scholarly journals Alveolar macrophages up-regulate a non-classical innate response toMycobacterium tuberculosisinfectionin vivo

2019 ◽  
Author(s):  
A. C. Rothchild ◽  
G. S. Olson ◽  
J. Nemeth ◽  
L. M. Amon ◽  
D. Mai ◽  
...  
Keyword(s):  
Immune Response ◽  
Bacterial Growth ◽  
Alveolar Macrophages ◽  
Binding Motif ◽  
Transcriptional Program ◽  
Innate Response ◽  
Transcription Factor Binding Motif ◽  
Knock Out

AbstractAlveolar macrophages (AMs) are the first cells to be infected duringMycobacterium tuberculosis(Mtb) infection. Thus the AM response to infection is the first of many steps leading to initiation of the adaptive immune response, which is required for efficient control of infection. A hallmark of Mtb infection is the delay of the adaptive response, yet the mechanisms responsible for this delay are largely unknown. We developed a system to identify, sort and analyze Mtb-infected AMs from the lung within the first 10 days of infection. In contrast to what has been previously described usingin vitrosystems, we find that Mtb-infected AMs up-regulate a cell-protective antioxidant transcriptional signature that is dependent on the lung environment and not dependent on bacterial virulence. Computational approaches including pathway analysis and transcription factor binding motif enrichment analysis identify Nrf2 as a master regulator of the response of AMs to Mtb infection. Using knock-out mouse models, we demonstrate that Nrf2 drives the expression of the cell protective transcriptional program and impairs the ability of the host to control bacterial growth over the first 10 days of infection. Mtb-infected AMs exhibit a highly delayed pro-inflammatory response, and comparisons with uninfected AMs from the same infected animals demonstrate that inflammatory signals in the lung environment are blocked in the Mtb-infected cells. Thus, we have identified a novel lung-specific transcriptional response to Mtb infection that impedes AMs from responding rapidly to intracellular infection and thereby hinders the overall immune response.One Sentence SummaryIn response to Mtb infectionin vivo, alveolar macrophages fail to up-regulate the canonical pro-inflammatory innate response and instead induce an Nrf2-dependent cell protective transcriptional program, which in turn impairs the host’s control of bacterial growth.

scholarly journals Evolution of the Equine Infectious Anemia Virus Long Terminal Repeat during the Alteration of Cell Tropism

2005 ◽  
Vol 79 (9) ◽  
pp. 5653-5664 ◽  
Author(s):  
Wendy Maury ◽  
Robert J. Thompson ◽  
Quentin Jones ◽  
Sarahann Bradley ◽  
Tara Denke ◽  
...  
Keyword(s):  
Long Terminal Repeat ◽  
Equine Infectious Anemia Virus ◽  
Terminal Repeat ◽  
Binding Motif ◽  
Cell Tropism ◽  
Transcription Factor Binding Motif ◽  
Equine Infectious Anemia ◽  
Anemia Virus

ABSTRACT Equine infectious anemia virus (EIAV) is a lentivirus with in vivo cell tropism primarily for tissue macrophages; however, in vitro the virus can be adapted to fibroblasts and other cell types. Tropism adaptation is associated with both envelope and long terminal repeat (LTR) changes, and findings strongly suggest that these regions of the genome influence cell tropism and virulence. Furthermore, high levels of genetic variation have been well documented in both of these genomic regions. However, specific EIAV nucleotide or amino acid changes that are responsible for cell tropism changes have not been identified. A study was undertaken with the highly virulent, macrophage-tropic strain of virus EIAVwyo to identify LTR changes associated with alterations in cell tropism. We found the stepwise generation of a new transcription factor binding motif within the enhancer that was associated with adaptation of EIAV to endothelial cells and fibroblasts. An LTR that contained the new motif had enhanced transcriptional activity in fibroblasts, whereas the new site did not alter LTR activity in a macrophage cell line. This finding supports a previous prediction that selection for new LTR genetic variants may be a consequence of cell-specific selective pressures. Additional investigations of the EIAVwyo LTR were performed in vivo to determine if LTR evolution could be detected over the course of a 3-year infection. Consistent with previous in vivo findings, we observed no changes in the enhancer region of the LTR over that time period, indicating that the EIAVwyo LTR was evolutionarily stable in vivo.

scholarly journals Peptides containing the PCNA interacting motif APIM bind to the β-clamp and inhibit bacterial growth and mutagenesis

2020 ◽  
Vol 48 (10) ◽  
pp. 5540-5554 ◽  
Author(s):  
Aina Nedal ◽  
Synnøve B Ræder ◽  
Bjørn Dalhus ◽  
Emily Helgesen ◽  
Rune J Forstrøm ◽  
...  
Keyword(s):  
Dna Replication ◽  
Bacterial Growth ◽  
Cell Penetrating Peptides ◽  
Infection Model ◽  
Binding Motif ◽  
Hydrophobic Pocket ◽  
Surface Plasmon Resonance Analysis ◽  
Bacterial Dna

Abstract In the fight against antimicrobial resistance, the bacterial DNA sliding clamp, β-clamp, is a promising drug target for inhibition of DNA replication and translesion synthesis. The β-clamp and its eukaryotic homolog, PCNA, share a C-terminal hydrophobic pocket where all the DNA polymerases bind. Here we report that cell penetrating peptides containing the PCNA-interacting motif APIM (APIM-peptides) inhibit bacterial growth at low concentrations in vitro, and in vivo in a bacterial skin infection model in mice. Surface plasmon resonance analysis and computer modeling suggest that APIM bind to the hydrophobic pocket on the β-clamp, and accordingly, we find that APIM-peptides inhibit bacterial DNA replication. Interestingly, at sub-lethal concentrations, APIM-peptides have anti-mutagenic activities, and this activity is increased after SOS induction. Our results show that although the sequence homology between the β-clamp and PCNA are modest, the presence of similar polymerase binding pockets in the DNA clamps allows for binding of the eukaryotic binding motif APIM to the bacterial β-clamp. Importantly, because APIM-peptides display both anti-mutagenic and growth inhibitory properties, they may have clinical potential both in combination with other antibiotics and as single agents.

scholarly journals Immuno informatics Approach in Designing a Novel Vaccine Using Epitopes from All the Structural Proteins of SARS-CoV-2

2020 ◽  
Vol 13 (4) ◽  
pp. 1845-1862
Author(s):  
Leana Rich M. Herrera
Keyword(s):  
Immune Response ◽  
Tertiary Structure ◽  
Cross Reactivity ◽  
Structural Proteins ◽  
In Vivo Studies ◽  
Binding Motif ◽  
Population Coverage ◽  
Viral Attachment

The rapid transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted to the death of hundreds of thousands of people worldwide. With the devastating effects on the economy and healthcare system of many countries, it is crucial to acceleratevaccine development against SARS-CoV-2. Thus, thisworkutilizedimmunoinformaticsto efficiently design a novel multi-epitope vaccine that can potentially induce immune response through the immunogenic, and abundantly expressed structural proteins in SARS-CoV-2. Epitopes were screened and evaluated using various immunoinformatics tools and databases. Antigenicity, allergenicity, and population coverage were assessed. Epitopes were adjoined to form a single vaccine construct (Covax),linked with 50S ribosomal protein as an adjuvant. Physicochemical properties, cross-reactivity, antigenicity,andallergenicityof Covax were evaluated. The tertiary structure of Covax was modeled, refined and validated for docking with toll-like receptor 4 (TLR4). Binding affinity of Covax-TLR4 was estimated and compared with TLR4-adjuvant as control. Lastly,the immune response with Covax was simulated and compared withadjuvant alone. Total of 33 epitopes from S (21), E (3), M (5),and N (4)proteins were merged in Covax. These include epitopes on thereceptor-binding motif (RBM) of S protein known to beessential in the viral attachment. In silico evaluations classified Covax as stable, antigenic, and non-allergenic. Epitopes were estimated to have large worldwide population coverage, especially in areas with high infection rates, indicating broad potential efficacy of Covax as a vaccine for the most affected populations.Results in this work showed that Covax can bind to TLR4 whichindicates potential immunogenicity and superior properties necessary for a successful vaccine. Overall, this work efficiently minimized time, effort and cost in designing a candidate vaccine against SARS-CoV-2. In vitro and in vivo studies on Covax are anticipated.

scholarly journals Functional recruitment of dynamin requires multimeric interactions for efficient endocytosis

2018 ◽  
Author(s):  
Morgane Rosendale ◽  
Thi Nhu Ngoc Van ◽  
Dolors Grillo-Bosch ◽  
Silvia Sposini ◽  
Léa Claverie ◽  
...  
Keyword(s):  
Binding Motif ◽  
Knock Out ◽  
Rich Domain ◽  
Single Motif ◽  
Src Homology ◽  
Domain 3 ◽  
And Function ◽  
Kinetics Of

AbstractDuring clathrin mediated endocytosis (CME), membrane scission is achieved by the concerted action of dynamin and its interacting partners. Essential interactions occur between the proline/arginine-rich domain of dynamin (dynPRD) and the Src-homology domain 3 (SH3) of various proteins including amphiphysins. Here we show that multiple SH3 domains must bind simultaneously to dynPRD through three adjacent motifs for dynamin’s efficient recruitment and function. First, we show in dynamin triple knock-out cells that mutant dynamins modified in a single motif, including the central amphiphysin SH3 (amphSH3) binding motif, are partially capable of rescuing CME. However, mutating two motifs largely prevents that ability. To support this observation, we designed divalent dynPRD-derived peptides. These ligands bind multimers of amphSH3 with >100-fold higher affinity than monovalent onesin vitro. Accordingly, dialyzing living cells with these divalent peptides through a patch-clamp pipette blocks CME 2 to 3 times more effectively than with monovalent ones. Finally, the frequency of endocytic events decreases with competing peptides or hypomorphic rescue mutants but the kinetics of dynamin recruitment is unaffected. This suggests that PRD-SH3 interactions act upstream of dynamin accumulation at the neck of nascent vesicles. We conclude from these data that dynamin drives vesicle scissionviamultivalent interactionsin vivo.

scholarly journals The Role of Antibodies Against the Crude Capsular Extract in the Immune Response of Porcine Alveolar Macrophages to In Vitro Infection of Various Serovars of Glaesserella (Haemophilus) parasuis

2021 ◽  
Vol 12 ◽  
Author(s):  
Katarína Matiašková ◽  
Lenka Kavanová ◽  
Pavel Kulich ◽  
Jan Gebauer ◽  
Kateřina Nedbalcová ◽  
...  
Keyword(s):  
Immune Response ◽  
Alveolar Macrophages ◽  
Specific Binding ◽  
Outer Membrane Proteins ◽  
Disease Outbreaks ◽  
Haemophilus Parasuis ◽  
Associated Proteins

In Glässer’s disease outbreaks, Glaesserella (Haemophilus) parasuis has to overcome the non-specific immune system in the lower respiratory tract, the alveolar macrophages. Here we showed that porcine alveolar macrophages (PAMs) were able to recognize and phagocyte G. parasuis with strain-to-strain variability despite the presence of the capsule in virulent (serovar 1, 5, 12) as well in avirulent strains (serovar 6 and 9). The capsule, outer membrane proteins, virulence-associated autotransporters, cytolethal distending toxins and many other proteins have been identified as virulence factors of this bacterium. Therefore, we immunized pigs with the crude capsular extract (cCE) from the virulent G. parasuis CAPM 6475 strain (serovar 5) and evaluated the role of the anti-cCE/post-vaccinal IgG in the immune response of PAMs to in vitro infection with various G. parasuis strains. We demonstrated the specific binding of the antibodies to the cCE by Western-blotting assay and immunoprecipitation as well as the specific binding to the strain CAPM 6475 in transmission electron microscopy. In the cCE, we identified several virulence-associated proteins that were immunoreactive with IgG isolated from sera of immunized pigs. Opsonization of G. parasuis strains by post-vaccinal IgG led to enhanced phagocytosis of G. parasuis by PAMs at the first two hours of infection. Moreover, opsonization increased the oxidative burst and expression/production of both pro- and anti-inflammatory cytokines. The neutralizing effects of these antibodies on the antioxidant mechanisms of G. parasuis may lead to attenuation of its virulence and pathogenicity in vivo. Together with opsonization of bacteria by these antibodies, the host may eliminate G. parasuis in the infection site more efficiently. Based on these results, the crude capsular extract is a vaccine candidate with immunogenic properties.

scholarly journals Sarcoma IL-12 overexpression facilitates NK cell immunomodulation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mary Jo Rademacher ◽  
Anahi Cruz ◽  
Mary Faber ◽  
Robyn A. A. Oldham ◽  
Dandan Wang ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Lines ◽  
Nk Cell ◽  
Autologous Tumor ◽  
Murine Models ◽  
Lentiviral Transduction ◽  
Ifn Γ ◽  
Human Sarcoma

AbstractInterleukin-12 (IL-12) is an inflammatory cytokine that has demonstrated efficacy for cancer immunotherapy, but systemic administration has detrimental toxicities. Lentiviral transduction eliciting IL-12-producing human sarcoma for autologous reintroduction provides localized delivery for both innate and adaptive immune response augmentation. Sarcoma cell lines and primary human sarcoma samples were transduced with recombinant lentivirus engineering expression of human IL-12 (hu-IL-12). IL-12 expressing sarcomas were assessed in vitro and in vivo following implantation into humanized NSG and transgenic human IL-15 expressing (NSG.Tg(Hu-IL-15)) murine models. Lentiviral transduction (LV/hu-IL-12) of human osteosarcoma, Ewing sarcoma and rhabdomyosarcoma cell lines, as well as low-passage primary human sarcomas, engendered high-level expression of hu-IL-12. Hu-IL-12 demonstrated functional viability, eliciting specific NK cell-mediated interferon-γ (IFN-γ) release and cytotoxic growth restriction of spheroids in vitro. In orthotopic xenograft murine models, the LV/hu-IL-12 transduced human sarcoma produced detectable IL-12 and elicited an IFN-γ inflammatory immune response specific to mature human NK reconstitution in the NSG.Tg(Hu-IL-15) model while restricting tumor growth. We conclude that LV/hu-IL-12 transduction of sarcoma elicits a specific immune reaction and the humanized NSG.Tg(Hu-IL-15) xenograft, with mature human NK cells, can define in vivo anti-tumor effects and systemic toxicities. IL-12 immunomodulation through autologous tumor transduction and reintroduction merits exploration for sarcoma treatment.

scholarly journals Splicing factor SRSF1 promotes breast cancer progression via oncogenic splice switching of PTPMT1

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Jun-Xian Du ◽  
Yi-Hong Luo ◽  
Si-Jia Zhang ◽  
Biao Wang ◽  
Cong Chen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Progression ◽  
High Risk Group ◽  
Clinical Samples ◽  
Binding Motif ◽  
Ki 67 ◽  
Tissue Samples

Abstract Background Intensive evidence has highlighted the effect of aberrant alternative splicing (AS) events on cancer progression when triggered by dysregulation of the SR protein family. Nonetheless, the underlying mechanism in breast cancer (BRCA) remains elusive. Here we sought to explore the molecular function of SRSF1 and identify the key AS events regulated by SRSF1 in BRCA. Methods We conducted a comprehensive analysis of the expression and clinical correlation of SRSF1 in BRCA based on the TCGA dataset, Metabric database and clinical tissue samples. Functional analysis of SRSF1 in BRCA was conducted in vitro and in vivo. SRSF1-mediated AS events and their binding motifs were identified by RNA-seq, RNA immunoprecipitation-PCR (RIP-PCR) and in vivo crosslinking followed by immunoprecipitation (CLIP), which was further validated by the minigene reporter assay. PTPMT1 exon 3 (E3) AS was identified to partially mediate the oncogenic role of SRSF1 by the P-AKT/C-MYC axis. Finally, the expression and clinical significance of these AS events were validated in clinical samples and using the TCGA database. Results SRSF1 expression was consistently upregulated in BRCA samples, positively associated with tumor grade and the Ki-67 index, and correlated with poor prognosis in a hormone receptor-positive (HR+) cohort, which facilitated proliferation, cell migration and inhibited apoptosis in vitro and in vivo. We identified SRSF1-mediated AS events and discovered the SRSF1 binding motif in the regulation of splice switching of PTPMT1. Furthermore, PTPMT1 splice switching was regulated by SRSF1 by binding directly to its motif in E3 which partially mediated the oncogenic role of SRSF1 by the AKT/C-MYC axis. Additionally, PTPMT1 splice switching was validated in tissue samples of BRCA patients and using the TCGA database. The high-risk group, identified by AS of PTPMT1 and expression of SRSF1, possessed poorer prognosis in the stage I/II TCGA BRCA cohort. Conclusions SRSF1 exerts oncogenic roles in BRCA partially by regulating the AS of PTPMT1, which could be a therapeutic target candidate in BRCA and a prognostic factor in HR+ BRCA patient.

scholarly journals 68Ga, 44Sc and 177Lu-labeled AAZTA5-PSMA-617: synthesis, radiolabeling, stability and cell binding compared to DOTA-PSMA-617 analogues

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Jean-Philippe Sinnes ◽  
Ulrike Bauder-Wüst ◽  
Martin Schäfer ◽  
Euy Sung Moon ◽  
Klaus Kopka ◽  
...  
Keyword(s):  
Human Serum ◽  
Room Temperature ◽  
Solid Phase ◽  
Negative Influence ◽  
Binding Motif ◽  
Lncap Cells ◽  
Cell Binding ◽  
Binding Characteristics

Abstract Background The AAZTA chelator and in particular its bifunctional derivative AAZTA5 was recently investigated to demonstrate unique capabilities to complex diagnostic and therapeutic trivalent radiometals under mild conditions. This study presents a comparison of 68Ga, 44Sc and 177Lu-labeled AAZTA5-PSMA-617 with DOTA-PSMA-617 analogues. We evaluated the radiolabeling characteristics, in vitro stability of the radiolabeled compounds and evaluated their binding affinity and internalization behavior on LNCaP tumor cells in direct comparison to the radiolabeled DOTA-conjugated PSMA-617 analogs. Results AAZTA5 was synthesized in a five-step synthesis and coupled to the PSMA-617 backbone on solid phase. Radiochemical evaluation of AAZTA5-PSMA-617 with 68Ga, 44Sc and 177Lu achieved quantitative radiolabeling of > 99% after less than 5 min at room temperature. Stabilities against human serum, PBS buffer and EDTA and DTPA solutions were analyzed. While there was a small degradation of the 68Ga complex over 2 h in human serum, PBS and EDTA/DTPA, the 44Sc and 177Lu complexes were stable at 2 h and remained stable over 8 h and 1 day. For all three compounds, i.e. [natGa]Ga-AAZTA5-PSMA-617, [natSc]Sc-AAZTA5-PSMA-617 and [natLu]Lu-AAZTA5-PSMA-617, in vitro studies on PSMA-positive LNCaP cells were performed in direct comparison to radiolabeled DOTA-PSMA-617 yielding the corresponding inhibition constants (Ki). Ki values were in the range of 8–31 nM values which correspond with those of [natGa]Ga-DOTA-PSMA-617, [natSc]Sc-DOTA-PSMA-617 and [natLu]Lu-DOTA-PSMA-617, i.e. 5–7 nM, respectively. Internalization studies demonstrated cellular membrane to internalization ratios for the radiolabeled 68Ga, 44Sc and 177Lu-AAZTA5-PSMA-617 tracers (13–20%IA/106 cells) in the same range as the ones of the three radiolabeled DOTA-PSMA-617 tracers (17–20%IA/106 cells) in the same assay. Conclusions The AAZTA5-PSMA-617 structure proved fast and quantitative radiolabeling with all three radiometal complexes at room temperature, excellent stability with 44Sc, very high stability with 177Lu and medium stability with 68Ga in human serum, PBS and EDTA/DTPA solutions. All three AAZTA5-PSMA-617 tracers showed binding affinities and internalization ratios in LNCaP cells comparable with that of radiolabeled DOTA-PSMA-617 analogues. Therefore, the exchange of the chelator DOTA with AAZTA5 within the PSMA-617 binding motif has no negative influence on in vitro LNCaP cell binding characteristics. In combination with the faster and milder radiolabeling features, AAZTA5-PSMA-617 thus demonstrates promising potential for in vivo application for theranostics of prostate cancer.

scholarly journals RBMX suppresses tumorigenicity and progression of bladder cancer by interacting with the hnRNP A1 protein to regulate PKM alternative splicing

Oncogene ◽  
2021 ◽  
Author(s):  
Qiuxia Yan ◽  
Peng Zeng ◽  
Xiuqin Zhou ◽  
Xiaoying Zhao ◽  
Runqiang Chen ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Alternative Splicing ◽  
Rna Binding ◽  
Aerobic Glycolysis ◽  
Histological Grade ◽  
Migration And Invasion ◽  
Binding Motif ◽  
Hnrnp A1

AbstractThe prognosis for patients with metastatic bladder cancer (BCa) is poor, and it is not improved by current treatments. RNA-binding motif protein X-linked (RBMX) are involved in the regulation of the malignant progression of various tumors. However, the role of RBMX in BCa tumorigenicity and progression remains unclear. In this study, we found that RBMX was significantly downregulated in BCa tissues, especially in muscle-invasive BCa tissues. RBMX expression was negatively correlated with tumor stage, histological grade and poor patient prognosis. Functional assays demonstrated that RBMX inhibited BCa cell proliferation, colony formation, migration, and invasion in vitro and suppressed tumor growth and metastasis in vivo. Mechanistic investigations revealed that hnRNP A1 was an RBMX-binding protein. RBMX competitively inhibited the combination of the RGG motif in hnRNP A1 and the sequences flanking PKM exon 9, leading to the formation of lower PKM2 and higher PKM1 levels, which attenuated the tumorigenicity and progression of BCa. Moreover, RBMX inhibited aerobic glycolysis through hnRNP A1-dependent PKM alternative splicing and counteracted the PKM2 overexpression-induced aggressive phenotype of the BCa cells. In conclusion, our findings indicate that RBMX suppresses BCa tumorigenicity and progression via an hnRNP A1-mediated PKM alternative splicing mechanism. RBMX may serve as a novel prognostic biomarker for clinical intervention in BCa.

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