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1.
Int J Mol Sci ; 18(10)2017 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-29019941

RESUMO

Laminitis, a highly debilitating disease of the foot in ungulates, is characterized by pathological changes of the complex lamellar structures that maintain the appendicular skeleton within the hoof. Laminitis is a multifactorial disease that involves perturbation of the vascular, hematological, and inflammatory homeostasis of the foot. Interestingly, the pathogenesis of the disease resembles what is observed in metabolic syndromes and sepsis-induced organ failure in humans and animals. We hypothesized that local administration of mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) might contribute to establishing an anti-inflammatory and pro-angiogenic environment, and could stimulate the injured tissue in order to restore its functional integrity. According to this assumption, an experimental protocol based on the local intravenous administration of adipose tissue-derived MSCs (aMSCs) in combination with PRP was developed for the treatment of horses affected by chronic laminitis. Nine horses with severely compromised venograms (showing grade III and IV laminitis) that had been unsuccessfully treated with conventional therapies were enrolled. aMSCs and PRP (15 × 106 cells resuspended in 15 mL of PRP) were injected into the lateral or medial digital vein three times, at one-month intervals. The first administration was performed with allogeneic aMSCs, while for the following administrations, autologous aMSCs were used. There was no adverse short-term reaction to the intravenous injection of aMSCs. In the long term, venograms outlined, in all subjects, a progressive amelioration of the vascularization of the foot. An improvement in the structure and function of the hoof was also observed. No adverse events were reported during the follow-up, and the horses returned to a comfortable quality of life. Although the number of animals enrolled in the study is limited, both clinical observations and venography demonstrated an enhancement in the condition of all horses, suggesting that the regenerative therapies in chronic laminitis could be useful, and are worthy of further investigation.


Assuntos
Tecido Adiposo/citologia , Doenças do Pé/veterinária , Doenças dos Cavalos/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Plasma Rico em Plaquetas , Administração Intravenosa , Animais , Doença Crônica , Doenças do Pé/terapia , Casco e Garras/patologia , Cavalos , Inflamação/terapia , Inflamação/veterinária , Qualidade de Vida , Medicina Regenerativa
2.
PLoS One ; 12(1): e0169391, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28046048

RESUMO

The key role of cell cultures in different scientific fields is worldwide recognized, both as in vitro research models alternative to laboratory animals and substrates for biological production. However, many safety concerns rise from the use of animal/human cell lines that may be tumorigenic, leading to potential adverse contaminations in cell-derived biologicals. In order to evaluate the suitability of 13 different cell lines for Poliovirus vaccine production, safety and quality, in vitro/in vivo tumorigenicity and Poliovirus propagation properties were evaluated. Our results revealed that non-human primate cell lines CYNOM-K1, FRhK-4, 4MBr-5 and 4647 are free of tumorigenic features and represent highly susceptible substrates for attenuated Sabin Poliovirus strains. In particular, FRhK-4 and 4647 cell lines are characterized by a higher in vitro replication, resulting indicated for the use in large-scale production field.


Assuntos
Carcinogênese/patologia , Transformação Celular Neoplásica/patologia , Poliovirus/fisiologia , Replicação Viral , Animais , Bioensaio , Linhagem Celular , Primatas
3.
Res Vet Sci ; 103: 176-8, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26679814

RESUMO

Traditionally, embryonated chicken eggs (ECE) are considered the gold standard for Influenza virus isolation and vaccine production. Nowadays, different biological systems have been improved and performed, in order to evaluate a feasible alternative to ECE. In fact, in a previous study, mammalian and avian cell cultures were successfully used for avian influenza viruses primary isolation from target tissues and virus propagation. This research is focused on the investigation of adaptive mutations that occur after influenza A virus amplification in ECE and cell cultures. The results of the study shows that avian influenza viruses after multiple passages in different biological systems undergo mutations, in particular, the largest number of amino acid substitutions occurred in all biological substrates in the hemagglutinin.


Assuntos
Substituição de Aminoácidos , Vírus da Influenza A/genética , Mutação , Proteínas Virais/genética , Adaptação Biológica , Vírus da Influenza A/metabolismo , Proteínas Virais/metabolismo , Cultura de Vírus/veterinária
6.
Antiviral Res ; 120: 16-22, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25986248

RESUMO

Influenza A virus is the principal agent responsible of the respiratory tract's infections in humans. Every year, highly pathogenic and infectious strains with new antigenic assets appear, making ineffective vaccines so far developed. The discovery of RNA interference (RNAi) opened the way to the progress of new promising drugs against Influenza A virus and also to the introduction of disease resistance traits in genetically modified animals. In this paper, we show that Madin-Darby Canine Kidney (MDCK) cell line expressing short hairpin RNAs (shRNAs) cassette, designed on a specific conserved region of the nucleoprotein (NP) viral genome, can strongly inhibit the viral replication of four viral strains sharing the target sequence, reducing the viral mRNA respectively to 2.5×10(-4), 7.5×10(-5), 1.7×10(-3), 1.9×10(-4) compared to the control, as assessed by real-time PCR. Moreover, we demonstrate that during the challenge with a viral strain bearing a single mismatch on the target sequence, although a weaker inhibition is observed, viral mRNA is still lowered down to 1.2×10(-3) folds in the shRNA-expressing clone compared to the control, indicating a broad potential use of this approach. In addition, we developed a highly predictive and fast screening test of siRNA sequences based on dual-luciferase assay, useful for the in vitro prediction of the potential effect of viral inhibition. In conclusion, these findings reveal new siRNA sequences able to inhibit Influenza A virus replication and provide a basis for the development of siRNAs as prophylaxis and therapy for influenza infection both in humans and animals.


Assuntos
Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/fisiologia , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas do Core Viral/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Animais , Aves , Cães , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Células Madin Darby de Rim Canino , Infecções por Orthomyxoviridae/veterinária , Infecções por Orthomyxoviridae/virologia , RNA Interferente Pequeno/genética , RNA Viral/análise , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Suínos , Proteínas do Core Viral/genética
7.
Stem Cells Dev ; 24(6): 677-85, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25517941

RESUMO

Mesenchymal stromal cells (MSCs), as advanced therapy products, must satisfy all the requirements for human use of medicinal products, aiming to maintain the quality and safety of the cells. The MSC manufacturing process for clinical use should comply with the principles of Good Manufacturing Practice (GMP). This ensures that cell preparations are produced and controlled, from the collection and manipulation of raw materials, through the processing of intermediate products, to the quality controls, storage, labeling and packaging, and release. The objective of this document is to provide the minimal quality requirements for the MSC production and its delivery for clinical use, so that the safety of the final cell therapy product will not be compromised. For this purpose, the document evaluates the most important steps of GMP-compliant MSC production: the isolation and expansion process; the validation phase of the process, including all quality controls for the characterization, functionality, potency, and safety of MSCs; and the quality control at the batch release to guarantee the safety of patient infusion.


Assuntos
Transplante de Células-Tronco Mesenquimais/normas , Células-Tronco Mesenquimais/citologia , Ensaios Clínicos como Assunto , Humanos , Técnicas In Vitro , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Controle de Qualidade
8.
Methods Mol Biol ; 1247: 43-60, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25399087

RESUMO

Biobanking is an essential tool for ensuring easy availability of high-quality biomaterial collections that combine essential samples and epidemiological, clinical, and research data for the scientific community. Specimen collection is an integral part of clinical research. Indeed, every year throughout the world, millions of biological samples are stored for diagnostics and research, but in many fields the lack of biological material and models is a major hindrance for ongoing research. A biobank facility provides suitable samples for large-scale screening studies and database repositories. Software dedicated to biological banks simplify sample registration and identification, the cataloging of sample properties (type of sample/specimen, associated diseases and/or therapeutic protocols, environmental information, etc.), sample tracking, quality assurance, and specimen availability characterized by well-defined features. Biobank facilities must adopt good laboratory practices (GLPs) and a stringent quality control system and also comply with ethical issues, when required. The creation of a veterinary network can be useful under different aspects: the first one is related to the importance of animal sciences itself to improve research and strategies in the different branches of the veterinary area, and the second aspect is related to the possibility of data management harmonization to improve scientific cooperation.


Assuntos
Bancos de Espécimes Biológicos , Medicina Veterinária , Doenças dos Animais/diagnóstico , Animais , Bancos de Espécimes Biológicos/normas , Preservação Biológica/métodos , Pesquisa , Manejo de Espécimes/métodos , Manejo de Espécimes/normas , Medicina Veterinária/normas
10.
Br J Haematol ; 160(6): 766-78, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23293837

RESUMO

Current leukaemia therapy focuses on increasing chemotherapy efficacy. Mesenchymal stromal cells (MSCs) have been proposed for carrying and delivery drugs to improve killing of cancer cells. We have shown that MSCs loaded with Paclitaxel (PTX) acquire a potent anti-tumour activity. We investigated the effect of human MSCs (hMSCs) and mouse SR4987 loaded with PTX (hMSCsPTX and SR4987PTX) on MOLT-4 and L1210, two leukaemia cell (LCs) lines of human and mouse origin, respectively. SR4987PTX and hMSCsPTX showed strong anti-LC activity. hMSCsPTX, co-injected with MOLT-4 cells or intra-tumour injected into established subcutaneous MOLT-4 nodules, strongly inhibited growth and angiogenesis. In BDF1-mice-bearing L1210, the intraperitoneal administration of SR4987PTX doubled mouse survival time. In vitro, both hMSCs and hMSCsPTX released chemotactic factors, bound and formed rosettes with LCs. In ultrastructural analysis of rosettes, hMSCsPTX showed no morphological alterations while the attached LCs were apoptotic and necrotic. hMSCs and hMSCsPTX released molecules that reduced LC adhesion to microvascular endothelium (hMECs) and down-modulated ICAM1 and VCAM1 on hMECs. Priming hMSCs with PTX is a simple procedure that does not require any genetic cell manipulation. Once the effectiveness of hMSCsPTX on established cancers in mice is proven, this procedure could be proposed for leukaemia therapy in humans.


Assuntos
Comunicação Celular/fisiologia , Leucemia/patologia , Leucemia/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/patologia , Paclitaxel/farmacologia , Animais , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Leucemia/tratamento farmacológico , Leucemia/cirurgia , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Nus , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Anticancer Agents Med Chem ; 13(3): 523-30, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22931415

RESUMO

The main goal in cancer chemotherapy is to drive the drug into the tumor microenvironment to kill as many cancer cells as possible while producing the lowest collateral toxicity. Previously, we have shown that human bone marrow derived mesenchymal stromal cells (hBM-MSCs) exposed to Paclitaxel (PTX) were able to uptake and subsequently release the drug in the culture medium. PTX primed hBM-MSCs (hBM-MSCsPTX) located in the vicinity of cancer cells produced a strong inhibition of tumor cell growth both in vitro and in vivo. To expand these observations, in the present study we exposed human skin derived fibroblasts (hSDFs) to 2,000 ng/ml of PTX and then tested both cells and their conditioned medium (CM) in vitro for their capacity to inhibit the proliferation of human tumor cell lines (MOLT-4, DU-145, U87-MG, SH-SY5Y(+) and LAN-5). We found that hSDFs primed with PTX (hSDFsPTX) were able to uptake and subsequently release PTX in a time dependent manner. hSDFsPTX-derived CM(hSDFsPTX-CM) from 1:4 to 1:10 dilutions produced a significant (p < 0.05) in vitro tumor growth inhibition. hSDFsPTX co-cultured with leukemia cells at 1:1 to 1:10 ratio, completely inhibited cells growth whereas no inhibition was induced by normal hSDFs cells. Our results demonstrate for the first time that hSDFs can be loaded in vitro with PTX and thus can acquire a potent anti-tumor activity. Since hSDFs can be easily isolated from skin biopsies without any particular pain and discomfort to donor patients, we conclude that hSDFs may represent a valid cell type option for carrying and delivering anti-cancer drugs.


Assuntos
Antineoplásicos/farmacologia , Preparações de Ação Retardada/farmacologia , Fibroblastos/metabolismo , Paclitaxel/farmacologia , Pele/metabolismo , Antineoplásicos/metabolismo , Transporte Biológico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Preparações de Ação Retardada/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Paclitaxel/metabolismo , Pele/citologia , Pele/efeitos dos fármacos , Microambiente Tumoral
12.
Vaccines (Basel) ; 1(4): 463-80, 2013 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-26344342

RESUMO

In veterinary medicine, there have been different experiences with the plasmid DNA vaccination. In this area and with the hypothesis to demonstrate the effectiveness of different plasmids encoding porcine respiratory and reproductive syndrome (PRRS), five DNA vaccines against PRRS were evaluated for their innocuity and efficacy in pigs. Eighteen animals were divided into five groups which were injected with five (A, B, C, D, E) different DNA vaccines. Albeit, none of the proposed vaccines were able to protect the animals against PRRS virus. Only vaccines A and B were able to reduce the clinical signs of the infection. ELISA IgM were detected 30 days after the first vaccination in the pigs injected by Vaccine A or B. ELISA IgG were detected 90 days after the first vaccination in the pigs injected by Vaccine B or C. Neutralizing antibody were detected Post Challenge Days 61 (PCD) in all groups. In the pigs inoculated with Vaccine C, IFN-g were detected 90 days after first vaccination, and after challenge exposure they increased. In the other groups, the IFN-g were detected after challenge infection. Pigs injected with each of the vaccines A, B, C, D and E showed a significantly higher level of CD4(-)CD8⁺ lymphocytes (p < 0.001) after infection in comparison with their controls.

13.
Lab Invest ; 92(9): 1297-309, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22732936

RESUMO

Hepatocellular carcinoma (HCC) is a very angiogenic and malignant cancer. Conventional chemotherapy is poorly effective because of the abnormal structural organization of HCC-infiltrating vessels. In previous work, we demonstrated that HCC angiogenesis is driven by transforming growth factor beta-1(TGF-ß1)/CD105 axis, stimulating liver-derived microvascular endothelial cells (Ld-MECs) migration. As TGF-ß1 also affects mural cells (MCs) recruitment and maturation, we asked whether it may contribute to HCC-induced vascular abnormalities. HCC and adjacent non-neoplastic liver (nNL) biopsies obtained from 12 patients were analyzed by immunohistochemistry for angiogenic markers CD105, TGF-ß1, CD44 and vascular endothelial growth factor-a (VEGFa) and for MC markers NG2, α-smooth muscle actin (αSMA) and neural cell adhesion molecule (NCAM). The same markers were also investigated by immunocytochemistry on cultured HCC-derived stromal cells (HCC-StCs) and nNL-derived StCs (nNL-StCs) isolated from the same liver biopsies. Angiogenic factors released by StCs were analyzed by ELISA and the interaction between StCs and Ld-MECs by adhesion assay. Compared with nNL, HCC biopsies showed increased angiogenic markers and αSMA that was localized in vessels. By contrast, NG2 and NCAM were substantially localized in tumor cells but absent in vessels and stroma. Cultured HCC-StCs showed less expression of NG2, αSMA and NCAM. They also demonstrated a lower capacity to release angiogenic factors and adhered on Ld-MECs. HCC-StCs and nNL-StCs treated with TGF-ß1 or with of HepG2 (a human hepatoma cell line) derived conditioned medium (CM), down-modulated NCAM expression, whereas anti-NCAM antibodies significantly reduced the adhesion of StCs to Ld-MECs. By further blocking TGF-ß1 with anti-TGF-ß1 antibodies or with Ly-364947 (a specific inhibitor TGF-ß1-receptor) adhesion to Ld-MECs and NCAM expression respectively was partially restored. TGF-ß1 contributes to HCC-induced vascular alterations by affecting the interaction between HCC-StCs and Ld-MECs through a down-modulation of NCAM expression.


Assuntos
Carcinoma Hepatocelular/metabolismo , Regulação para Baixo , Neoplasias Hepáticas/metabolismo , Microvasos/anormalidades , Moléculas de Adesão de Célula Nervosa/fisiologia , Fator de Crescimento Transformador beta1/fisiologia , Biomarcadores/metabolismo , Carcinoma Hepatocelular/irrigação sanguínea , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/irrigação sanguínea , Neoplasias Hepáticas/patologia , Neovascularização Patológica
14.
J Virol Methods ; 185(1): 82-8, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22728276

RESUMO

Influenza outbreaks are widespread in swine and avian populations. Disease control is jeopardized by the extreme antigenic variability of virus strains. Primary isolation of Influenza virus is performed using embryonated chicken eggs (ECE), but alternatives to ECE are badly needed. Although various cultured cells have been used for propagating Influenza A viruses, few types of cells can efficiently support virus replication. One of the most commonly cell lines used in order to isolate Influenza A virus, is represented by the Madin Darby Canine Kidney (MDCK) cell line, but cells derived from primary swine organs (kidney, testicle, lung and trachea) can also be employed. The aim of this study was the evaluation of NSK, MDCK, UMNSAH/DF1 cell lines suitability, compared to ECE for isolation and propagation of Avian and Swine virus subtypes. The results indicated both NSK and MDCK could provide an appropriate substrate for cultivating either Avian (AIV) or Swine (SIV) Influenza virus strains, especially for high pathogenicity Avian Influenza ones. Furthermore, NSK appeared more susceptible than MDCK cells for primary isolation of AIV. In contrast, UMNSAH/DF1 cell line seemed to be less permissive to support Avian virus growth. Furthermore, no SIV replication was detected except for one subtype. Additionally, the results of this study indicated that not all virus strains seemed to adapt with the same efficiency to the different cell lines. On the contrary, chicken embryos were shown to be the most suitable biological system for AIV isolation.


Assuntos
Especificidade de Hospedeiro , Vírus da Influenza A/fisiologia , Animais , Linhagem Celular , Galinhas , Cães , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/patogenicidade , Suínos , Cultura de Vírus/métodos
15.
Biologicals ; 40(1): 31-5, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22014410

RESUMO

Investigations into immune responses are often based upon recovery of peripheral blood mononuclear cells (PBMC). To this purpose, the recovery of PBMC by gradient centrifugation is labour-intensive and requires a reasonable level of skill by the laboratory technician. Thus, we set out to determine whether laboratory automation equipment could be used for the recovery of PBMC from blood samples of horses, pigs and cattle, based on the Ficoll-Paque gradient centrifugation technique. Mixing of blood samples with PBS, layering of diluted blood onto Ficoll-Paque gradients, recovery of separated PBMC in RPMI 1640 medium were performed using an automated robotic system, the SBF200 (AM Robotic Systems, Warrington, UK) under laminar air flow conditions. Tubes were tagged with bar codes and manually placed after gradient centrifugation into a tube reader to measure the volume and position of the PBMC layer. The results of the automated procedure compared very well to those of the manual one in terms of percent cell recovery, sterility and cell viability. Also, a high throughput of samples could be implemented: with the integration of cell counting it should be possible for 96 blood samples to be processed, including the production of aliquots, by one person in a day.


Assuntos
Separação Celular/instrumentação , Separação Celular/métodos , Leucócitos Mononucleares/citologia , Robótica/instrumentação , Robótica/métodos , Animais , Bovinos , Sobrevivência Celular , Estudos de Avaliação como Assunto , Cavalos , Suínos
16.
Biopreserv Biobank ; 10(3): 276-81, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24835066

RESUMO

The advent of stem cells and stem cell-based therapies for specific diseases requires particular knowledge of laboratory procedures, which not only guarantee the continuous production of cells, but also provide them an identity and integrity as close as possible to their origin. Their cryopreservation at temperatures below -80°C and typically below -140°C is of paramount importance. This target can be achieved by incorporating high molar concentrations of cryoprotectant mixtures that preserve cells from deleterious ice crystal formation. Usually, dimethyl sulfoxide (DMSO) and animal proteins are used as protectant reagents, but unexpected changes in stem cell fate and downstream toxicity effects have been reported, limiting their wide use in clinical settings. In scientific reviews, there are not much data regarding viability of mesenchymal stromal cells (MSCs) after the freezing/thawing process. During our routine analysis, a poor resistance to cryopreservation of these cells was observed, as well as their weak ability to replicate. This is an important point in the study of MSCs; moreover, it represents a limit for preservation and long-term storage. For this reason, MSCs isolated from equine, ovine, and rodent bone marrow and equine adipose tissue were compared using different cryopreservation solutions for this study of vitality. Our findings showed the best results regarding cell viability using a solution of fetal bovine serum with addition of 10% DMSO. In particular, we noted an increase in survival of equine bone marrow MSCs. This parameter has been evaluated by Trypan blue staining at fixed times (0, 24, and 48 hours post-thaw). This result highlights the fact that equine bone marrow MSCs are the frailest we analyzed. Therefore, it could be useful to delve further into this topic in order to improve the storage possibility for these cells and their potential use in cell-based therapies.


Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Células-Tronco Mesenquimais/citologia , Soluções para Preservação de Órgãos/química , Tecido Adiposo/citologia , Animais , Sobrevivência Celular , Células Cultivadas , Cavalos , Ratos , Ovinos , Bancos de Tecidos
17.
PLoS One ; 6(12): e28321, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22205945

RESUMO

BACKGROUND: Mesenchymal stromal cells may represent an ideal candidate to deliver anti-cancer drugs. In a previous study, we demonstrated that exposure of mouse bone marrow derived stromal cells to Doxorubicin led them to acquire anti-proliferative potential towards co-cultured haematopoietic stem cells (HSCs). We thus hypothesized whether freshly isolated human bone marrow Mesenchymal stem cells (hMSCs) and mature murine stromal cells (SR4987 line) primed in vitro with anti-cancer drugs and then localized near cancer cells, could inhibit proliferation. METHODS AND PRINCIPAL FINDINGS: Paclitaxel (PTX) was used to prime culture of hMSCs and SR4987. Incorporation of PTX into hMSCs was studied by using FICT-labelled-PTX and analyzed by FACS and confocal microscopy. Release of PTX in culture medium by PTX primed hMSCs (hMSCsPTX) was investigated by HPLC. Culture of Endothelial cells (ECs) and aorta ring assay were used to test the anti-angiogenic activity of hMSCsPTX and PTX primed SR4987(SR4987PTX), while anti-tumor activity was tested in vitro on the proliferation of different tumor cell lines and in vivo by co-transplanting hMSCsPTX and SR4987PTX with cancer cells in mice. Nevertheless, despite a loss of cells due to chemo-induced apoptosis, both hMSCs and SR4987 were able to rapidly incorporate PTX and could slowly release PTX in the culture medium in a time dependent manner. PTX primed cells acquired a potent anti-tumor and anti-angiogenic activity in vitro that was dose dependent, and demonstrable by using their conditioned medium or by co-culture assay. Finally, hMSCsPTX and SR4987PTX co-injected with human cancer cells (DU145 and U87MG) and mouse melanoma cells (B16) in immunodeficient and in syngenic mice significantly delayed tumor takes and reduced tumor growth. CONCLUSIONS: These data demonstrate, for the first time, that without any genetic manipulation, mesenchymal stromal cells can uptake and subsequently slowly release PTX. This may lead to potential new tools to increase efficacy of cancer therapy.


Assuntos
Antineoplásicos/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Paclitaxel/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Transporte Biológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Cinética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Neoplasias/irrigação sanguínea , Neoplasias/metabolismo , Neovascularização Patológica/tratamento farmacológico , Paclitaxel/metabolismo , Paclitaxel/uso terapêutico , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Ann Thorac Med ; 6(4): 217-20, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21977067

RESUMO

BACKGROUND: Non-invasive measurement of oxygenation is a routine procedure in clinical practice, but transcutaneous monitoring of PCO(2)(PtCO(2)) is used much less than expected. METHODS: The aim of our study was to analyze the value of a commercially available combined SpO(2)/PtCO(2) monitor (TOSCA-Linde Medical System, Basel, Switzerland) in adult non-invasive ventilated patients with acute respiratory failure. Eighty critically ill adult patients, requiring arterial blood sample gas analyses, underwent SpO(2) and PtCO(2) measurements (10 min after the probe was attached to an earlobe) simultaneously with arterial blood sampling. The level of agreement between PaCO(2) - PtCO(2) and SaO(2) - SpO(2)was assessed by Bland-Altman analyses. RESULTS: Both, SaO(2) from blood gas analysis and SpO(2) from the transcutaneous monitor, and PaCO(2) and PtCO(2) were equally useful. No measurements were outside of the acceptable clinical range of agreement of ± 7.5 mmHg. CONCLUSIONS: The accuracy of estimation of the TOSCA transcutaneous electrode (compared with the "gold standard" blood sample gas analysis) was generally good. Moreover, TOSCA presents the advantage of the possibility of continuous non-invasive measurement. The level of agreement of the two methods of measurement allows us to state that the TOSCA sensor is useful in routine monitoring of adults admitted to an intermediate respiratory unit and undergoing non-invasive ventilation.

19.
Res Vet Sci ; 90(2): 218-25, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20598328

RESUMO

The purpose of this study was to evaluate the time-course of the immune response to a field Porcine Respiratory and Reproductive Syndrome virus (PRRSV) strain in PRRS-naïve, untreated pigs, as well as in four groups of age and breed-matched pigs injected with a live attenuated PRRS vaccine, its adjuvant, an inactivated PRRS vaccine and an irrelevant, inactivated Porcine Circovirus type 2 (PCV2) vaccine, respectively. PRRSV infection was confirmed in all groups by PCR and antibody assays. The antibody response measured by ELISA took place earlier in pigs injected with the live attenuated vaccine, which also developed a much stronger serum-neutralizing antibody response to the vaccine strain. Yet, no clear protection was evidenced in terms of viremia against the field virus strain, which showed 11.1% nucleotide divergence in ORF7 from the vaccine strain. In vitro, the interferon (IFN)-γ response to PRRSV was almost absent on PVD 60 in all groups under study, whereas the prevalence of interleukin (IL)-10 responses to PRRSV was fairly high in PCV2-vaccinated animals, only. Results indicate that distinct patterns of immune response to a field PRRSV strain can be recognized in PRRS-vaccinated and naïve pigs, which probably underlies fundamental differences in the development and differentiation of PRRSV-specific immune effector cells.


Assuntos
Síndrome Respiratória e Reprodutiva Suína/prevenção & controle , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes , Células Cultivadas , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática/veterinária , Regulação da Expressão Gênica/imunologia , Imunidade Celular , Imunoglobulina G/sangue , Leucócitos/metabolismo , Síndrome Respiratória e Reprodutiva Suína/imunologia , Suínos , Viremia
20.
Vaccine ; 29(5): 867-72, 2011 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-21115049

RESUMO

Increasingly effective vaccination strategies are needed to counteract the high incidence of contagious diseases associated with intensive swine breeding. Recombinant viral vaccines are a promising new avenue in this direction. Key features of viral vectors suitable for immunoprophylaxis are safety, ease of manipulation and the ability to replicate in a variety of hosts. Most of the above requirements are met by bovine herpesvirus 4 (BoHV-4), a non-pathogenic dsDNA virus capable of infecting a broad range of cell types in vitro. Here we report the results of an exploratory study using an engineered BoHV-4 virus (eBoHV-4) expressing two unrelated glycoprotein antigens from bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1), to assess the potential of recombinant BoHV-4 as a self-adjuvanted immunogen in pigs. Free eBoHV-4 virions and virions preloaded into homologous swine adipose-derived stromal cells (SADSC) were tested. Neither virus formulation elicited neutralizing anti-BoHV-4 antibodies, nor any disease symptom, yet both induced specific immune responses against the heterologous antigens. However, a much earlier (18 vs 28 days post-infection) and more robust neutralizing response against BVDV and BoHV-1 viruses was elicited by eBoHV-4-preinfected SADSCs compared to free virions. The data validate BoHV-4 as a safe and effective heterologous antigen carrier/producer and identify SADSCs as helpful tools for the formulation of increasingly efficacious recombinant immunogens for pig vaccination.


Assuntos
Anticorpos Antivirais/sangue , Vírus da Diarreia Viral Bovina/imunologia , Vetores Genéticos , Herpesvirus Bovino 4/genética , Células Estromais/imunologia , Vacinação/métodos , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Antígenos Virais/genética , Antígenos Virais/imunologia , Vírus da Diarreia Viral Bovina/genética , Herpesvirus Bovino 4/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Células Estromais/metabolismo , Suínos , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
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