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Emergence of antimicrobial resistance mediated through New Delhi metallo-ß-lactamases (NDMs) is a serious therapeutic challenge. Till date, 16 different NDMs have been described. In this study, we report the molecular and structural characteristics of NDM-5 isolated from an Escherichia coli isolate (KOEC3) of bovine origin. Using PCR amplification, cloning and sequencing of full blaNDM gene, we identified the NDM type as NDM-5. Cloning of full gene in E. coli DH5α and subsequent assessment of antibiotic susceptibility of the transformed cells indicated possible role of native promoter in expression blaNDM-5. Translated amino acid sequence had two substitutions (Val88Leu and Met154Leu) compared to NDM-1. Theoretically deduced isoelectric pH of NDM-5 was 5.88 and instability index was 36.99, indicating a stable protein. From the amino acids sequence, a 3D model of the protein was computed. Analysis of the protein structure elucidated zinc coordination and also revealed a large binding cleft and flexible nature of the protein, which might be the reason for broad substrate range. Docking experiments revealed plausible binding poses for five carbapenem drugs in the vicinity of metal ions. In conclusion, results provided possible explanation for wide range of antibiotics catalyzed by NDM-5 and likely interaction modes with five carbapenem drugs.
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AIM: Mineral trioxide aggregate (MTA) is a biomaterial with numerous clinical applications in the field of endodontics. The properties of this material come closest to the requirements of an ideal endodontic repair material. Delayed setting time though may limit the use of MTA in endodontic procedures. Many chemical based additives have influenced the setting time of MTA. The aim of this paper was to evaluate the effect of a natural additive in the form of human dentin powder on setting properties of ProRoot MTA (Dentsply/Tulsa Dental, Tulsa, OK). METHODS: Thirty extracted human teeth were taken to obtain fine dentin powder. The methodology used to obtain dentin powder for the present study was same as performed by Haapsalo et al. The specimens were divided into two groups. The samples were placed in standardized stainless steel ring moulds. The setting time of MTA alone and in equal proportions with dentin powder as an additive was evaluated using Vicats apparatus. RESULTS: MTA mixed with dentin powder showed faster setting time (70 minutes) in comparison to MTA without dentin powder (120 minutes) (P<0.5). CONCLUSION: Although MTA is an ideal retrograde material with increased efficacy due to reduced setting on addition of dentin powder. A combination of MTA with dentin powder might be a viable option in procedures where fast setting of the MTA is critical.
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Compostos de Alumínio , Compostos de Cálcio , Dentina , Óxidos , Materiais Restauradores do Canal Radicular , Silicatos , Extratos de Tecidos/farmacologia , Compostos de Alumínio/química , Compostos de Cálcio/química , Combinação de Medicamentos , Humanos , Técnicas In Vitro , Teste de Materiais , Óxidos/química , Pós , Materiais Restauradores do Canal Radicular/química , Silicatos/química , Fatores de Tempo , Extratos de Tecidos/isolamento & purificaçãoRESUMO
BACKGROUND AND PURPOSE: Radiotherapy for bulky tumors often results in palliation with suboptimal outcomes. The prognosis is worsened by immunosuppression caused by radio-chemotherapy, negatively impacting on survival. Novel Partial Tumor Irradiation (PTI) was designed to spare the Peritumoral Immune Microenvironment (PIM) and to be delivered synchronously with immune activity peaks, thus enhancing both local and distant tumor control through immunostimulation. MATERIALS AND METHODS: Present proof-of-principle trial enrolled 26 patients with bulky tumors, comparing outcomes between treatments administered at immune activity peaks versus troughs. The primary endpoint was local-bystander and distal-abscopal response-rate. Secondary endpoints included overall-, progression-free-, cancer-specific survival, neoadjuvant and immunomodulatory potential. RESULTS: All measured outcomes were significantly influenced by treatment-timing. The bystander and abscopal response rates were 77% and 41%, respectively. PTI significantly upregulated pro-inflammatory and cell-death-inducing pathways improving the efficacy of radiotherapy by highly complex tumors. CONCLUSIONS: This study highlights the profound impact PTI can have on a highly palliative patient cohort previously deemed beyond therapeutic hope. With 41 % of these patients still alive after a median follow-up of 50 months, PTI offers a potential lifeline for those facing advanced, treatment-resistant cancers. This approach generated also distant immunogenic anti-tumor responses, offering a promising new avenue for the treatment of advanced cancers.
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â¢Improvement of therapeutic ratio by novel unconventional radiotherapy approaches.â¢Immunomodulation using high-dose spatially fractionated radiotherapy.â¢Boosting radiation anti-tumor effects by adding an immune-mediated cell killing.
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The aim of this study is to validate the prognostic and predictive value of the non-synonymous cytidine deaminase (CDA) Lys²7Gln polymorphism for hematological toxicity and survival using a randomized phase III adjuvant trial (Radiation Therapy Oncology Group (RTOG) 9704) in pancreatic cancer in which one treatment arm received gemcitabine. CDA is involved in gemcitabine inactivation, and there is conflicting data on the role of the non-synonymous CDA Lys²7Gln polymorphism in predicting toxicity and survival in cancer patients treated with gemcitabine. RTOG 9704 randomized 538 patients after pancreatic resection to receive radiotherapy with either 5-fluorouracil (5-FU) or gemcitabine. CDA Lys²7Gln polymorphism genotype was analyzed. We tested an association between CDA single-nucleotide polymorphism genotype and the survival outcome by the Cox proportional hazard model adjusting for other covariates, as well as toxicity by the logistic regression model. There is statistically significant more severe hematological toxicity in patients treated with gemcitabine with either the homozygote wild-type genotype (Lys/Lys) alone (odds ratio (OR)=0.06, P=0.01), or in combination with the heterozygote (Lys/Gln; OR=0.14, P=0.03) when compared with homozygote variant genotype (Gln/Gln) when adjusted for other covariates. This was not seen in the non-gemcitabine treated arm. There are no genotype differences with respect to survival outcome. In conclusion, in this prospective randomized adjuvant study of patients with pancreatic cancer, the CDA Lys²7Gln polymorphism is validated as a predictive marker of gemcitabine hematological toxicity, but not with treatment response or survival.
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Citidina Desaminase/genética , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamento farmacológico , Polimorfismo de Nucleotídeo Único/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Farmacológicos , Citidina Desaminase/metabolismo , Desoxicitidina/administração & dosagem , Desoxicitidina/toxicidade , Intervalo Livre de Doença , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/toxicidade , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/cirurgia , GencitabinaRESUMO
PURPOSE: Immunotherapy is now a first-line treatment for metastatic non-small cell lung cancer (NSCLC) and melanomaQuery. It is important to understand the relationship between immunotherapy and radiation to the brain. The aim of this study was to assess the role of stereotactic radiosurgery (SRS) or WBRT in addition to immunotherapy in patients with melanoma or NSCLC metastatic to the brain. METHODS/PATIENTS: Using the National Cancer Database, 2951 patients with NSCLC and 936 patients with melanoma treated with immunotherapy were identified. Patients were classified as having received immunotherapy alone, immunotherapy with SRS, or immunotherapy with whole-brain radiation therapy (WBRT). Kaplan-Meier, multivariate Cox regression analyses, and propensity matching were performed to evaluate the impact of adding SRS to immunotherapy on overall survival (OS). Immortal survival bias was accounted for by only including patients who received radiation before immunotherapy and time zero was defined as the start of immunotherapy. RESULTS: 205(6.9%) and 75(8.0%) patients received immunotherapy with no radiation, 822(27.9%) and 326(34.8%) received SRS and immunotherapy, and 1924(65.2%) and 535(57.2%) received WBRT and immunotherapy for NSCLC and melanoma, respectively. Adding SRS to immunotherapy was associated with improved OS in multivariate analyses (NSCLC HR = 0.81, 95% CI 0.66-0.99, p = 0.044; melanoma HR = 0.63, 95% CI 0.45-0.90, p = 0.011). The addition of WBRT to immunotherapy did not improve OS in patients with melanoma nor NSCLC. CONCLUSIONS: This analysis suggests that treatment with SRS and immunotherapy is associated with improved OS compared to immunotherapy alone for patients with melanoma or NSCLC metastatic to the brain.
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Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/terapia , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/terapia , Imunoterapia , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/terapia , Melanoma/mortalidade , Melanoma/terapia , Radiocirurgia , Idoso , Neoplasias Encefálicas/secundário , Carcinoma Pulmonar de Células não Pequenas/secundário , Terapia Combinada , Bases de Dados Factuais , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Melanoma/secundário , Pessoa de Meia-Idade , Estudos Retrospectivos , Taxa de Sobrevida , Estados UnidosRESUMO
Radiation therapy is traditionally used for the local control of tumour growth, but recent studies suggest that radiation therapy can have immunomodulatory properties that can be applied in combination therapy with immunotherapeutic agents. The paradigm of using radiation therapy for immunomodulation in cancer treatment is a rapidly progressing field, with multiple ongoing clinical trials exploring its use in combination with immune checkpoint blockades to induce an abscopal effect. Permutations of radiation therapy regimens, including variations in radiation dosing, radiation planning parameters and radiation modality, are being tested with varying degrees of success. The relative biological effectiveness was a concept introduced in the early days of radiation biology that allows the comparison of local tumour control across various radiation modalities and energies. Similarly, there remains a need for a new concept of comparing the immunological effectiveness of various radiation modalities. In this review, we will provide an overview of immunobiological models for preclinical and clinical monitoring of radiation therapy regimens and introduce the concept of relative immunological effectiveness to compare and screen for immune-activating functions of these regimens.
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Imunoterapia , Neoplasias , Terapia Combinada , Humanos , Oncologia , Neoplasias/radioterapiaAssuntos
Cisto Dermoide/patologia , Laparoscopia/métodos , Neoplasias Ovarianas/patologia , Ovariectomia/métodos , Antígeno Prostático Específico , Teratoma/patologia , Cisto Dermoide/metabolismo , Cisto Dermoide/fisiopatologia , Cisto Dermoide/cirurgia , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Ovarianas/fisiopatologia , Neoplasias Ovarianas/cirurgia , Gravidez , Antígeno Prostático Específico/análise , Antígeno Prostático Específico/metabolismo , Teratoma/metabolismo , Teratoma/fisiopatologia , Teratoma/cirurgia , Anormalidade Torcional/diagnóstico por imagem , Anormalidade Torcional/cirurgia , Resultado do Tratamento , Adulto JovemRESUMO
PurposeImmunotherapy is now a first-line treatment for metastatic non-small cell lung cancer (NSCLC) and melanomaQuery. It is important to understand the relationship between immunotherapy and radiation to the brain. The aim of this study was to assess the role of stereotactic radiosurgery (SRS) or WBRT in addition to immunotherapy in patients with melanoma or NSCLC metastatic to the brain.Methods/patientsUsing the National Cancer Database, 2951 patients with NSCLC and 936 patients with melanoma treated with immunotherapy were identified. Patients were classified as having received immunotherapy alone, immunotherapy with SRS, or immunotherapy with whole-brain radiation therapy (WBRT). KaplanMeier, multivariate Cox regression analyses, and propensity matching were performed to evaluate the impact of adding SRS to immunotherapy on overall survival (OS). Immortal survival bias was accounted for by only including patients who received radiation before immunotherapy and time zero was defined as the start of immunotherapy.Results205(6.9%) and 75(8.0%) patients received immunotherapy with no radiation, 822(27.9%) and 326(34.8%) received SRS and immunotherapy, and 1924(65.2%) and 535(57.2%) received WBRT and immunotherapy for NSCLC and melanoma, respectively. Adding SRS to immunotherapy was associated with improved OS in multivariate analyses (NSCLC HR = 0.81, 95% CI 0.660.99, p = 0.044; melanoma HR = 0.63, 95% CI 0.450.90, p = 0.011). The addition of WBRT to immunotherapy did not improve OS in patients with melanoma nor NSCLC.ConclusionsThis analysis suggests that treatment with SRS and immunotherapy is associated with improved OS compared to immunotherapy alone for patients with melanoma or NSCLC metastatic to the brain.
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Humanos , Ciências da Saúde , Radiocirurgia , Imunoterapia , Melanoma , Neoplasias Pulmonares , Neoplasias , Metástase NeoplásicaRESUMO
The incidence of melanoma in the United States continues to rise, with metastatic lesions notoriously recalcitrant to therapy. There are limited effective treatment options available and a great need for more effective therapies that can be rapidly integrated in the clinic. In this study, we demonstrate that the combination of RGD-targeted adeno-associated virus phage (RGD-AAVP-TNF) with hypofractionated radiation therapy results in synergistic inhibition of primary syngeneic B16 melanoma in a C57 mouse model. Furthermore, this combination appeared to modify the tumor microenvironment, resulting in decreased Tregs in the draining LN and increased tumor-associated macrophages within the primary tumor. Finally, there appeared to be a reduction in metastatic potential and a prolongation of overall survival in the combined treatment group. These results indicate the use of targeted TNF gene therapy vector with radiation treatment could be a valuable treatment option for patients with metastatic melanoma.
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Dependovirus/genética , Dependovirus/metabolismo , Vetores Genéticos/genética , Melanoma/genética , Melanoma/patologia , Oligopeptídeos/metabolismo , Fator de Necrose Tumoral alfa/genética , Animais , Linhagem Celular Tumoral , Terapia Combinada , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/efeitos adversos , Melanoma/metabolismo , Melanoma/terapia , Melanoma Experimental , Camundongos , Metástase Neoplásica , Neovascularização Patológica/genética , Neovascularização Patológica/imunologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/terapia , Radioterapia/métodos , Radioterapia Guiada por Imagem , Análise de Sobrevida , Resultado do Tratamento , Carga Tumoral/genética , Carga Tumoral/imunologia , Carga Tumoral/efeitos da radiação , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
An ineffective tumor-specific immune response from inadequate/incompetent antigen presentation could contribute to the failure in tumor control and its dissemination. Dendritic cells (DCs) have been shown to present antigen from apoptotic cells. We hypothesized that Flt3-ligand (Flt3L) therapy, which expands DCs in vivo, in combination with local tumor radiotherapy (RT), should improve antigen presentation from dying, irradiated tumor cells. RT + Flt3L reduced pulmonary metastases in a murine model of Lewis lung carcinoma and significantly improved survival in C57Bl/6 mice with established footpad tumors. Mice treated with Flt3L alone showed delayed tumor growth but eventually succumbed to tumor progression. The combination therapy of RT + Flt3L failed to impact survival in immunodeficient athymic mice, implicating the role of T cells in prolonging survival. These results support an attractive strategy of sequential RT and immunotherapy with Flt3L to enhance tumor antigen presentation, which may produce therapeutic responses against disseminated cancer and improvement in survival.
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Antineoplásicos/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Proteínas de Membrana/uso terapêutico , Animais , Terapia Combinada , Modelos Animais de Doenças , Neoplasias Pulmonares/diagnóstico por imagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Metástase Neoplásica/prevenção & controle , Transplante de Neoplasias , RadiografiaRESUMO
Hepatic tumors often recur in the liver after surgical resection. Postoperative radiotherapy (RT) could improve survival, but curative RT may induce delayed life-threatening radiation-induced liver damage. Because RT inhibits liver regeneration, we hypothesized that unirradiated, transplanted hepatocytes would proliferate preferentially in a partially resected and irradiated liver, providing metabolic support. We subjected F344 rats to hepatic RT and partial hepatectomy with/without a single intrasplenic, syngeneic hepatocyte transplantation. Hepatocyte transplantation ameliorated radiation-induced liver damage and improved survival of rats receiving RT after partial hepatectomy. We further demonstrated that transplanted hepatocytes extensively repopulate and function in a heavily irradiated rat liver.
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Transplante de Células , Regeneração Hepática , Fígado/citologia , Fígado/efeitos da radiação , Animais , Hepatectomia , Fígado/patologia , Masculino , Ratos , Ratos Endogâmicos F344 , Baço , Transplante Heterólogo , Transplante IsogênicoRESUMO
OBJECTIVE: To evaluate the feasibility and accuracy of using cone beam CT (CBCT) scans obtained in radiation studies using the small-animal radiation research platform to perform semi-automatic tumour segmentation of pre-clinical tumour volumes. METHODS: Volume measurements were evaluated for different anatomical tumour sites, the flank, thigh and dorsum of the hind foot, for a variety of tumour cell lines. The estimated tumour volumes from CBCT and manual calliper measurements using different volume equations were compared with the "gold standard", measured by weighing the tumours following euthanasia and tumour resection. The correlation between tumour volumes estimated with the different methods, compared with the gold standard, was estimated by the Spearman's rank correlation coefficient, root-mean-square deviation and the coefficient of determination. RESULTS: The semi-automatic CBCT volume segmentation performed favourably compared with manual calliper measures for flank tumours ≤2 cm(3) and thigh tumours ≤1 cm(3). For tumours >2 cm(3) or foot tumours, the CBCT method was not able to accurately segment the tumour volumes and manual calliper measures were superior. CONCLUSION: We demonstrated that tumour volumes of flank and thigh tumours, obtained as a part of radiation studies using image-guided small-animal irradiators, can be estimated more efficiently and accurately using semi-automatic segmentation from CBCT scans. ADVANCES IN KNOWLEDGE: This is the first study evaluating tumour volume assessment of pre-clinical subcutaneous tumours in different anatomical sites using on-board CBCT imaging. We also compared the accuracy of the CBCT method to manual calliper measures, using various volume calculation equations.
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Tomografia Computadorizada de Feixe Cônico , Neoplasias/diagnóstico por imagem , Neoplasias/patologia , Carga Tumoral , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Estudos de Viabilidade , Camundongos , Interpretação de Imagem Radiográfica Assistida por ComputadorRESUMO
Treatment failure after radiation therapy of prostate cancer (PC) could be a significant problem. Our objective is to design genetic radiosensitizing strategies for the treatment of PC. Cells from individuals with the genetic disorder ataxia telangiectasia (AT) are hypersensitive to ionizing radiation. Therefore, we examined whether attenuation of the AT gene product, AT mutated (ATM), in PC cells could result in an increased intrinsic radiosensitivity. A p53-mutant PC cell line, PC-3 was infected with adenoviral vectors, expressing antisense ATM RNA to various domains of the ATM gene. Immunoblot analyses of cellular extracts from antisense ATM-transfected PC-3 cells showed attenuated expression of the ATM protein within 2 days of viral infection. Compared with cells infected with an adeno-beta-galactosidase vector, antisense ATM-transfected PC-3 cells showed aberrant control of S-phase cell-cycle checkpoints after exposure to ionizing radiation. Under these conditions, the intrinsic radiosensitivity of the PC-3 cells was enhanced. Consequently antisense ATM gene therapy could serve as a paradigm for strategies that target the cellular survival mechanisms of an irradiated tumor cell and may provide therapeutic benefit to patients undergoing radiation therapy for PC.
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Adenoviridae/genética , Neoplasias da Próstata/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Antissenso/genética , Tolerância a Radiação/genética , Transfecção , Células Tumorais Cultivadas/efeitos da radiação , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular , Proteínas de Ciclo Celular , Divisão Celular , Sobrevivência Celular/efeitos da radiação , Dano ao DNA/efeitos da radiação , Primers do DNA/química , Proteínas de Ligação a DNA , Relação Dose-Resposta à Radiação , Citometria de Fluxo , Vetores Genéticos , Humanos , Immunoblotting , Masculino , Mutação , Fenótipo , Reação em Cadeia da Polimerase , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Serina-Treonina Quinases/genética , Radiossensibilizantes/uso terapêutico , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de TumorRESUMO
Advances in the understanding of hepatocyte engraftment and repopulation of the host liver have already led to the use of hepatocyte transplantation (HT) with some success in the treatment of inherited and acquired liver diseases. Wider application of HT is severely limited by the unavailability of large number of transplantable hepatocytes and difficulties associated with transplanting an adequate number of cells for achieving therapeutically satisfactory levels of metabolic correction. Therefore, there is a need for preparative regimens that provide a growth advantage to the transplanted (healthy) hepatocytes over the host's own (diseased) hepatocytes so that the former can repopulate the host liver. We have recently shown that when the liver of recipient rats was subjected to radiotherapy and partial hepatectomy before HT, the transplanted hepatocytes engrafted in and massively repopulated the liver, and also ameliorated the adverse clinical and histopathological changes associated with hepatic irradiation. This protocol was then used as a preparative regimen for transplanting normal hepatocytes into jaundice mutant rats (Gunn strain), which lack hepatic bilirubin-uridinediphosphoglucuronate glucuronosyltransferase and is a model of Crigler-Najjar syndrome Type I. The results showed long-term correction of the metabolic abnormality, suggesting that the transplanted hepatocytes repopulated an irradiated liver and were metabolically functional. This strategy could be useful in the treatment of various genetic, metabolic, or malignant diseases of the liver.
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Hepatócitos/transplante , Hepatopatias/terapia , Fígado/efeitos da radiação , Condicionamento Pré-Transplante/métodos , Animais , Sobrevivência Celular , Previsões , Sobrevivência de Enxerto , Hepatectomia/métodos , Hepatócitos/fisiologia , Hepatócitos/efeitos da radiação , Hepatopatias/radioterapia , Neoplasias Hepáticas/radioterapia , Neoplasias Hepáticas/terapia , Modelos Animais , Ratos , Ratos Endogâmicos F344RESUMO
An important consideration in application of hepatocyte transplantation is whether the number of engrafted hepatocytes is sufficient to achieve the desired effect. Here we have evaluated the proliferative potential of transplanted primary hepatocytes during regeneration of hepatic lobes. Two million hepatocytes isolated from congeneic normal Wistar-RHA rats were injected into the main portal vein of deficient, jaundiced Gunn rats. The right branch of the portal vein was ligated 24 hr before hepatocyte transplantation (group A) or transiently clamped during hepatocyte injection (group B) or 24 hr after hepatocyte injection (group C). In these groups, the three lobes supplied by the right branch of the portal vein rapidly atrophied and disappeared in 4 days, whereas the remaining lobes proliferated, as shown by size increase and 5-bromo-2-deoxy-uridine uptake. Two control groups received 2 million (group D) or 20 million hepatocytes (group E) without ligation. Hepatocyte engraftment occurred in all groups. The greatest hypobilirubinemic effect was observed in group A, in which serum bilirubin concentrations were reduced to 1.7+/-0.45 mg/dl from pretransplantation levels of 6.9+/-1.2 mg/dl. This effect was even greater than that observed after transplantation of 20 times more hepatocytes without ligation (group E). Specific endonuclease digestion of a polymerase chain reaction-amplified segment of the ugt1 gene from hepatic DNA showed that up to 25% of the DNA was of donor origin. This paralleled the hepatic bilirubin-UDP-glucuronosyltransferase activity, which was above 50% of normal. The results indicate that the transplanted hepatocytes proliferate preferentially within the regenerating lobes, replacing more than 20% of the liver mass with the progeny of the transplanted phenotypically normal hepatocytes.
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Transplante de Células/fisiologia , Fígado/citologia , Veia Porta/cirurgia , Animais , Apoptose/fisiologia , Atrofia/etiologia , Pigmentos Biliares/análise , Bilirrubina/sangue , Divisão Celular , Genótipo , Glucuronosiltransferase/análise , Glucuronosiltransferase/genética , Immunoblotting , Ligadura , Fígado/patologia , Regeneração Hepática , Ratos , Ratos Gunn , Ratos Wistar , Doadores de TecidosRESUMO
We have utilized enzyme-linked immunosorbent assay (ELISA) to quantitate PCR-amplified DNA. This method was used to measure mRNA for the vitamin D-binding protein (Gc), beta-actin and the transferrin receptor (TR) gene in the Hep3B cell line. Total RNA from Hep3B cells was reverse transcribed to obtain cDNA, which was amplified in the presence of digoxigenin-dUTP by PCR. The PCR products were then hybridized in liquid phase to a biotinylated, nested capture probe for the respective sequences. The hybridized products were bound to a streptavidin-coated ELISA plate and were detected by an alkaline-phosphatase-conjugated antibody to digoxigenin. ELISA standard curves for Gc and control genes, beta-actin and TR, were obtained after PCR amplification of serial dilutions of Hep3B total RNA. As an external standard, an ELISA standard curve for Gc was obtained after PCR amplification of serial dilutions of a full-length Gc cDNA insert obtained from a recombinant plasmid. Thus, we were able to develop a non-isotopic quantitation assay for PCR-amplified DNA that is highly sensitive and has the specificity of hybridization-based methods.
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DNA/análise , Ensaio de Imunoadsorção Enzimática , Reação em Cadeia da Polimerase , Proteína de Ligação a Vitamina D/genética , Actinas/genética , Sequência de Bases , Biotina , Sondas de DNA , DNA Complementar/análise , Immunoblotting , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , RNA Mensageiro/análise , Receptores da Transferrina/genética , Células Tumorais CultivadasRESUMO
In this study, eight Escherichia coli isolates were obtained from milk samples of dairy cattle suffering from clinical/subclinical mastitis. Isolates were characterized for antimicrobial resistance traits and virulence genes. Results revealed that one isolate was harbouring New Delhi metallo-beta-lactamase gene (blaNDM ). Cloning and sequencing of the PCR amplicon confirmed the identity of the gene (GenBank accession no. KC769583) having 100% homology with blaNDM-5 (GenBank accession no. JN104597.1), and this isolate was susceptible to colistin, chloramphenicol and tetracycline only. Moreover, another isolate carried extended-spectrum beta-lactamase (ESBL) gene - blaCTX-M , and all isolates possessed blaTEM gene. Of the eight isolates, only one isolate was positive for shiga toxin gene (stx2), and none were harbouring stx1 gene. Occurrence of New Delhi metallo-beta-lactamase (blaNDM ) in one E. coli isolate and ESBL genes in other isolates poses a potential threat to human health following possible entry and spread through food chain.