RESUMO
Mammary tumors are the most frequent type of neoplasms in intact female dogs. New therapies that target neoplastic cells without affecting normal cells are highly sought. The Bacillus anthracis toxin has been reengineered to target tumor cells that express urokinase plasminogen activators and metalloproteinases. In previous studies carried out in our laboratory, the reengineered anthrax toxin had inhibitory effects on canine oral mucosal melanoma and canine osteosarcoma cells. In this study, five canine neoplastic epithelial cell lines (four adenocarcinomas and one adenoma) and one non-neoplastic canine mammary epithelial cell line were treated with different concentrations of reengineered anthrax toxin components. Cell viability was quantified using an MTT assay and half-maximal inhibitory concentration (IC50) values. Cell lines were considered sensitive when the IC50 was lower than 5000 ng/ml. One canine mammary adenocarcinoma cell line and one mammary adenoma cell line showed significantly decreased viability after treatment, whereas the non-neoplastic cell line was resistant. We conclude that the reengineered anthrax toxin may be considered a targeted therapy for canine mammary neoplasms while preserving normal canine mammary epithelial cells.
Assuntos
Antígenos de Bactérias , Toxinas Bacterianas , Doenças do Cão , Neoplasias Mamárias Animais , Animais , Cães , Neoplasias Mamárias Animais/tratamento farmacológico , Toxinas Bacterianas/farmacologia , Feminino , Antígenos de Bactérias/farmacologia , Linhagem Celular Tumoral , Doenças do Cão/tratamento farmacológico , Células Epiteliais/efeitos dos fármacos , Adenocarcinoma/veterinária , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Sobrevivência Celular/efeitos dos fármacos , Adenoma/veterinária , Adenoma/tratamento farmacológico , Adenoma/patologiaRESUMO
Canine and human osteosarcomas (OSA) share similarities. Novel therapies are necessary for these tumours. The Bacillus anthracis toxin was reengineered to target and kill cells with high expressions of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA). Since canine OSA express MMPs and uPA, we assessed whether the reengineered toxin could show efficacy against these tumours. Two OSA cell lines (canine D17 and human MG63) and a non-neoplastic canine osteoblastic cell line (COBS) were used. Cells were treated with different concentrations of the reengineered anthrax toxin and cell viability was quantified using MTT assay. The cell cycle, apoptosis, and necrosis were analysed by flow cytometry. The wound-healing assay was performed to quantify the migration capacity of treated cells. D17 and MG63 cells had significantly decreased viability after 24 h of treatment. Cell cycle analysis revealed that OSA cells underwent apoptosis when treated with the toxin, whereas COBS cells arrested in the G1 phase. The wound-healing assay showed that D17 and MG63 cells had a significantly reduced migration capacity after treatment. These results point for the first time towards the in vitro inhibitory effects of the reengineered anthrax toxin on OSA cells; this reengineered toxin could be further tested as a new therapy for OSA.
Assuntos
Antígenos de Bactérias/farmacologia , Antineoplásicos/farmacologia , Toxinas Bacterianas/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Osteossarcoma/tratamento farmacológico , Adolescente , Animais , Antígenos de Bactérias/genética , Apoptose/efeitos dos fármacos , Toxinas Bacterianas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Masculino , Metaloproteinases da Matriz/metabolismo , Proteínas de Membrana/metabolismo , Invasividade Neoplásica , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Engenharia de ProteínasRESUMO
Canine oral mucosal melanomas (OMM) are the most common oral malignancy in dogs and few treatments are available. Thus, new treatment modalities are needed for this disease. Bacillus anthracis (anthrax) toxin has been reengineered to target tumor cells that express urokinase plasminogen activator (uPA) and metalloproteinases (MMP-2), and has shown antineoplastic effects both, in vitro and in vivo. This study aimed to evaluate the effects of a reengineered anthrax toxin on canine OMM. Five dogs bearing OMM without lung metastasis were included in the clinical study. Tumor tissue was analyzed by immunohistochemistry for expression of uPA, uPA receptor, MMP-2, MT1-MMP and TIMP-2. Animals received either three or six intratumoral injections of the reengineered anthrax toxin prior to surgical tumor excision. OMM samples from the five dogs were positive for all antibodies. After intratumoral treatment, all dogs showed stable disease according to the canine Response Evaluation Criteria in Solid Tumors (cRECIST), and tumors had decreased bleeding. Histopathology has shown necrosis of tumor cells and blood vessel walls after treatment. No significant systemic side effects were noted. In conclusion, the reengineered anthrax toxin exerted inhibitory effects when administered intratumorally, and systemic administration of this toxin is a promising therapy for canine OMM.
Assuntos
Antígenos de Bactérias/uso terapêutico , Antineoplásicos/uso terapêutico , Toxinas Bacterianas/uso terapêutico , Doenças do Cão/tratamento farmacológico , Melanoma/tratamento farmacológico , Neoplasias Bucais/tratamento farmacológico , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/farmacologia , Antineoplásicos/farmacologia , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacologia , Doenças do Cão/metabolismo , Doenças do Cão/patologia , Cães , Feminino , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Melanoma/veterinária , Mucosa Bucal/efeitos dos fármacos , Mucosa Bucal/metabolismo , Mucosa Bucal/patologia , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Neoplasias Bucais/veterinária , Engenharia de Proteínas , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Pneumococcal Surface Protein A (PspA) has been successfully tested as vaccine candidate against Streptococcus pneumoniae infections. Vaccines able to induce PspA-specific antibodies and Th1 cytokines usually provide protection in mice. We have shown that the whole cell pertussis vaccine (wP) or components from acellular pertussis vaccines, such as Pertussis Toxin or Filamentous Hemagglutinin (FHA), are good adjuvants to PspA, suggesting that combined pertussis-PspA vaccines would be interesting strategies against the two infections. Here, we evaluated the potential of wP as a delivery vector to PspA. Bordetella pertussis strains producing a PspA from clade 4 (PspA4Pro) fused to the N-terminal region of FHA (Fha44) were constructed and inactivated with formaldehyde for the production of wPPspA4Pro. Subcutaneous immunization of mice with wPPspA4Pro induced low levels of anti-PspA4 IgG, even after 3 doses, and did not protect against a lethal pneumococcal challenge. Prime-boost strategies using wPPspA4Pro and PspA4Pro showed that there was no advantage in using the wPPspA4Pro vaccine. Immunization of mice with purified PspA4Pro induced higher levels of antibodies and protection against pneumococcal infection than the prime-boost strategies. Finally, purified Fha44:PspA4Pro induced high levels of anti-PspA4Pro IgG, but no protection, suggesting that the antibodies induced by the fusion protein were not directed to protective epitopes.
Assuntos
Adesinas Bacterianas/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Proteínas de Bactérias/farmacologia , Vacina contra Coqueluche/administração & dosagem , Infecções Pneumocócicas/prevenção & controle , Fatores de Virulência de Bordetella/administração & dosagem , Animais , Antígenos de Bactérias/farmacologia , Antígenos de Superfície/farmacologia , Portadores de Fármacos/administração & dosagem , Feminino , Camundongos , Camundongos Endogâmicos BALB C , VacinaçãoRESUMO
Exercise training has been shown to reduce symptoms and exacerbations in COPD patients; however, the exercise effect on patients' immune response is poorly known. We thus verified if an exercise program (EP) impacted on proliferative T cell response of COPD patients. Fourteen non-O2 dependent COPD patients on standard treatment were studied. EP consisted in 24 sessions of aerobic and muscular training. Peripheral blood mononuclear cells were stimulated with the mitogen phytohemagglutinin and antigens from Haemophilus influenzae and cytomegalovirus, and the lymphocyte proliferative response (LPR) was assessed through the expression of Ki67 before and after the EP. The Quality of life [COPD assessment test (CAT)], dyspnea [(modified Medical Research Council scale (mMRC)], and 6-min walk distance were also assessed. The EP program increased significantly the LPR of TCD4+ lymphocytes to phytohemagglutinin and cytomegalovirus and H. influenzae antigens, but with TCD8+ lymphocytes the increase was less marked. Consistent with this, a higher proportion of TCD8+ than TCD4+ cells did not express the costimulatory molecule CD28. The EP also resulted in improvement of the quality of life, dyspnea, and physical capacity. The improvement in TCD4+ cell function may represent an additional mechanism through which the EP results in less exacerbations and hospitalizations.
Assuntos
Proliferação de Células/fisiologia , Terapia por Exercício , Linfócitos/imunologia , Doença Pulmonar Obstrutiva Crônica/reabilitação , Linfócitos T/imunologia , Idoso , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/farmacologia , Antígenos Virais/imunologia , Antígenos Virais/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células/efeitos dos fármacos , Citomegalovirus/imunologia , Dispneia , Feminino , Volume Expiratório Forçado , Haemophilus influenzae/imunologia , Humanos , Antígeno Ki-67/efeitos dos fármacos , Antígeno Ki-67/metabolismo , Masculino , Pessoa de Meia-Idade , Fito-Hemaglutininas/imunologia , Fito-Hemaglutininas/farmacologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Qualidade de Vida , Linfócitos T/efeitos dos fármacos , Capacidade Vital , Teste de CaminhadaRESUMO
OBJECTIVE: New tests for diagnosing active tuberculosis (aTB) are urgently needed, and TB antigen-specific cell-mediated immunity can be expected to develop new testing methods of aTB. MATERIALS AND METHODS: Rv0183 protein, the only monoglyceride lipase identified in mycobacteria, was used to stimulate freshly heparin-treated whole blood. The Rv0183-specific cytokines/chemokines response associated with aTB was screened firstly with 4 aTB patients and 4 LTBIs, and further evaluated in 192 suspected aTB patients and 372 healthy individuals. RESULTS: Out of 71 cytokines/chemokines, the response of IL-6 against Rv0183 protein was found to be associated with aTB. The Rv0183-specific IL-6 response was significantly higher in aTB patients (n = 128) than in those with non-TB lung disease (n = 64) and in healthy individuals (n = 327) (p < 0.0001), and not affected by latent TB infection. In IGRA+ suspected active TB patients, the sensitivity, specificity, PPV and NPV of IL-6 response (with cutoff of 235.2 pg/ml) were 85.7%, 100%, 100% and 51.5% for diagnosing aTB, respectively. While in IGRA- ones, they were 87.5%, 80.5%, 60.9% and 95.0% with 174.2 pg/ml IL-6 response as cutoff, respectively. CONCLUSIONS: These results clearly show that the Rv0183 antigen-specific IL-6 response has the potential to be used as an immune-diagnosis test for active TB in clinical practice.
Assuntos
Antígenos de Bactérias/farmacologia , Testes Imunológicos/métodos , Interleucina-6/imunologia , Tuberculose/diagnóstico , Tuberculose/imunologia , Adulto , Antígenos de Bactérias/imunologia , Biomarcadores/sangue , Citocinas/análise , Citocinas/imunologia , Diagnóstico Diferencial , Feminino , Humanos , Imunidade Celular , Testes de Liberação de Interferon-gama , Tuberculose Latente/diagnóstico , Tuberculose Latente/imunologia , Leucócitos Mononucleares/imunologia , Masculino , Mycobacterium tuberculosis , Sensibilidade e Especificidade , Teste TuberculínicoRESUMO
Background & objectives: The nature of the rickettsial antigens and the immune response generated by them, have been the subject of exhaustive research so that a suitable vaccine can be developed. Till date evaluations of Rickettsia rickettsii antigens that induce both humoral and cellular responses in animal models have only shown partial protection and short-term immunological memory. This study was aimed to evaluate the immune response induced by DNA plasmids generated from the OmpA and OmpB genes of R. rickettsii in peripheral blood mononuclear cells of rickettsial (sensitized) patients compared to healthy subjects. Methods: Plasmids OmpA-49, OmpB-15 and OmpB-24 were generated in the pVAX vector. Macrophages derived from the THP-1 cell line were transfected in vitro with the plasmids and were co-cultured with T-lymphocytes from sensitized subjects and healthy subjects to evaluate cell proliferation and cytokine production. Results: The OmpB-24 plasmid induced proliferative response in human lymphocytes, with production of IL-2, IFN-γ, IL-12p70, IL-6 and TNF-α, likely due to the presence of conserved epitopes among R. rickettsii, R. typhi and R. felis (differing from 1 to 3 amino acids) during the construction of the plasmids. Interpretation & conclusion: DNA sequences of rickettsial epitopes can be cloned into the pVAX vector. Constructed plasmids can generate a proliferative response and produce cytokines in vitro, in co-culture of transfected macrophages with sensitized human lymphocytes. Plasmid OmpB-24 proved to be the most immunogenic with respect to plasmids OmpA-49 and OmpB-15.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Rickettsia rickettsii/imunologia , Adolescente , Adulto , Idoso , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/farmacologia , Proteínas da Membrana Bacteriana Externa/farmacologia , Citocinas/biossíntese , Citocinas/efeitos dos fármacos , Feminino , Humanos , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Plasmídeos/imunologia , Rickettsia rickettsii/química , Adulto JovemRESUMO
During periodontitis, alveolar bone resorption is associated with activation of T helper type 17 (Th17) lymphocytes and receptor activator of nuclear factor-κB ligand (RANKL) -induced osteoclasts. We previously reported that serotype b of Aggregatibacter actinomycetemcomitans has a higher capacity to trigger Th17-type differentiation and function in activated T lymphocytes and its lipopolysaccharide is a more potent immunogen compared with the other serotypes. This study aimed to investigate whether serotype b of A. actinomycetemcomitans induces higher Th17-associated RANKL production, RANKL-induced osteoclast activation, and antigen-specific memory T lymphocyte proliferation. On naive CD4(+) T lymphocytes stimulated with autologous dendritic cells primed with different A. actinomycetemcomitans serotypes, RANKL production, T-bet, GATA-3, RORC2 and Foxp3 expression, RORC2/RANKL intracellular double-expression, TRAP(+) osteoclast activation, and bone resorption were quantified. The frequency of proliferating memory T lymphocytes in response to A. actinomycetemcomitans serotypes was determined in periodontitis and healthy subjects. Naive CD4(+) T lymphocytes stimulated by serotype b-primed dendritic cells elicited higher levels of RANKL, RORC2, TRAP(+) osteoclasts, and bone resorption than the same cells stimulated with the other serotypes. RANKL positively correlated and co-expressed with RORC2. Memory T lymphocytes responding to serotype b were more frequently detected in periodontitis patients than healthy subjects. These results indicate that serotype b of A. actinomycetemcomitans is associated with higher production of RANKL and these increased levels are associated with Th17 lymphocyte induction, osteoclast activation, and bone resorption.
Assuntos
Aggregatibacter actinomycetemcomitans/imunologia , Osteoclastos/imunologia , Linfócitos T/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/farmacologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Diferenciação Celular/imunologia , Periodontite Crônica/imunologia , Periodontite Crônica/microbiologia , Células Dendríticas/imunologia , Fatores de Transcrição Forkhead/biossíntese , Fator de Transcrição GATA3/biossíntese , Humanos , Memória Imunológica/imunologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/biossíntese , Ligante RANK/imunologia , Sorogrupo , Linfócitos T/microbiologiaRESUMO
Ovine brucellosis caused by Brucella ovis is considered one of the most important reproductive diseases of rams worldwide. This study aimed to characterize the kinetics of infection of a ΔabcAB B. ovis mutant strain in rams. Twelve 1-year-old crossbred rams were used. Six rams were challenged with 2 mL of a suspension containing 1.2×10(9) CFU/mL of B. ovis strain ATCC25840 (wild type) by intraprepucial inoculation and additional 50 µL in each conjunctival sac of a suspension containing 1.2×10(10) CFU/mL of the same strain. The other six rams were challenged with an equivalent number of CFU of the mutant strain ΔabcAB B. ovis through the same routes. Serum samples for serology and semen and urine samples for bacteriologic culture and PCR were collected weekly during 24 weeks. At 24 weeks post infection, tissue samples were collected for bacteriologic culture and PCR. All rams inoculated with wild type or the ΔabcAB strain seroconverted at the fourth week post infection, remaining positive up to the 16th week post infection. PCR and bacteriology demonstrated that only rams inoculated with the wild type strain shed the organism in semen and urine. Lymphocytes from rams inoculated with wild type or ΔabcAB B. ovis had significantly higher proliferation in response to B. ovis antigens when compared with unstimulated controls. Tissue bacteriology and PCR detected B. ovis in all rams challenged with the wild type strain, whereas only one ΔabcAB-infected ram had a positive iliac lymph node sample by PCR.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Brucella ovis/genética , Brucella ovis/imunologia , Brucelose/veterinária , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/microbiologia , Transportadores de Cassetes de Ligação de ATP/imunologia , Animais , Antígenos de Bactérias/farmacologia , Brucelose/imunologia , Brucelose/microbiologia , Proliferação de Células/efeitos dos fármacos , Imunidade Humoral , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Masculino , Mutação , Sêmen/microbiologia , Ovinos , Urina/microbiologiaRESUMO
Introduction. Rheumatoid arthritis patients under treatment with anti-TNF-α are at a high risk of developing active tuberculosis, and therefore, screening for latent tuberculosis infection is recommended before anti-TNF-α therapy. Objective. To compare the tuberculin test and IFNγ production induced by culture filtrate proteins(CFPs) and Mycobacterium tuberculosis-specific CFP-10 antigens in rheumatoid arthritis patients. Materials and methods. An analytic transversal study was conducted in rheumatoid arthritis patients treated at Hospital Universitario San Vicente Fundación between January and December 2007. IFNγ production in response to CFPs and CFP-10 was measured in the supernatants of whole blood cultures and evaluated for correlations with tuberculin reactivity. The degree of concordance between both tests was also established. Results. Forty-five patients were included, of which 14 (31.1%) had a tuberculin reaction of ≥10 mm of induration, 9 (20%) produced IFNγ in response to CFP-10, and 7 were positive for both tests. The correlation between tests was r=0.53 (IC 95%:0.28-0.72), and the global concordance between tests was80%, with a Kappa coefficient of 0.48 (IC95%:0.20-0.76). Conclusions. Only two tuberculin (-)/CFP-10+ "anergic" patients were observed. By contrast, six tuberculin +/CFP-10(-) "tuberculin false-positive" patients were observed. These data suggest that the tuberculin test is not an appropriate tool for determining the need for tuberculosis prophylaxis.
Introducción. Los pacientes con artritis reumatoide bajo tratamiento con anti-TNFα están en alto riesgo de desarrollar tuberculosis activa, por lo cual se recomienda hacer la tamización para infección latente de tuberculosis, antes de iniciar el tratamiento. Objetivo. Comparar la prueba de tuberculina y la producción de IFNγ inducida por antígenos CFP (Culture Filtrate Protein) y antígenos específicos de Mycobacterium tuberculosis (CFP-10) para el diagnóstico de infección latente de tuberculosis en pacientes con artritis reumatoide. Materiales y métodos. Se llevó a cabo un estudio transversal analítico en pacientes con artritis reumatoide atendidos en el Hospital Universitario San Vicente Fundación, entre enero y diciembre de 2007, a los cuales se les determinó la producción de IFNγ en respuesta a CFP y CFP-10 en sobrenadantes de cultivos de sangre total, y se correlacionó con la reacción en la prueba de tuberculina. Además, se estableció el grado de concordancia entre ambas pruebas. Resultados. Se incluyeron 45 pacientes, de los cuales, 14 (31,1 %) tuvieron un diámetro de induración ≥10 mm (tuberculina positiva), nueve (20 %) produjeron IFNγ en respuesta a CFP-10, y siete fueron positivos para ambas pruebas. La correlación entre las pruebas fue de r=0,53 (IC95%: 0,28-0,72) y la concordancia global entre pruebas fue de 80 %, con un coeficiente kappa de 0,48 (IC95%: 0,20-0,76). Conclusiones. Solo se observaron dos pacientes con tuberculina positiva y CFP-10 positivo "anérgicos" y se encontraron seis pacientes con tuberculina positiva y CFP-10 negativa "falsos positivos para tuberculina", lo cual sugiere que la prueba de la tuberculina no es la más adecuada para indicar profilaxis para tuberculosis.