RESUMO
BACKGROUND: Osteosarcoma (OS) is a rare cancer with a bimodal age distribution that peaks in children and young adults. It has been shown that the expression of programmed cell death protein 1 (PD-1) and programmed death ligand-1 (PD-L1) on tumor-infiltrating immune cells negatively correlates with prognosis of OS patients. However, a comprehensive assessment of the tumor-infiltrating immune cells in OS and their function has not been performed. METHODS: CD8+ T cells were isolated from biopsy tissue samples collected from OS patients and control subjects. Mass cytometry, Treg suppression assay, mixed lymphocyte reaction assay, and effector T cell functional assay were performed to analyze the function of tumor-infiltrating T cells. A xenograft metastasis model was established in BALB/c nude mice. RESULTS: Macrophages and CD3+ T cells comprised most of the tumor-infiltrating immune cells in OS, with a disproportionately higher number of helper CD4+ T cells than effector CD8+ T cells. Whereas the tumor-infiltrating regulatory T cells were functionally intact, the CD8+ T cells showed increased expression of the immune checkpoint receptor (ICR) PD-1 and T cell immunoglobulin and mucin-domain containing 3 (TIM3) and were functionally inactive. TIM3 blockade using a monoclonal antibody restored the T cell alloreactive function of the CD8+ T cells ex vivo. TIM3 blockade in a xenograft model of OS impaired tumor growth in vivo. TIM3 blockade decreased the number of tumor-infiltrating CD4+ T cells while increasing the numbers and functional activation of tumor-infiltrating CD8+ T cells in vivo. CONCLUSIONS: These results highlight that TIM3 blockade might be a viable therapeutic option and should be tested in additional preclinical models.
RESUMO
Most RNA positive controls currently used for monitoring the quality of RT-PCR assays have some disadvantages, such as instability, inability to monitor the quality of the relevant primers and/or causing indifferentiable false positives. To avoid these disadvantages, a simple method to prepare stable and differentiable RNA positive controls is now demonstrated with a real-time RT-PCR assay for the detection of Nipah virus (NiV). A DNA sequence which was shorter than its counterpart in the NiV genome and contained the binding sites of the primers of the RT-PCR assay was designed, synthesized and inserted into a vector, and then amplified by PCR with two vector-specific primers both of which contained a T7 promoter at the 5' terminal. The RNA positive control was the dsRNA in vitro transcribed from the PCR amplicons flanked by two T7 promoters. The RNA positive control was stable and able to monitor the quality of the whole concerned RT-PCR assay. False positives caused by contaminations of the RNA positive control or its amplicons could be easily identified because the amplicons of the RNA positive control were obviously shorter than those of real positive samples. Thus, the RNA positive control reported in this study avoided some common disadvantages of current RNA positive controls.
Assuntos
Biotecnologia/métodos , Vírus Nipah/genética , RNA Viral/genética , RNA/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Técnicas Genéticas , Modelos Genéticos , Plasmídeos/metabolismo , RNA de Cadeia Dupla/química , RNA Viral/análise , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Radiation-induced lung toxicity (RILT), leading to radiation pneumonia or fibrosis, is a primary problem of radiation therapy. The pathogenesis of RILT remains unclear. In this study, we used a rat model of RILT to examine the expression of aquaporins (AQPs) after radiation injury. Sprague Dawley rats were given a single dose of 17 Gy (dose rate of 3.0 Gy/min) of X-irradiation to the thorax. Rats that survived acute pneumonitis (at 1-4 weeks) were evaluated weekly for the expression of AQP1 and AQP5 in the lung by immunohistochemical and reverse transcription polymerase chain reaction (RT-PCR) analyses. Immunohistochemical analysis showed that AQP1 protein was expressed in the capillary endothelium, and its level was significantly decreased after irradiation. AQP5 protein was expressed in the alveolar epithelium, and its level was increased between Days 7 and 14 after irradiation but decreased at Day 28, compared with the sham group. The RT-PCR results were consistent with the immunohistochemical analysis results. In summary, this study provides the first report of AQP1 and AQP5 expression in a model of radiation-induced pulmonary inflammation and edema. Decreased levels of AQP1 and AQP5 after irradiation suggest that these proteins play a role in the pathogenesis of RILT.
Assuntos
Aquaporina 1/metabolismo , Aquaporina 5/metabolismo , Pulmão/metabolismo , Pulmão/efeitos da radiação , Animais , Aquaporina 1/genética , Aquaporina 5/genética , Expressão Gênica/efeitos da radiação , Imuno-Histoquímica , Pulmão/patologia , Lesão Pulmonar/etiologia , Lesão Pulmonar/genética , Lesão Pulmonar/metabolismo , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Lesões Experimentais por Radiação/genética , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/patologia , Ratos , Ratos Sprague-DawleyRESUMO
Mutation in any of five key amino acid residues (at positions 26, 27, 30, 31 and 34) within the M2 protein of influenza A viruses leads to resistance against the amantodine class of anti-influenza drugs. In this study, a pyrosequencing method was described to rapidly detect established five molecular markers of resistance to M2 blockers, amantadine. The residues L26, V27, A30, S31 and G34 in the M2 protein were targeted for pyrosequencing, and 94 avian influenza viruses were used to perform the amantadine resistance analysis. Our results showed that most of avian influenza viruses were amantadine resistant, Mutations V27I and S31N were founded in these isolates.
Assuntos
Amantadina/uso terapêutico , Antivirais/uso terapêutico , Farmacorresistência Viral/genética , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/genética , Animais , Galinhas , Influenza Aviária/tratamento farmacológico , Influenza Aviária/virologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Thirteen isolates of Class I Newcastle disease virus obtained from healthy poultry in China during 2008 were characterized genotypically in this study. All the isolates were proved to be lentogenic strains based on the deduced amino acid sequence of the Fusion protein gene. Molecular epidemiological analysis showed that 13 isolates could be subdivided into 2 distinct genotypes, 11 isolates belonged to genotype 2, and other 2 isolates belonged to genotype 3. Results indicated two genotypes of Class I Newcastle disease virus might widely exist in domestic poultry in China.
Assuntos
Epidemiologia Molecular/métodos , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/genética , Animais , Aves , China/epidemiologia , Genótipo , Humanos , Doença de Newcastle/epidemiologia , Vírus da Doença de Newcastle/classificação , Vírus da Doença de Newcastle/patogenicidade , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Virais de Fusão/genéticaRESUMO
OBJECTIVE: To design and rapidly evaluate a TaqMan assay for detecting influenza A viruses. METHODS: The probe and the primers of the assay were designed with the software packages of DNA Star and Primer Premier 5.0. Their specificity and conservation were verified through Blast in GenBank and electronic hybridization. The assay's sensitivity was compared with the standard RT-PCR. RESULTS: The designed primers and probe were confirmed to be very specific and conserved. The assay was 3-27 folds more sensitive than the standard RT-PCR. The RT and PCR steps could be simplified into one step. CONCLUSION: The TaqMan Real-time PCR assay is specific, sensitive and easy to perform.