive lesions (60%). Of 21 cases with keratosis and no dysplasia, 11 were positive for HPV DNA (52%) and 4 showed L1 staining (36%). By contrast, L1 positivity was revealed only in two lesions with moderate dysplasia, confirming that fully productive HPV infection is strictly dependent on epithelial differentiation and surface keratinization. The probability that HPV is a cofactor in the malignant progression of these lesions is suggested by the fact that 3/4 patients who developed cancer within 50 months were positive for HPV DNA.
Assuntos
Neoplasias Laríngeas/virologia , Laringe/virologia , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/virologia , Lesões Pré-Cancerosas/virologia , Infecções Tumorais por Vírus/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , DNA Viral/análise , Humanos , Hiperplasia/patologia , Hiperplasia/virologia , Imuno-Histoquímica , Ceratose/virologia , Neoplasias Laríngeas/patologia , Laringe/patologia , Pessoa de Meia-Idade , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/classificação , Reação em Cadeia da Polimerase/métodos , Lesões Pré-Cancerosas/patologiaRESUMO
Iron may be important in catalyzing excessive production of reactive oxygen species (ROS). Cellular iron homeostasis is regulated by iron regulatory proteins (IRPs), which bind to iron-responsive elements (IRE) of mRNAs for ferritin and transferrin receptor (TfR) modulating iron uptake and sequestration, respectively. Although iron is the main regulator of IRP activity, IRP is also influenced by other factors, including the redox state. Therefore, IRP might be sensitive to pathophysiological alterations of redox state caused by ROS. However, previous studies have produced diverging evidence on the effect of oxidative injury on IRP. Results obtained in an animal model close to a pathophysiological condition, such as ischemia reperfusion of the liver as well as in a cell-free system involving an enzymatic source of O2 and H2O2, indicate that IRP is downregulated by oxidative stress. In fact, IRP activity is inhibited at early times of post-ischemic reperfusion. Moreover, the concerted action of O2 and H2O2 produced by xanthine oxidase in a cell-free system caused a remarkable inhibition of IRP activity. IRP seems a direct target of ROS; in fact, in vivo inhibition can be prevented by the antioxidant N-acetylcysteine and by interleukin-1 receptor antagonist. In addition, modulation of iron levels of the cell-free assay did not affect the downregulation imposed by xanthine oxidase. Conceivably, downregulation of IRP activity by O2 and H2O2 may facilitate iron sequestration into ferritin, thus limiting the pro-oxidant challenge of iron.