The transcriptome of lung tumor-infiltrating dendritic cells reveals a tumor-supporting phenotype and a microRNA signature with negative impact on clinical outcome

Targeting immunomodulatory pathways has ushered a new era in lung cancer therapy. Further progress requires deeper insights into the biology of immune cells in the lung cancer micro-environment. Dendritic cells (DCs) represent a heterogeneous and highly plastic immune cell system with a central role in controlling immune responses. The intratumoral infiltration and activation status of DCs are emerging as clinically relevant parameters in lung cancer. In this study, we used an orthotopic preclinical model of lung cancer to dissect how the lung tumor micro-environment affects tissue-resident DCs and extract novel biologically and clinically relevant information. Lung tumor-infiltrating leukocytes expressing generic DC markers were found to predominantly consist of CD11b(+) cells that, compare with peritumoral lung DC counterparts, strongly overexpress the T-cell inhibitory molecule PD-L1 and acquire classical surface markers of tumor-associated macrophages (TAMs). Transcriptome analysis of these CD11b(+) tumor-infiltrating DCs (TIDCs) indicates impaired antitumoral immunogenicity, confirms the skewing toward TAM-related features, and indicates exposure to a hypoxic environment. In parallel, TIDCs display a specific microRNA (miRNA) signature dominated by the prototypical lung cancer oncomir miR-31. In vitro, hypoxia drives intrinsic miR-31 expression in CD11b(+) DCs. Conditioned medium of miR-31 overexpressing CD11b(+) DCs induces pro-invasive lung cancer cell shape changes and is enriched with pro-metastatic soluble factors. Finally, analysis of TCGA datasets reveals that the TIDC-associated miRNA signature has a negative prognostic impact in non-small cell lung cancer. Together, these data suggest a novel mechanism through which the lung cancer micro-environment exploits the plasticity of the DC system to support tumoral progression

The transcriptome of lung tumor-infiltrating dendritic cells reveals a tumor-supporting phenotype and a microRNA signature with negative impact on clinical outcome

Targeting immunomodulatory pathways has ushered a new era in lung cancer therapy. Further progress requires deeper insights into the biology of immune cells in th

Lung tumours reprogram pulmonary dendritic cell immunogenicity at the microRNA level

Lung cancer arises in a context of tumour-induced immune suppression. Dendritic cells (DCs) are central players in the induction of anti-tumoural immunity, providing critical signals that drive the induction of cytotoxic T-cell responses. Meanwhile, microRNAs are associated with tumour development as well as immune regulation. We postulated that lung tumours escape immune control by reprogramming DC immunogenicity at the microRNA level. Using an orthotopic model of lung cancer, we first identified the DC population responsible for transport and cross-presentation of lung tumour-derived antigens to naive T cells in the draining mediastinal lymph nodes (LNs). Profiling the full microRNA repertoire of these DCs revealed a restricted set of microRNAs that was consistently dysregulated in the presence of lung tumours, with miR-301a as one of the top upregulated transcripts. Overexpression of miR-301a in DCs suppressed IL-12 secretion, decreased IFN-gamma release from antigen-specific cytotoxic T cells, and shifted antigen-specific T helper cytokine profile away from IFN-gamma towards IL-13 and IL-17A-secreting T cells. Strikingly, DC-selective Dicer1 gene deletion resulted in delayed lung tumour growth and a survival benefit. Taken together, our data reveal that lung tumours induce an immunosuppressive microRNA signature in pulmonary DCs. Interfering with the DC-intrinsic capacity to remodel microRNA repertoires affects lung tumour outcome

A sensitive in-house RT-PCR genotyping system for combined detection of plasma HIV-1 and assessment of drug resistance

Quantification of the viral burden and identification of drug resistant mutations are important laboratory tools in the management of HIV-1 infected patients. However, widespread use of assays for viral load determination and genotyping is still hampered by the high cost. Here, an in-house RT-PCR-sequencing assay for HIV-1 drug resistance monitoring with the potential to be used both as a qualitative assay to detect the virus in plasma and as a genotyping system is described. A total of 377 clinical samples, collected from 374 HIV-infected patients of diverse geographic origin, were tested. The nested RT-PCR for amplification of the protease reverse transcriptase gene was found positive for 350 (92.8%) and 346 (91.8%) of 377 samples, respectively. All amplification-failures were due to viral loads of below 500 copies/ml. However, low viral load does not exclude amplification since 80.2 and 76% of 121 samples with viral loads of less than 500 copies/ml were amplified successfully for protease and reverse transcriptase, respectively. The high sensitivity of the assay was independent of the HIV-subtype, with a broad range of different HIV-1 subtypes tested. In conclusion the RT-PCR-direct sequencing method is convenient for the sensitive detection and subsequent genotyping of plasma RNA from a broad range of different HIV-1 subtypes. The assay enables the accurate follow-up of patients under treatment at a significantly reduced cost compared to the currently available commercial assays for viral load assessment and genotyping

Evaluation of two commercially available alternatives for HIV-1 viral load testing in resource-limited settings

OBJECTIVE: To evaluate respiratory muscle strength, oxygenation and chest pain in patients undergoing off-pump coronary artery bypass (OPCAB) using internal thoracic artery grafts comparing pleural drain insertion site at the subxyphoid region versus the lateral region. METHODS: Forty patients were randomized into two groups in accordance with the pleural drain site. Group II (n = 19) -pleural drain exteriorized in the intercostal space; group (SI) (n = 21) chest tube exteriorized at the subxyphoid region. All patients underwent assessment of respiratory muscle strength (inspiratory and expiratory) on the pre, 1, 3 and 5 postoperative days (POD). Arterial blood gas analysis was collected on the pre and POD1. The chest pain sensation was measured 1, 3 and 5 POD. RESULTS: A significant decrease in respiratory muscle strength (inspiratory and expiratory) was seen in both groups until POD5 (P OBJETIVO: Avaliar a força muscular respiratória, oxigenação e dor torácica em pacientes submetidos à cirurgia de revascularização miocárdica (RM) sem circulação extracorpórea (CEC) comparando o local de inserção do dreno pleural na região subxifoidea versus lateral. MÉTODOS: Quarenta pacientes foram randomizados em dois grupos Grupo (II - n = 19) - dreno pleural exteriorizado na região intercostal; Grupo (SI - n = 21) dreno pleural exteriorizado na região subxifoidea. Os pacientes foram submetidos à avaliação da força muscular respiratória no pré, 1º, 3ºe 5º dias de pós-operatório (PO). Gasometria arterial foi coletada no pré e 1º dia do PO. A dor torácica foi avaliada no 1º, 3º e 5º dias de PO. RESULTADOS: Ambos os grupos apresentaram diminuição significante da força muscular respiratória até o quinto dia do PO (P <0,05). A diferença entre os grupos manteve-se significante com maior decréscimo no grupo II (P <0,05). Houve queda na pressão arterial de oxigênio em ambos os grupos (P <0,05), mas quando comparado à queda foi maior no grupo II (P <0,05). A dor torácica no 1º, 2º e 5º dia do PO foi maior grupo II (P <0,05). O tempo de intubação orotraqueal e permanência hospitalar no PO foram maiores no grupo II (P<0,05). CONCLUSÃO: Pacientes submetidos a drenagem pleural subxifoidea apresentaram menor queda na força muscular respiratória, melhor preservação da oxigenação arterial e menos dor comparado aos pacientes com inserção do dreno na região intercostal no PO precoce de cirurgia de RM sem CEC

Drug-resistant variants that evolve during nonsuppressive therapy persist in HIV-1-infected peripheral blood mononuclear cells after long-term highly active antiretroviral therapy

The aim of this study was to determine whether drug-resistant virus persists in peripheral blood mononuclear cells (PBMCs) after long-term suppression of virus replication. Proviral DNA was extracted from the PBMCs of I I patients on long-term highly active antiretroviral therapy (HAART). Genotyping of the reverse transcriptase (RT) and protease gene of several proviral variants was performed using limiting dilution polyincrase chain reaction and single-copy sequencing. All patients were on successful HAART for a mean period of 59 months but had a history of suboptimal therapy and genotypic drug resistance before. Comparison of the amino acid sequence of the RT and protease gene in the different proviral variants, with that of the plasma virus isolated before HAART treatment, revealed that the different drug-resistant viral variants that evolved during the process of gradually building up resistance were still detectable in the PBMCs in 10 of the 11 patients tested. The proportion of resistant variants was found to correlate with the time that the resistant variants had been able to replicate. These data clearly show that virus variants that are able to replicate for a certain period enter the latent reservoir and remain archived in the PBMCs for a very long period

Diversity of the Human Immunodeficiency Virus Type 1 (HIV-1) env Sequence after Vertical Transmission in Mother-Child Pairs Infected with HIV-1 Subtype A

Although several virologic and immunologic factors associated with an increased risk of perinatal human immunodeficiency virus type 1 (HIV-1) transmission have been described, the mechanism of mother-to-child transmission is still unclear. More specifically, the question of whether selective pressures influence the transmission remains unanswered. The aim of this study was to assess the genetic diversity of the transmitted virus after in utero transmission and after peripartum transmission and to compare the viral heterogeneity in the child with the viral heterogeneity in the mother. To allow a very accurate characterization of the viral heterogeneity in a single sample, limiting-dilution sequencing of a 1,016-bp fragment of the env gene was performed. Thirteen children were tested, including 6 with in utero infections and 7 with peripartum infections. Samples were taken the day after birth and at the ages of 6 and 14 weeks. A homogeneous virus population was seen in six (46.2%) infants, of whom two were infected in utero and four were infected peripartum. A more heterogeneous virus population was detected in seven infants (53.8%), four infected in utero and three infected peripartum. The phylogenetic trees of the mother-child pairs presented a whole range of different tree topologies and showed infection of the child by one or more maternal variants. In conclusion, after HIV-1 transmission from mother to child a heterogeneous virus population was detected in approximately one-half of the children examined. Heterogeneous virus populations were found after peripartum infection as well as after in utero infection. Phylogenetic tree topologies argue against selection processes as the major mechanism driving mother-to-child transmission but support the hypothesis that virus variability is mainly driven by the inoculum level and/or exposure time

An accelerated, clinical-grade protocol to generate high yields of type 1-polarizing messenger RNA loaded dendritic cells for cancer vaccination

Background: Many efforts have been devoted to improve the performance of dendritic cell (DC) based cancer vaccines. Ideally, a DC vaccine should induce robust type 1 polarized T-cell responses and efficiently expand antigen (Ag)-specific cytotoxic T-cells, while being applicable regardless of patient human leukocyte antigen (HLA) type. Production time should be short, while maximally being good manufacturing practice (GMP) compliant. We developed a method that caters to all of these demands and demonstrated the superiority of the resulting product compared with DCs generated using a well-established "classical" protocol. Methods: Immunomagnetically purified monocytes were cultured in a closed system for 3 days in GMP-compliant serum-free medium and cytokines, and matured for 24 h using monophosphoryl lipid A (MPLA)+ interferon -gamma (IFN-y). Mature DCs were electroporated with messenger RNA (mRNA) encoding full-length antigen and cryopreserved. "Classical" DCs were cultured for 8 days in flasks, with one round of medium and cytokine supplementation, and matured with tumor necrosis factor alpha (TNF-alpha) + prostaglandin E2 (PGE2) during the last 2 days. Results: Four-day MPLA/IFN-y matured DCs were superior to 8-day TNF-alpha/PGE2 matured DCs in terms of yield, co-stimulatory/coinhibitory molecule expression, resilience to electroporation and cryopreservation and type 1 polarizing cytokine and chemokine release after cell thawing. Electroporated and cryopreserved DCs according to our protocol efficiently present epitopes from tumor antigen-encoding mRNA, inducing a strong expansion of antigen-specific CD8+ T-cells with full cytolytic capacity. Conclusion: We demonstrate using a GMP-compliant culture protocol the feasibility of generating high yields of mature DCs in a short time, with a superior immunogenic profile compared with 8-day TNF-alpha/PGE2 matured DCs, and capable of inducing vigorous cytotoxic T-cell responses to antigen from electroporated mRNA. This method is now being applied in our clinical trial program

A case of dendritic cell, monocyte, B and NK lymphoid deficiency (DCML)

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