RESUMO
Bovicola ovis, commonly known as the sheep-biting louse, is an ectoparasite that adversely affects the sheep industry. Sheep louse infestation lowers the quality of products, including wool and leather, causing a loss of approximately AUD 123M per annum in Australia alone. The lack of a high-quality genome assembly for the sheep-biting louse, as well as any closely related livestock lice, has hindered the development of louse research and management control tools. In this study, we present the assembly of B. ovis with a genome size of ~123 Mbp based on a nanopore long-read sequencing library and Illumina RNA sequencing, complemented with a chromosome-level scaffolding using the Pore-C multiway chromatin contact dataset. Combining multiple alignment and gene prediction tools, a comprehensive annotation on the assembled B. ovis genome was conducted and recalled 11,810 genes as well as other genomic features including orf, ssr, rRNA and tRNA. A manual curation using alignment with the available closely related louse species, Pediculus humanus, increased the number of annotated genes to 16,024. Overall, this study reported critical genetic resources and biological insights for the advancement of sheep louse research and the development of sustainable control strategies in the sheep industry.
Assuntos
Sequenciamento por Nanoporos , Animais , Sequenciamento por Nanoporos/métodos , Ovinos/parasitologia , Anotação de Sequência Molecular , Cromossomos/genética , Doenças dos Ovinos/parasitologia , GenomaRESUMO
East Coast fever (ECF), caused by Theileria parva, is the most important tick-borne disease of cattle in sub-Saharan Africa. Practical disadvantages associated with the currently used live-parasite vaccine could be overcome by subunit vaccines. An 80-aa polypeptide derived from the C-terminal portion of p67, a sporozoite surface Ag and target of neutralizing Abs, was the focus of the efforts on subunit vaccines against ECF and subjected to several vaccine trials with very promising results. However, the vaccination regimen was far from optimized, involving three inoculations of 450 µg of soluble p67C (s-p67C) Ag formulated in the Seppic adjuvant Montanide ISA 206 VG. Hence, an improved formulation of this polypeptide Ag is needed. In this study, we report on two nanotechnologies that enhance the bovine immune responses to p67C. Individually, HBcAg-p67C (chimeric hepatitis B core Ag virus-like particles displaying p67C) and silica vesicle (SV)-p67C (s-p67C adsorbed to SV-140-C18, octadecyl-modified SVs) adjuvanted with ISA 206 VG primed strong Ab and T cell responses to p67C in cattle, respectively. Coimmunization of cattle (Bos taurus) with HBcAg-p67C and SV-p67C resulted in stimulation of both high Ab titers and CD4 T cell response to p67C, leading to the highest subunit vaccine efficacy we have achieved to date with the p67C immunogen. These results offer the much-needed research depth on the innovative platforms for developing effective novel protein-based bovine vaccines to further the advancement.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Nanotecnologia/métodos , Vacinas Protozoárias/imunologia , Theileria parva/fisiologia , Theileriose/imunologia , Doenças Transmitidas por Carrapatos/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Bovinos , Vírus da Hepatite B/química , Vírus da Hepatite B/genética , Camundongos , Óleo Mineral/administração & dosagem , Nanopartículas/química , Proteínas de Protozoários/genética , Vacinas Protozoárias/genética , Células RAW 264.7 , Dióxido de Silício/química , Carrapatos , Vacinação , Vacinas de Subunidades Antigênicas , Proteínas do Core Viral/química , Proteínas do Core Viral/genéticaRESUMO
Persistent human papillomavirus (HPV) infection is recognized as a major risk factor for cervical cancer. Women with persistent HPV and negative cytology are at greater risk of CIN2+ than women with negative infection. The diagnosis becomes more complicated when the woman has a type 3 transformation zone at colposcopy. The aim of this study was to determine the prevalence of CIN2+ in women with persistent HPV, negative cytology and TZ3; how to stratify the risk of CIN2+; and what the best diagnostic strategy is, given TZ3. METHODS: In a multicenter retrospective cohort study, we enrolled women with negative cytology and TZ3 among the 213 women referred for colposcopy for persistent HPV. The average age of the women was 53 years; in particular, 83% were postmenopausal women. In the presence of a TZ3, the entire transformation zone cannot be explored, making colposcopy and targeted biopsy useless and inadequate, with great risks of underdiagnosis or missed diagnosis. Women with TZ3 underwent diagnostic LEEP to ensure correct diagnoses. RESULTS: The study highlighted 19% (16/84) of CIN2+ lesions, a higher frequency of non-HPV 16/18 genotypes (76.2%), and 50% of CIN2+ lesions being due to non-HPV 16/18 genotypes. Furthermore, more than half of the women (80.9%) had normal histopathological results in the LEEP sample. CONCLUSION: Women with viral persistence, negative cytology, and TZ3 have a 19% risk of CIN2+; genotyping helps stratify risk, but extensive genotyping is necessary instead of partial genotyping (16/18), referring to a population of women over 50 years old in which the prevalence of genotypes 16,18 decreases and the prevalence of other genotypes increases; diagnostic LEEP is excessive (only 16 cases of CIN2+ out of 48 cases treated), even though 83% of women had viral clearance after LEEP; p16/Ki67 double staining could be a potential risk marker, which would only highlight women at risk of CIN2+ to undergo LEEP. To individualize the diagnostic workup and treatment and minimize the risk of under diagnosis and overtreatment, future studies should explore the use of extended genotyping and new biomarkers for individual risk stratification.
RESUMO
The purpose of this study was to evaluate the incidence of AIS and AC in the histological cone of women treated for CIN3. Furthermore, through the study of the specific HR HPV genotypes, we obtained more information on the possible different nature between the single CIN3 lesion and the CIN3 coexisting with the glandular lesion. METHODS: A sample of 414 women underwent LEEP for CIN3. The study sample consisted of 370 women with a CIN3 lesion alone and 44 women with a CIN3 lesion coexisting with AIS or adenocarcinoma. We studied the individual HR HPV genotypes and their frequency in the two groups under study. Furthermore, the therapeutic results and follow-ups for the population were studied on the entire study sample. RESULTS: In patients with a single CIN3 lesion, 11 high-risk genotypes were detected; in patients with CIN3 associated with AIS or AC, only 4 different genotypes were detected (16, 18, 45, 33). Overall, the frequency of HPV 18 was significantly higher in CIN3 coexisting with AIS compared to solitary CIN3 lesions, χ2 = 27.73 (p < 0.001), while the frequency of other high-risk genotypes was significantly higher in patients with a single CIN3 than in patients with CIN3 coexisting with AIS. In our study population, mixed lesions (CIN3 coexisting with AIS), unlike their squamous counterparts (single CIN3 lesions), were characterized by skip lesions, which demonstrate more aggressive behavior and a higher rate of viral persistence and recurrence. CONCLUSION: A relatively high rate (10.7%) of AIS-AC was found in women treated for CIN3. Our study confirms the multifocal biological nature of the CIN3 lesion coexisting with AIS compared to the single CIN3 lesion. All this justifies the different treatments to which CIN3 lesions coexisting with AIS are addressed; in fact, the latter are treated with hysterectomy, while CIN3 is treated with conization alone.
RESUMO
Immunization to the model protein antigen ovalbumin (OVA) is investigated using MCM-41 mesoporous silica nanoparticles as a novel vaccine delivery vehicle and adjuvant system in mice. The effects of amino surface functionalization and adsorption time on OVA adsorption to nanoparticles are assessed. Amino-functionalized MCM-41 (AM-41) shows an effect on the amount of OVA binding, with 2.5-fold increase in binding capacity (72 mg OVA/g AM-41) compared to nonfunctionalized MCM-41 (29 mg OVA/g MCM-41). Immunization studies in mice with a 10 µg dose of OVA adsorbed to AM-41 elicits both antibody and cell-mediated immune responses following three subcutaneous injections. Immunizations at a lower 2 µg dose of OVA adsorbed to AM-41 particles results in an antibody response but not cell-mediated immunity. The level of antibody responses following immunization with nanoformulations containing either 2 µg or 10 µg of OVA are only slightly lower than that in mice which receive 50 µg OVA adjuvanted with QuilA, a crude mixture of saponins extracted from the bark of the Quillaja saponaria Molina tree. This is a significant result, since it demonstrates that AM-41 nanoparticles are self-adjuvanting and elicit immune responses at reduced antigen doses in vivo compared to a conventional delivery system. Importantly, there are no local or systemic negative effects in animals injected with AM-41. Histopathological studies of a range of tissue organs show no changes in histopathology of the animals receiving nanoparticles over a six week period. These results establish the biocompatible MCM-41 silica nanoparticles as a new method for vaccine delivery which incorporates a self-adjuvant effect.
Assuntos
Adjuvantes Imunológicos/química , Nanopartículas/química , Ovalbumina/química , Dióxido de Silício/química , Animais , CamundongosRESUMO
BACKGROUND: The aim of this study was to determine the hemodynamic and clinical changes after occlusion of polytetrafluoroethylene (PTFE) femorotibial grafts. METHODS: Twenty-seven patients were randomly selected from all patients who underwent femorotibial bypass grafting in our department. In 10 patients, the reversed autologous saphenous vein was used as graft, and in 17 patients a PTFE prosthesis was used. Out of the latter 17 patients, 10 began long-term aspirin therapy and 7 began oral anticoagulation with warfarin. RESULTS: Nine out of the 10 patients with occluded PTFE grafts and who received only aspirin therapy had a critical ischemia after occlusion of the graft, and 4 underwent major amputation. Among the 10 patients with occluded autologous vein bypass, critical ischemia was present in only 4 patients, and only 2 required some form of surgical therapy with no case of major amputation. CONCLUSIONS: After occlusion of a PTFE femorotibial graft, there is a condition of critical ischemia that is less common after occlusion of a vein graft. Oral anticoagulation seems to prevent these negative changes.
Assuntos
Prótese Vascular , Artéria Femoral/cirurgia , Pé/irrigação sanguínea , Oclusão de Enxerto Vascular/fisiopatologia , Isquemia/cirurgia , Politetrafluoretileno , Veia Safena/transplante , Artérias da Tíbia/cirurgia , Idoso , Amputação Cirúrgica , Índice Tornozelo-Braço , Anticoagulantes/uso terapêutico , Feminino , Pé/fisiopatologia , Humanos , Masculino , RecidivaRESUMO
BACKGROUND: The goal of this study was to describe and review the results of a technique in which reconstruction of the common and profunda femoral arteries is combined with a femoro-femoral crossover graft using the same synthetic graft. A synthetic bifurcated graft (such as the ones used for aortobifemoral reconstruction), in which one limb is cut off, is used, leaving an enlarging patch at the end where the proximal anastomosis will be fashioned. METHODS: From January 1972 to January 2000, 6 patients underwent this reconstruction for severe limb ischemia. Patients were followed up in the outpatient clinic every 6 months. RESULTS: No postoperative mortality and no major complications were seen. One patient had a superficial wound infection, which resolved with conservative treatment. Five patients had a patent graft at an average follow-up of 39 months. CONCLUSIONS: Using the same synthetic graft allows angioplasty of the common and profunda femoral arteries of the donor side and revascularization of the opposite lower limb, with good short- and long-term results.
Assuntos
Prótese Vascular , Artéria Femoral/cirurgia , Procedimentos Cirúrgicos Vasculares/métodos , HumanosRESUMO
The risk of overtreatment or not treating an occult carcinoma exists in women at risk of residual disease after a LEEP excision for CIN3. Our goal was to discover an efficient method to select patients requiring a second LEEP from those requiring a FU only through an mRNA-detection test. In a population of 686 women undergoing a LEEP excision for CIN 3, we selected 285 women at risk of residual disease and subjected them to a search for E6/E7 mRNA HPV. The women with negative mRNA were subjected to a follow up, while the women with positive mRNA were subjected to a second LEEP. The histological examination of the second cone revealed 120 (85.7%) cases of residual disease in the mRNA-positive women: 40 cases of CIN2, 51 cases of CIN3, 11 cases of squamous microinvasive carcinoma, 7 cases of squamous carcinoma, 9 cases of AIS (adenocarcinoma in situ) and 2 cases of adenocarcinoma. Among the mRNA-negative women undergoing a follow up, there were only five cases of residual disease. During the follow-up period of about 6 years, we witnessed the regression of the residual disease and the elimination of the virus, just as predicted by the negative result of the mRNA test. Testing patients for E6/E7 mRNA allowed us to identify women with residual disease (CIN2+) and treat them appropriately.
RESUMO
Lumbar hernia is a rare form of abdominal hernia, which has been recognized later along the early development of the modern surgery. it has been, on many occasions, the object of heavy debate regarding its anatomical background and as well its etiology. The authors reports the historical aspects of this rare pathology, focusing on the earliest descriptions of hernia arising in lumbar regions, on the first reports of surgical repair, and on the anatomical description of the lumbar weakness areas, that are currently named Petit's triangle and Grynfeltt and Lesshaft's triangle.
Assuntos
Hérnia/história , História do Século XVII , História do Século XVIII , História do Século XIX , História do Século XX , Humanos , Região LombossacralRESUMO
BACKGROUND: Protein expression in Escherichia coli may result in the recombinant protein being expressed as insoluble inclusion bodies. In addition, proteins purified from E. coli contain endotoxins which need to be removed for in vivo applications. The structural protein, E2, from Bovine Viral Diarrhoea Virus (BVDV) is a major immunogenic determinant, and is an ideal candidate as a subunit vaccine. The E2 protein contains 17 cysteine residues creating difficulties in E. coli expression. In this report we outline a procedure for successfully producing soluble and endotoxin-free BVDV E2 protein from inclusion bodies (IB). RESULTS: The expression of a truncated form of BVDV-E2 protein (E2-T1) in E. coli resulted in predominantly aggregated insoluble IB. Solubilisation of E2-T1 with high purity and stability from IB aggregates was achieved using a strong reducing buffer containing 100 mM Dithiothreitol. Refolding by dialysis into 50 mM Tris (pH 7.0) containing 0.2% Igepal CA630 resulted in a soluble but aggregated protein solution. The novel application of a two-phase extraction of inclusion body preparations with Triton X-114 reduced endotoxin in solubilised E2-T1 to levels suitable for in vivo use without affecting protein yields. Dynamic light scattering analyses showed 37.5% of the protein was monomeric, the remaining comprised of soluble aggregates. Mice immunised with E2-T1 developed a high titre antibody response by ELISA. Western hybridisation analysis showed E2-T1 was recognised by sera from immunised mice and also by several BVDV-E2 polyclonal and monoclonal antibodies. CONCLUSION: We have developed a procedure using E. coli to produce soluble E2-T1 protein from IB, and due to their insoluble nature we utilised a novel approach using Triton X-114 to efficiently remove endotoxin. The resultant protein is immunogenic and detectable by BVDV-E2 specific antibodies indicating its usefulness for diagnostic applications and as a subunit vaccine. The optimised E. coli expression system for E2-T1 combined with methodologies for solubilisation, refolding and integrated endotoxin removal presented in this study should prove useful for other vaccine applications.
Assuntos
Vírus da Diarreia Viral Bovina/metabolismo , Endotoxinas/isolamento & purificação , Corpos de Inclusão/metabolismo , Proteínas do Envelope Viral/biossíntese , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Bovinos , Clonagem Molecular , Escherichia coli/metabolismo , Corpos de Inclusão/genética , Camundongos , Octoxinol , Polietilenoglicóis/química , Redobramento de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Substâncias Redutoras/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologiaRESUMO
MATERIAL: A total of 10 patients randomly selected from those who had reversed saphenous vein femoropopliteal bypass at our Institution and who had a postoperative angiogram were included into the study. METHODS: The occurrence of degenerative changes at the control angiography was correlated to the quality of the distal runoff as seen at the preoperative angiography and to the level of the index of distal resistances, calculated as the difference between the upper calf pressure and that at the ankle, divided by the arm pressure. RESULTS: Three patients had degenerative changes which were more common in case of poor distal runoff and high index of distal resistances. CONCLUSIONS: Degenerative changes in reversed saphenous vein grafts are more common in case of poor distal runoff and high distal resistance. These factors should be added to the many risk factors that have been analyzed previously.
Assuntos
Artéria Femoral/cirurgia , Oclusão de Enxerto Vascular/etiologia , Doença Arterial Periférica/cirurgia , Artéria Poplítea/cirurgia , Veia Safena/transplante , Enxerto Vascular/efeitos adversos , Artéria Femoral/diagnóstico por imagem , Artéria Femoral/fisiopatologia , Oclusão de Enxerto Vascular/diagnóstico por imagem , Oclusão de Enxerto Vascular/fisiopatologia , Humanos , Itália , Doença Arterial Periférica/diagnóstico por imagem , Doença Arterial Periférica/fisiopatologia , Artéria Poplítea/diagnóstico por imagem , Artéria Poplítea/fisiopatologia , Radiografia , Fatores de Risco , Veia Safena/diagnóstico por imagem , Fatores de Tempo , Resultado do TratamentoRESUMO
INTRODUCTION: Primary hyperparathyroidism (pHPT) is caused by a single monoclonal adenoma in more than 80% of patients. Biomolecular mechanisms causing pHPT are still not completely known, even if a great amount of studies have been developed recently, mainly regarding angiogenesis and growth factors. Among the latter, insulin-like growth factor 1 (IGF-1), basic fibroblastic growth factor (bFGF), vascular endothelial growth factor (VEGF), and transforming growth factor beta 1 (TGF-beta1) and their effects have been extensively evaluated in different kinds of endocrine disease. METHODS: Parathyroid cell cultures were prepared from six human adenomatous parathyroid glands that were surgically removed. After 7 days of culture, the cells were refed with DMEM supplemented with 2% FCS alone (control group), or containing hrTGFbeta1, or hrIGF-I, or hrbFGF, or hrVEGF. Then, after 48-hour incubation, cell count was performed by a particle count and size analyzer, and prevalence of cell cycle was analyzed by using a flow cytometer. RESULTS: Cell count (x10000) in the control group was 3.73 +/- 0.32. Low-dose TGF-beta1 stimulation resulted in 5.25 +/- 0.38 cells, and high-dose TGF-beta1 stimulation resulted in 2.35 +/- 0.37 cells. IGF-1 stimulation resulted in 5.4 +/- 0.65 cells, bFGF stimulation in 5.68 +/- 0.86 cells, and VEGF stimulation resulted in 6.03 +/- 1.03 cells. Statistical analysis revealed significant differences in the control group compared with the growth factor-stimulated groups. Cytometry showed different results in the percentage of cells in S-phase, in particular 22.65 +/- 4.98% of IGF-1-stimulated cells were found in S-phase compared with 7.55 +/- 3.2% of control group cells (p < 0.0001). CONCLUSIONS: Growth factors seem to play an important role in parathyroid adenoma cell proliferation; IGF-1, bFGF, VEGF, and low-dose TGF-beta1 promote cell proliferation, whereas high-dose TGF-beta1 inhibits these phenomena.
Assuntos
Adenoma/patologia , Proliferação de Células/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Neoplasias das Paratireoides/patologia , Fator de Crescimento Transformador beta1/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Adenoma/cirurgia , Análise de Variância , Células Cultivadas , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Pessoa de Meia-Idade , Neoplasias das Paratireoides/cirurgia , Fator de Crescimento Transformador beta1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Endovascular techniques are often applied, but they have occasionally been reported in the treatment of popliteal vascular entrapment (PVE). A case of bilateral PVE is presented with an acute occlusion of the right popliteal artery. This was twice unsuccessfully treated with arterial recanalization and stenting at another Institution. The patient required an arterial reconstruction with his reversed saphenous vein, in addition to resection of the medial gastrocnemius muscle laterally inserted on his right limb. The left limb was treated with a simple myotomy. Recanalization and stenting is not recommended for PVE treatment.
Assuntos
Angioplastia com Balão/instrumentação , Arteriopatias Oclusivas/terapia , Claudicação Intermitente/terapia , Artéria Poplítea/cirurgia , Veia Safena/transplante , Stents , Enxerto Vascular , Adulto , Arteriopatias Oclusivas/diagnóstico , Arteriopatias Oclusivas/cirurgia , Constrição Patológica , Humanos , Claudicação Intermitente/diagnóstico , Claudicação Intermitente/cirurgia , Angiografia por Ressonância Magnética , Masculino , Músculo Esquelético/cirurgia , Artéria Poplítea/diagnóstico por imagem , Radiografia , Falha de TratamentoRESUMO
Lumbar hernias, which are rare hernias of the posterolateral abdominal wall, can be divided into two groups: primary lumbar hernias, often the expression of a congenital defect, which typically arise in two areas of weakness, the superior triangle and inferior triangle and acquired (or diffuse) lumbar hernias which are usually due to previous lumbar trauma or surgery. Clinical examination may be adjuvated by ultrasound or CT scan, which can reveal the abdominal wall defect with the hernia content (viscera or extraperitoneal tissue). Surgical repair of lumbar hernias, both primary and acquired, has rapidly developed through recent years, similarly to the treatment of more frequent kinds of hernia (groin, epigastric), evolving from direct repair to mini-invasive techniques, even if, since the rarity of these hernias, precise knowledge of this complex anatomic region is required. Nowadays there are two valid alternatives: open tension-free repair (with use of mesh), and mini-invasive repair. Both are safe and effective, even if smaller hernias can be treated by open approach, with loco-regional anesthesia and good cosmetic effect. Larger hernias, or hernias with suspected viscera involvement, should require larger incisions and viscera exploration. For this reason laparoscopic access would be preferable.
Assuntos
Hérnia Ventral/cirurgia , Humanos , Laparoscopia , Procedimentos Cirúrgicos Minimamente Invasivos , Telas CirúrgicasRESUMO
The selective blockage of platelet-derived growth factor BB (PDGF-BB), basic fibroblast growth factor (bFGF), and transforming growth factor beta1 (TGF-beta1) by specific antibodies coated into expanded polytetrafluoroethylene (ePTFE) grafts may diminish neointimal hyperplasia. Sixty pigs were divided into two groups (n = 30 each) and then further divided into five subgroups. Group 1 had a bilateral iliac artery ePTFE interposition graft precoated with Matrigel. Three subgroups (A, B, and C) received a specific monoclonal antibody against PDGF-BB, bFGF, or TGF-beta1. One (D) received all antibodies, and one served as control (nonimmune immunoglobulin G [IgG] isotypes) (E). Group 2 had a bilateral iliac artery endothelial cell (EC)-seeded ePTFE interposition graft precoated with Matrigel. Three subgroups (A, B, and C) received a specific antibody against PDGF-BB, bFGF, or TGF-beta1. One (D) received all antibodies, and one served as control (nonimmune IgG isotypes) (E). Light microscopy and immunohistochemical stain showed that neointimal hyperplasia formation was significantly reduced in subgroups D compared to the others (p < 0.05). In subgroups D, the different precoating influenced neointimal hyperplasia formation. It was more pronounced in the prosthesis precoated with EC and Matrigel (p < 0.05). In organ culture, the amount of PDGF-BB, bFGF, and TGF-beta1 release was reduced in subgroup D animals compared to the others (p < 0.05). In subgroups D, the release of PDGF-BB, bFGF, and TGF-beta1 depended on ePTFE seeding. A higher amount of these growth factors was released in the prostheses precoated with EC and Matrigel (p < 0.05), and the bromodeoxyuridine labeling index confirmed higher incorporation in this subgroup (p < 0.001). The combined use of locally administered anti-PDGF-BB, bFGF, and TGF-beta1 monoclonal antibodies reduces neointimal hyperplasia formation.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Implante de Prótese Vascular/instrumentação , Prótese Vascular , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Artéria Ilíaca/cirurgia , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Politetrafluoretileno , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Túnica Íntima/cirurgia , Animais , Becaplermina , Western Blotting , Proliferação de Células , Células Cultivadas , Materiais Revestidos Biocompatíveis , Colágeno , Combinação de Medicamentos , Células Endoteliais/metabolismo , Células Endoteliais/transplante , Ensaio de Imunoadsorção Enzimática , Feminino , Fator 2 de Crescimento de Fibroblastos/imunologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Hiperplasia , Artéria Ilíaca/metabolismo , Artéria Ilíaca/ultraestrutura , Imuno-Histoquímica , Laminina , Microscopia Eletrônica de Varredura , Modelos Animais , Técnicas de Cultura de Órgãos , Fator de Crescimento Derivado de Plaquetas/imunologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Desenho de Prótese , Proteoglicanas , Proteínas Proto-Oncogênicas c-sis , Suínos , Fator de Crescimento Transformador beta1/imunologia , Fator de Crescimento Transformador beta1/metabolismo , Túnica Íntima/metabolismo , Túnica Íntima/ultraestruturaRESUMO
Atherosclerosis and neointimal hyperplasia formation are induced by alterations in the homeostatic balance between cell growth and cell death. Apoptosis is a physiological cell death process that, when deregulated, may be involved in many pathological conditions. Cigarette smoking is a primary risk factor for vascular disease and nicotine seems to exert its atherogenic effects in part through the increase of smooth muscle cell (SMC) proliferation. The aim of this study was to investigate the effect of nicotine on SMC apoptosis. Nicotine added for 24 and 72 h to serum deprived cell cultures resulted in a decrease of apoptotic SMCs. The inhibition was direct and not mediated by platelet-derived growth factor, basic fibroblast growth factor, and transforming growth factor beta(1), autocrinally released by nicotine-treated SMCs, because it was not influenced by addition of specific neutralizing antibodies. Apoptosis inhibition as well as the proliferation increase, and basic fibroblast growth factor expression on nicotine-treated SMCs were blocked by nicotinic acetylcholine receptor antagonists, including alpha-bungarotoxin, a competitive antagonist of alpha subunits of nicotinic receptor. In conclusion, we propose that nicotine could lead to the increase of neointimal SMCs in vascular lesions by inducing the inhibition of physiological SMC apoptosis and the increase of SMC proliferation. We also showed that nicotine signaling occurs as a result of activation of the classical nicotine receptor pathways.
Assuntos
Apoptose/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Animais , Anexina A5 , Bungarotoxinas , Bovinos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Dactinomicina/análogos & derivados , Ensaio de Imunoadsorção Enzimática , Fatores de Crescimento de Fibroblastos/metabolismo , Corantes Fluorescentes , Miócitos de Músculo Liso/metabolismo , Nicotina/metabolismo , Agonistas Nicotínicos/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
S-adenosylmethionine is a metabolite regulating many biological processes; S-adenosylmethionine effect on ubiquitin-proteasome system (UPS) has not been studied yet. We investigated S-adenosylmethionine effects on UPS activity both in vitro, by inhibitor screening assay, and in rat vascular smooth muscle cells, by Western Blot of proteasomal targets. We found that S-adenosylmethionine inhibited UPS activity.
Assuntos
Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Inibidores de Proteassoma , S-Adenosilmetionina/farmacologia , Ubiquitina/antagonistas & inibidores , Animais , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/enzimologia , Células Cultivadas , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ratos , Ubiquitina/metabolismoRESUMO
Anaplasma marginale is a devastating tick-borne pathogen causing anaplasmosis in cattle and results in significant economic loss to the cattle industry worldwide. Currently, there is no widely accepted vaccine against A. marginale. New generation subunit vaccines against A. marginale, which are much safer, more efficient and cost-effective, are in great need. The A. marginale outer membrane protein VirB9-1 is a promising antigen for vaccination. We previously have shown that soluble recombinant VirB9-1 protein can be expressed and purified from Escherichia coli and induce a high level of humoral and cellular immunity in mice. In this study, we re-formulated the nanovaccines using the partially-purified VirB9-1 protein as the antigen and hollow nano-size silica vesicles (SV-100) as the adjuvant. We simplified the purification method to obtain the partially-purified antigen VirB9-1 with a six-fold higher yield. The new formulations using the partially-purified VirB9-1 protein achieved higher antibody and cell-mediated immune responses compared to the purified ones. This finding suggests that the partially-purified VirB9-1 protein performs better than the purified ones in the vaccination against A. marginale, and a certain level of contaminants in the protein antigen can be self-adjuvant and boost immunogenicity together with the nanoparticle adjuvant. This may lead to finding a "Goldilocks" level of contaminants. The new nanovaccine formulation using partially-purified antigens along with nanoparticle adjuvants offers an alternative strategy for making cheaper veterinary vaccines.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Anaplasma marginale/imunologia , Anaplasmose/prevenção & controle , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Doenças dos Bovinos/prevenção & controle , Dióxido de Silício/administração & dosagem , Animais , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/isolamento & purificação , Bovinos , Feminino , Camundongos Endogâmicos C57BL , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/isolamento & purificaçãoRESUMO
Increase in plasma low density lipoprotein (LDL) levels and/or decrease in high density lipoprotein (HDL) levels are major risk factors for the development of atherosclerosis. An oxidative modification of LDL represents a key process in atherogenesis. It is well known that the LDL/HDL ratio is more important than the individual LDL and HDL levels to predict atherosclerosis. The purpose of our study was to investigate the effects of mildly oxidized LDL (minimally modified LDL: MM-LDL) and HDL, administrated alone or in combination, on the production and release of basic fibroblast growth factor (bFGF) by bovine aortic smooth muscle cells (SMCs) in culture. MM-LDL and HDL have opposite effects on aortic SMCs: MM-LDL increases both bFGF production and release and SMC proliferation, while HDL decreases both bFGF production and release and SMC proliferation. The effects of either MM-LDL or HDL on SMCs are mediated through a Gi-protein-coupled receptor. The simultaneous treatment of SMCs with MM-LDL and HDL (MM-LDL/HDL ratio=4.0) produced the inhibition of MM-LDL effects. Our data suggest that the protective role of HDL could also be exerted through the inhibition of the pro-atherosclerotic effects of MM-LDL on SMCs.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos , Lipoproteínas HDL/farmacologia , Lipoproteínas LDL/farmacologia , Músculo Liso Vascular/metabolismo , RNA Mensageiro/genética , Animais , Aorta Torácica/citologia , Aorta Torácica/metabolismo , Aterosclerose/etiologia , Aterosclerose/metabolismo , Aterosclerose/prevenção & controle , Bovinos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados , Fator 2 de Crescimento de Fibroblastos/biossíntese , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/genética , Mitose/efeitos dos fármacos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Oxirredução , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: We investigated the role and influence of platelet derived growth factor (PDGF) and transforming growth factor beta1 (TGF) in the pathologic mechanism at the basis of plaque instability regulating the expression of matrix metalloproteinases (MMPs). METHODS: Plaques obtained from 70 patients who underwent carotid endarterectomy were classified histologically as stable or unstable. Serum levels of PDGF and TGF were measured pre- and postoperatively. The serum activities of MMP-2 and MMP-9 were also analyzed. Human umbilical artery smooth muscle cells (HUASMCs) were stimulated in vitro with PDGF at various concentrations (20 and 50 ng/mL) and TGF (2 and 5 ng/mL) in a serum-free medium. The release of MMPs in the conditioned medium was assessed by enzyme-linked immunosorbent assay. Release of the MMPs was confirmed by Western blot analysis; their activity and expression were determined by zymography and reverse transcription-polymerase chain reaction. Specific inhibition tests were performed on HUASMCs to evaluate the role of these growth factors. RESULTS: Forty-two (60%) patients had an unstable carotid plaque and 28 (40%) a stable plaque. Preoperatively, patients affected with unstable carotid plaques had higher PDGF and lower TGF plasma levels than patients with stable carotid plaques (P < .001); the levels returned to normal at 1 and 30 days postoperatively, compared with 20 non-operated healthy volunteers. Release, activity, protein level, and expression of MMPs in PDGF-stimulated HUASMCs were greater than in the controls (P < .001), whereas these values in the TGF-stimulated HUASMCs were lower (P < .001). The addition of monoclonal anti-PDGF antibodies decreased the release, activity, protein level, and expression of MMPs, whereas the addition of monoclonal anti-TGF antibodies increased the release, activity, protein level and expression of MMPs (P < .001). CONCLUSIONS: TGF seems to be an important stabilizing factor and prevents plaque rupture through the decrease of MMPs.