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1.
Med Glas (Zenica) ; 17(1)2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31359742

RESUMO

Aim To present combined measles cases data and phylogenetic analysis of the virus circulated in 2018 in the Federation of Bosnia and Herzegovina (FB&H, the entity of Bosnia and Herzegovina), in order to analyse endemic transmission patterns of circulating strains and its implications for elimination efforts. Methods The data were derived from epidemiological case investigations and laboratory diagnoses based on serology, molecular detection and genotyping of the measles virus. Results During 2018 16 measles cases were reported in FB&H, of which five were classified as laboratory confirmed cases, one was an epidemiologically linked case and 10 were clinically compatible cases. Among them 12 (75.00%) cases were unvaccinated or had unknown vaccination status. The most affected population was up to 14 years of age (13/16; 81.25%). None of the cases was fully vaccinated. Viruses of other genetic lineages had been introduced in FB&H in the recent period. Two virus lineages of genotype B3 were identified. Phylogenetic analysis indicated the presence of a unique sequence of measles B3 virus in FB&H (Sarajevo). Conclusion Further strengthening of measles surveillance system and renewed efforts to increase vaccination levels are necessary to prevent disease and for elimination setting.

2.
Rev Environ Contam Toxicol ; 249: 153-197, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30900073

RESUMO

Lead (Pb) is an extremely toxic metal for all living forms including plants. It enters plants through roots from soil or soil solution. It is considered as one of the most eminent examples of anthropogenic environmental pollutant added in environment through mining and smelting of lead ores, coal burning, waste from battery industries, leaded paints, metal plating, and automobile exhaust. Uptake of Pb in plants is a nonselective process and is driven by H+/ATPases. Translocation of Pb metal ions occurs by apoplastic movement resulting in deposition of metal ions in the endodermis and is further transported by symplastic movement. Plants exposed to high concentration of Pb show toxic symptoms due to the overproduction of reactive oxygen species (ROS) through Fenton-Haber-Weiss reaction. ROS include superoxide anion, hydroxyl radical, and hydrogen peroxide, which reach to macro- and micro-cellular levels in the plant cells and cause oxidative damage. Plant growth and plethora of biochemical and physiological attributes including plant growth, water status, photosynthetic efficiency, antioxidative defense system, phenolic compounds, metal chelators, osmolytes, and redox status are adversely influenced by Pb toxicity. Plants respond to toxic levels of Pb in varied ways such as restricted uptake of metal, chelation of metal ions to the root endodermis, enhancement in activity of antioxidative defense, alteration in metal transporters expression, and involvement of plant growth regulators.


Assuntos
Chumbo/toxicidade , Fenômenos Fisiológicos Vegetais , Plantas/efeitos dos fármacos , Poluentes do Solo/toxicidade , Antioxidantes , Espécies Reativas de Oxigênio
3.
Rev Environ Contam Toxicol ; 251: 109-129, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31289937

RESUMO

One of the fastest-growing global food sectors is the bivalve aquaculture industry. Bivalves particularly oysters, mussels and clams are important sources of animal protein (Tan and Ransangan 2016a, b). Bivalve aquaculture represents 14-16% of the average per capita animal protein for 1.5 billion people and supports over 200,000 livelihoods, mostly in developing countries (FAO 2018). Most of the bivalves produced around the world (89%) are from aquaculture (FAO 2016). To date, mollusc aquaculture have accounted for 21.42% (17.14 million tonnes) of the total aquaculture production, with Asia being the largest contributor (92.27%) (FAO 2018).


Assuntos
Bivalves , Mudança Climática , Animais , Aquicultura
4.
Ann Lab Med ; 40(1): 1-6, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31432632

RESUMO

BACKGROUND: JL1, a CD43 epitope and mucin family cell surface glycoprotein, is expressed on leukemic cells. An anti-JL1 antibody combined with a toxic substance can have targeted therapeutic effects against JL1-positive leukemia; however, JL1 expression on bone marrow (BM) lymphoma cells has not been assessed using flow cytometry. We investigated JL1 expression on BM lymphoma cells from patients with non-Hodgkin lymphoma (NHL) to assess the potential of JL1 as a therapeutic target. METHODS: Patients with BM involvement of mature B-cell (N=44) or T- and natural killer (NK)-cell (N=4) lymphomas were enrolled from May 2015 to September 2016. JL1 expression on BM lymphoma cells was investigated using flow cytometry. Clinical, pathological, and cytogenetic characteristics, and treatment responses were compared according to JL1 expression status. RESULTS: Of the patients with NHL and BM involvement, 37.5% (18/48) were JL1-positive. Among mature B-cell lymphomas, 100%, 38.9%, 33.3%, 100%, and 25.0% of Burkitt lymphomas, diffuse large B-cell leukemias, mantle cell leukemias, Waldenstrom macroglobulinemia, and other B-cell lymphomas, respectively, were JL1-positive. Three mature T- and NK-cell NHLs were JL1-positive. JL1 expression was associated with age (P=0.045), complete response (P=0.004), and BM involvement at follow-up (P=0.017), but not with sex, performance status, the B symptoms, packed marrow pattern, cytogenetic abnormalities, or survival. CONCLUSIONS: JL1 positivity was associated with superior complete response and less BM involvement in NHL following chemotherapy.

5.
Ann Lab Med ; 40(1): 7-14, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31432633

RESUMO

BACKGROUND: Rapid and accurate diagnosis of acute myocardial infarction (AMI) is critical for initiating effective treatment and achieving better prognosis. We investigated the performance of copeptin for early diagnosis of AMI, in comparison with creatine kinase myocardial band (CK-MB) and troponin I (TnI). METHODS: We prospectively enrolled 271 patients presenting with chest pain (within six hours of onset), suggestive of acute coronary syndrome, at an emergency department (ED). Serum CK-MB, TnI, and copeptin levels were measured. The diagnostic performance of CK-MB, TnI, and copeptin, alone and in combination, for AMI was assessed by ROC curve analysis by comparing the area under the curve (AUC). Sensitivity, specificity, negative predictive value, and positive predictive value of each marker were obtained, and the characteristics of each marker were analyzed. RESULTS: The patients were diagnosed as having ST elevation myocardial infarction (STEMI; N=43), non-ST elevation myocardial infarction (NSTEMI; N=25), unstable angina (N=78), or other diseases (N=125). AUC comparisons showed copeptin had significantly better diagnostic performance than TnI in patients with chest pain within two hours of onset (AMI: P=0.022, ≤1 hour; STEMI: P=0.017, ≤1 hour and P=0.010, ≤2 hours). In addition, TnI and copeptin in combination exhibited significantly better diagnostic performance than CK-MB plus TnI in AMI and STEMI patients. CONCLUSIONS: The combination of TnI and copeptin improves AMI diagnostic performance in patients with early-onset chest pain in an ED setting.

6.
Ann Lab Med ; 40(1): 15-20, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31432634

RESUMO

BACKGROUND: Carbapenem-resistant K. pneumoniae 2297, isolated from a patient treated with tigecycline for pneumonia, developed tigecycline resistance, in contrast to carbapenem-resistant isolate 1215, which was collected four months prior to the 2297 isolate. Mechanisms underlying tigecycline resistance were elucidated for the clinical isolates. METHODS: The tigecycline minimum inhibitory concentration (MIC) was determined using the broth microdilution method, with or without phenylalanine-arginine ß-naphthylamide (PABN), and whole-genome sequencing was carried out by single-molecule real-time sequencing. The expression levels of the genes acrA, oqxA, ramA, rarA, and rpoB were determined by reverse-transcription quantitative PCR. RESULTS: Both isolates presented identical antibiograms, except for tigecycline, which showed an MIC of 0.5 mg/L in 1215 and 2 mg/L in 2297. The addition of PABN to tigecycline-resistant 2297 caused a four-fold decrease in the tigecycline MIC to 0.5 mg/L, although acrA expression (encoding the AcrAB efflux pump) was upregulated by 2.5 fold and ramA expression (encoding the pump activator RamA) was upregulated by 1.4 fold. We identified a 6,096-bp fragment insertion flanking direct TATAT repeats that disrupted the romA gene located upstream of ramA in the chromosome of K. pneumoniae 2297; the insertion led the ramA gene promoter replacement resulting in stronger activation of the gene. CONCLUSIONS: The K. pneumoniae isolate developed tigecycline resistance during tigecycline treatment. It was related to the overexpression of the AcrAB resistance-nodulation-cell division efflux system due to promoter replacement.

7.
Ann Lab Med ; 40(1): 21-26, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31432635

RESUMO

BACKGROUND: The existing modified carbapenem inactivation methods (mCIMs) recommended by the CLSI for detecting carbapenemase production have not been applicable for Acinetobacter baumannii. We evaluated the influence of matrices used in mCIMs and CIMTris on the stability of the disks for detecting carbapenemase producers and suggested optimal mCIM conditions for detecting carbapenemase-producing A. baumannii. METHODS: Seventy-three A. baumannii isolates characterized for antimicrobial susceptibility and carbapenemase encoding genes were tested for carbapenemase production using mCIM and CIMTris. The influence of the matrices (Tryptic soy broth [TSB] and Tris-HCl) used in these methods on the stability of the meropenem (MEM) disk was also evaluated. The mCIM conditions were adjusted to enhance screening sensitivity and specificity for detecting carbapenemase-producing A. baumannii. RESULTS: The matrices had an impact on the stability of the MEM disk after the incubation period (two or four hrs). TSB nutrient broth is an appropriate matrix for mCIM compared with Tris-HCl pH 7.6, which leads to the loss of MEM activity in CIMTris. The sensitivity and the specificity of the optimal mCIM were both 100%. CONCLUSIONS: We established optimal mCIM conditions for simple, accurate, and reproducible detection of carbapenemase-producing A. baumannii.

8.
Ann Lab Med ; 40(1): 27-32, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31432636

RESUMO

BACKGROUND: Mutations in the quinolone resistance-determining regions (QRDRs) of Acinetobacter baumannii DNA gyrase (gyrA) and topoisomerase IV (parC) are linked to fluoroquinolone (FQ) resistance. We developed a mismatched PCR-restriction fragment length polymorphism (RFLP) assay to detect mutations in the gyrA and parC QRDRs associated with FQ resistance in A. baumannii. METHODS: Based on the conserved sequences of A. baumannii gyrA and parC, two primer sets were designed for mismatched PCR-RFLP to detect mutations in gyrA (codons 83 and 87) and parC (codons 80 and 84) by introducing an artificial restriction enzyme cleavage site into the PCR products. This assay was evaluated using 58 A. baumannii strains and 37 other Acinetobacter strains that have been identified by RNA polymerase ß-subunit gene sequence analysis. RESULTS: PCR amplification of gyrA and parC was successful for all A. baumannii strains. In 11 FQ -susceptible strains, the gyrA and parC PCR products were digested by the selected restriction enzymes at the site containing gyrA (codons 83 and 87) and parC (codons 80 and 84). PCR products from 47 FQ-resistant strains containing mutations in gyrA and parC were not digested by the restriction enzymes at the site containing the mutation. As for the non-baumannii Acinetobacter strains, although amplification products for gyrA were obtained for 28 strains, no parC amplification product was obtained for any strain. CONCLUSIONS: This assay specifically amplified gyrA and parC from A. baumannii and detected A. baumannii gyrA and parC mutations with FQ resistance.

9.
Ann Lab Med ; 40(1): 33-39, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31432637

RESUMO

BACKGROUND: The interferon-gamma (IFN-γ) releasing assay (IGRA) is widely used for latent tuberculosis infection (LTBI) diagnosis. We evaluated the analytical performance of a new automated chemiluminescent immunoanalyzer-based IGRA (CLIA-IGRA), AdvanSure I3 (LG Life Sciences, Seoul, Korea) and compared it with that of the QuantiFERON-TB Gold In-Tube (QFT-GIT) assay. METHODS: Repeatability and reproducibility were evaluated at four levels. Detection capability, including limit of blank (LoB), limit of detection (LoD), and limit of quantification (LoQ), was evaluated using IFN-γ standard material (National Institute for Biological Standards and Control code: 87/586). Agreement between the results of two assays was evaluated using 341 blood samples from healthcare workers and patients at a tertiary care hospital. To determine the cut-off value of CLIA-IGRA for diagnosing LTBI, the ROC curve was analyzed. RESULTS: Repeatability and reproducibility were 4.86-7.00% and 6.36-7.88% CV, respectively. LoB, LoD, and LoQ were 0.022, 0.077, and 0.249 IU/mL, respectively. IFN-γ values between CLIA-IGRA and QFT-GIT showed a strong correlation within the analytical measurable range of both assays, especially when the value was low. Qualitative comparison of the two assays yielded a 99.1% overall agreement (kappa coefficient=0.98). A cut-off value of 0.35 IU/mL was appropriate for diagnosing LTBI. CONCLUSIONS: CLIA-IGRA is a reliable assay for LTBI diagnosis, with performance similar to that of QFT-GIT.

10.
Ann Lab Med ; 40(1): 40-47, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31432638

RESUMO

BACKGROUND: Tumor markers are useful for detection and preoperative evaluation of ovarian tumors. We evaluated the clinical usefulness of cancer antigen (CA) 125, human epididymis 4 (HE4), and CA72-4 levels and Risk of Ovarian Malignancy Algorithm (ROMA) values for differential diagnosis of malignant and borderline tumors among suspected ovarian tumors, and the effects of endometriosis on these tumor markers. METHODS: In a total of 266 patients (213, 14, and 39 with benign, borderline and malignant tumors, respectively), CA125, HE4, and CA72-4 levels were measured, and ROMA values were calculated. Medians of each marker were compared among the three groups. The area under the ROC curve (AUC), sensitivity, and specificity were calculated to analyze the diagnostic performance of each marker. RESULTS: All markers were significantly higher in the malignant group than in the benign group. HE4 levels and ROMA values were significantly higher in the malignant group than in the borderline group. ROMA value had the highest AUC for distinguishing the malignant and borderline groups from the benign group in premenopausal (0.773) and postmenopausal (0.927) patients. CA125 level was significantly higher in patients with endometriosis than in those without (P<0.001), whereas HE4 and CA72-4 levels were not affected by endometriosis (P=0.128 and 0.271, respectively). CONCLUSIONS: ROMA value is the best marker to distinguish malignant and borderline tumors from benign tumors in pre- and postmenopausal patients. HE4 and CA72-4 levels provide information on possible CA125 elevation due to endometriosis.

11.
Ann Lab Med ; 40(1): 48-56, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31432639

RESUMO

BACKGROUND: Anti-carbohydrate antibody responses, including those of anti-blood group ABO antibodies, are yet to be thoroughly studied in humans. Because anti-ABO antibody-mediated rejection is a key hurdle in ABO-incompatible transplantation, it is important to understand the cellular mechanism of anti-ABO responses. We aimed to identify the main human B cell subsets that produce anti-ABO antibodies by analyzing the correlation between B cell subsets and anti-ABO antibody titers. METHODS: Blood group A-binding B cells were analyzed in peritoneal fluid and peripheral blood samples from 43 patients undergoing peritoneal dialysis and 18 healthy volunteers with blood group B or O. The correlation between each blood group A-specific B cell subset and anti-A antibody titer was then analyzed using Pearson's correlation analysis. RESULTS: Blood group A-binding B cells were enriched in CD27+CD43+CD1c- B1, CD5+ B1, CD11b+ B1, and CD27+CD43+CD1c+ marginal zone-B1 cells in peripheral blood. Blood group A-specific B1 cells (P=0.029 and R=0.356 for IgM; P=0.049 and R=0.325 for IgG) and marginal zone-B1 cells (P=0.011 and R=0.410 for IgM) were positively correlated with anti-A antibody titer. Further analysis of peritoneal B cells confirmed B1 cell enrichment in the peritoneal cavity but showed no difference in blood group A-specific B1 cell enrichment between the peritoneal cavity and peripheral blood. CONCLUSIONS: Human B1 cells are the key blood group A-specific B cells that have a moderate correlation with anti-A antibody titer and therefore constitute a potential therapeutic target for successful ABO-incompatible transplantation.

12.
Ann Lab Med ; 40(1): 57-62, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31432640

RESUMO

As various linezolid resistance mechanisms have been identified in methicillin-resistant Staphylococcus aureus (MRSA), we investigated the molecular characteristics of MRSA with elevated linezolid minimum inhibitory concentrations (MICs), using the VITEK 2 system (bioMérieux, Marcy-l'Étoile, France). Twenty-seven MRSA isolates from 14 patients exhibiting linezolid MICs ≥8 µg/mL were examined by broth microdilution (BMD) test as well as by sequencing for mutations in the 23S rRNA gene or ribosomal proteins (L3, L4, and L22) and the presence of the optrA, cfr, and cfr(B) genes. Of the 27 isolates, four (14.8%) from one patient were confirmed as linezolid resistant by BMD and harbored a 23S rRNA T2500A mutation. The remaining 23 were confirmed as linezolid susceptible, indicating that the linezolid-resistant results were major errors generated by VITEK 2. The most commonly detected mutation (19/27, 70.4%), L3 Gly152Asp, was detected in only linezolid-susceptible isolates. No isolates contained optrA, cfr, or cfr(B) or any L4 or L22 protein alterations. Our results show that the 23S rRNA T2500A mutation was mainly associated with linezolid resistance, while the L3 Gly152Asp mutation was not related to linezolid resistance. A confirmatory test is recommended for VITEK 2 linezolid-resistant results owing to the high probability of false resistant results.

13.
Ann Lab Med ; 40(1): 63-67, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31432641

RESUMO

As 16S ribosomal RNA (rRNA)-targeted sequencing can detect DNA from non-viable bacteria, it can be used to identify pathogens from clinical samples even in patients pretreated with antibiotics. We compared the results of 16S rRNA-targeted sequencing and culture for identifying bacterial species in normally sterile body fluid (NSBF): cerebrospinal, pericardial, peritoneal and pleural fluids. Over a 10-year period, a total of 312 NSBF samples were evaluated simultaneously using 16S rRNA-targeted sequencing and culture. Results were concordant in 287/312 (92.0%) samples, including 277 (88.8%) negative and 10 (3.2%) positive samples. Of the 16 sequencing-positive, culture-negative samples, eight showed clinically relevant isolates that included Fusobacterium nucleatum subsp. nucleatum, Streptococcus pneumoniae, and Staphylococcus spp. All these samples were obtained from the patients pretreated with antibiotics. The diagnostic yield of 16S rRNA-targeted sequencing combined with culture was 11.2%, while that of culture alone was 6.1%. 16S rRNA-targeted sequencing in conjunction with culture could be useful for identifying bacteria in NSBF samples, especially when patients have been pretreated with antibiotics and when anaerobic infection is suspected.

14.
Ann Lab Med ; 40(1): 68-71, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31432642

RESUMO

Evaluation of diagnostic tests requires reference standards, which are often unavailable. Latent class analysis (LCA) can be used to evaluate diagnostic tests without reference standards, using a combination of observed and estimated results. Conditionally independent diagnostic tests for Helicobacter pylori infection are required. We used LCA to construct a reference standard and evaluate the capability of non-invasive tests (stool antigen test and serum antibody test) to diagnose H. pylori infection compared with the conventional method, where histology is the reference standard. A total of 96 healthy subjects with endoscopy histology results were enrolled from January to July 2016. Sensitivity and specificity were determined for the LCA approach (i.e., using a combination of three tests as the reference standard) and the conventional method. When LCA was used, sensitivity and specificity were 83.8% and 99.4% for histology, 80.0% and 81.9% for the stool antigen test, and 63.6% and 89.3% for the serum antibody test, respectively. When the conventional method was used, sensitivity and specificity were 75.8% and 71.1% for the stool antigen test and 77.7% and 60.7% for the serum antibody test, respectively. LCA can be applied to evaluate diagnostic tests that lack a reference standard.

15.
Ann Lab Med ; 40(1): 72-75, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31432643

RESUMO

Accurate detection of BCR-ABL fusion transcripts at and below molecular response (MR) 4 (0.01% International Scale [IS]) is required for disease monitoring in patients with chronic myeloid leukemia (CML). We evaluated the analytical performance of the QXDx BCR-ABL %IS (Bio-Rad, Hercules, CA, USA) droplet digital PCR (ddPCR) assay, which is the first commercially available ddPCR-based in vitro diagnostics product. In precision analysis, the %CV was 9.3% and 3.0%, with mean values of 0.031% IS and 9.4% IS, respectively. The assay was linear in the first order, ranging from 0.032% IS to 20% IS. The manufacturer-claimed limit of blank, limit of detection, and limit of quantification were verified successfully. There was a very strong correlation between the results of the QXDx BCR-ABL %IS ddPCR assay and the ipsogen BCR-ABL1 Mbcr IS-MMR (Qiagen, Hilden, Germany) real-time quantitative PCR assay (r=0.996). In conclusion, the QXDx BCR-ABL %IS ddPCR assay can provide reliable results for CML patients.

16.
Methods Mol Biol ; 2058: 1-6, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31486028

RESUMO

Oncolytic viruses exploit key hallmarks of cancer for replication in malignant cells, leading to tumor cell lysis, modulation of the tumor microenvironment and in situ vaccination effects. Diverse virus platforms have been developed as oncolytic vectors and designed for improved tumor specificity, intratumoral spread, therapeutic gene delivery and especially as targeted cancer immunotherapeutics. This chapter provides a concise overview of the basic principles as well as current progress in preclinical and clinical studies of oncolytic virotherapy.

17.
Methods Mol Biol ; 2058: 7-29, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31486029

RESUMO

The optimal clinical exploitation of viruses as gene therapy or oncolytic vectors will require them to be administered intravenously. Strategies must therefore be deployed to enable viruses to survive the harsh neutralizing environment of the bloodstream and achieve deposition within and throughout target tissues or tumor deposits. This chapter describes the genetic and chemical engineering approaches that are being developed to overcome these challenges.

18.
Methods Mol Biol ; 2058: 31-49, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31486030

RESUMO

Tumor-selectively replicating "oncolytic" adenoviruses based on serotype 5 are promising tools for the treatment of solid tumors. However, their effective delivery to the tumor by systemic administration remains challenging. Several strategies of molecular retargeting have been pursued to equip adenoviruses with molecular features that facilitate their efficient uptake by tumors and to protect healthy tissue from damage. Transductional retargeting can be conveniently achieved using bispecific molecular adapter proteins based on the ectodomain of the coxsackievirus and adenovirus receptor linked to tumor ligands of choice. In this chapter, we describe methods for their design, purification, and application.

19.
Methods Mol Biol ; 2058: 51-75, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31486031

RESUMO

This chapter describes the development of recombinant oncolytic measles viruses (MeV) that selectively enter and destroy tumor cells. The envelope of MeV is a favorable targeting substrate because receptor attachment and membrane fusion functions are separated on two proteins: the hemagglutinin (H) that binds receptors, and the fusion (F) protein that fuses the viral envelope with the cell membrane. The cell entry process, which depends on receptor recognition and occurs at the plasma membrane at neutral pH, results in the delivery of encapsidated genomes to the cytoplasm, where they replicate. Towards improving cancer specificity of oncolytic MeV, two types of cell entry targeting have been achieved. First, entry has been redirected through cancer-specific cell surface proteins. This was done by displaying specificity domains on H while also ablating binding to its natural receptors. Second, activation of the F protein was made dependent on secreted cancer proteases, while also interfering with F cleavage/activation by a ubiquitous intracellular protease. This chapter describes how entry-targeted MeV are produced: In short, gene cassettes with modified H or F coding regions are generated, and then introduced into the viral genome available on plasmid DNA. Such full-length genome plasmids are transfected in cell lines expressing, stably or transiently, the three viral proteins necessary for genome replication. Infectious centers form among these "rescue" cells, which allow isolation of clonal recombinant viruses. These are amplified, characterized in vitro, and then evaluated for their oncolytic activity in appropriate preclinical animal models.

20.
Methods Mol Biol ; 2058: 77-94, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31486032

RESUMO

Although a variety of oncolytic viruses under clinical investigation have proven to be safe, the overall efficacy of oncolytic viruses as monotherapies has been suboptimal. While responses to combination therapies are much more promising, the development of oncolytic virus monotherapies with enhanced potency is imperative. With this initiative comes the need for improved mechanisms of virus targeting to prevent off-target toxicities. MicroRNA-detargeting has emerged as an invaluable tool for preventing unwanted toxicities of oncolytic viruses, particularly for picornaviruses. Here we describe methods to test the genetic stability of microRNA response elements in vitro and to evaluate the detargeting efficiency and therapeutic index of a microRNA-detargeted picornavirus in vivo. Although the methods described herein are specific to picornaviruses, microRNA-detargeting and these assays can be adapted for different classes of oncolytic viruses.

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