scholarly journals Lack of Kcnn4 improves mucociliary clearance in muco-obstructive lung disease

2020 ◽  
Author(s):  
Génesis Vega ◽  
Anita Guequén ◽  
Amber R. Philp ◽  
Ambra Gianotti ◽  
Lilian Arzola ◽  
...  
Keyword(s):  
Lung Disease ◽  
Obstructive Lung Disease ◽  
Mucociliary Clearance ◽  
Ex Vivo ◽  
Neutrophil Infiltration ◽  
Genetically Engineered ◽  
Anion Secretion ◽  
Obstructive Disease ◽  
Genetically Engineered Mouse Model

ABSTRACTAirway mucociliary clearance (MCC) is the main mechanism of lung defense keeping the airways free of infection and mucus obstruction. Airways surface liquid (ASL) volume, ciliary beating and mucus are central for proper MCC, and are critically regulated by sodium (Na+) absorption and anion secretion. Impaired MCC is a key feature of muco-obstructive disease. The calcium-activated potassium (K+)channel KCa.3.1, encoded by the Kcnn4 gene, participates in intestinal ion secretion and previous studies showed that its activation increase Na+ absorption in airway epithelia, suggesting that hyperpolarization induced by KCa3.1 was sufficient to drive Na+ absorption. However, its role in airway epithelial function is not fully understood. We therefore aimed to elucidate the role of KCa3.1 in MCC in a genetically engineered mouse model. We show that KCa3.1 inhibition reduced Na+ absorption in mouse and human airway epithelium. Furthermore, the genetic deletion of Kcnn4 enhanced cilia beating frequency (CBF) and MCC ex vivo and in vivo. Kcnn4 was silenced in the Scnn1b-transgenic mouse (Scnn1btg/+), a model of muco-obstructive lung disease triggered by increased epithelial Na+-absorption, leading to improvements in MCC and reduction of Na+-absorption. KCa3.1 deletion did not change the amount of mucus but did reduce mucus adhesion, neutrophil infiltration and emphysema. Our data support that KCa3.1 inhibition attenuated muco-obstructive disease in the Scnn1btg/+ mice. K+-channel modulation may be a novel therapeutic strategy to treat muco-obstuctive lung diseases.

scholarly journals STAT3 Serine phosphorylation is required for TLR4 metabolic reprogramming and IL-1β expression

2020 ◽  
Author(s):  
Jesse Balic ◽  
Hassan Albargy ◽  
Kevin Luu ◽  
Francis J Kirby ◽  
W. Samantha N. Jayasekara ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Aerobic Glycolysis ◽  
Genetically Engineered ◽  
Serine Phosphorylation ◽  
Metabolic Reprogramming ◽  
Inflammatory Cytokine Production ◽  
Genetically Engineered Mouse Model ◽  
Tlr4 Activation ◽  
Signalling Cascade

ABSTRACTDetection of microbial components such as lipopolysaccharide (LPS) by Toll-like receptor (TLR)-4 expressed on macrophages induces a robust pro-inflammatory response which has recently been shown to be dependent on metabolic reprogramming 1, 2, 3, 4. These innate metabolic changes have been compared to the Warburg effect (also known as aerobic glycolysis) described in tumour cells 5, 6. However, the mechanisms by which TLR4 activation leads to mitochondrial and glycolytic reprogramming remain unknown. Here we show that TLR4 activation induces a signalling cascade recruiting TRAF6 and TBK-1, while TBK-1 phosphorylates STAT3 on S727. Using a genetically engineered mouse model incapable of undergoing STAT3 Ser727 phosphorylation, we show both ex vivo and in vivo that STAT3 Ser727 phosphorylation is critical for LPS-induced glycolytic reprogramming, the production of the central immune-metabolite succinate and inflammatory cytokine production in a model of LPS-induced inflammation. Our study identifies non-canonical STAT3 activation as the crucial signalling intermediary for TLR4-induced glycolysis, macrophage metabolic reprogramming and inflammation.

scholarly journals Differential effect of anesthetics on mucociliary clearance in vivo in mice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kyle S. Feldman ◽  
Eunwon Kim ◽  
Michael J. Czachowski ◽  
Yijen Wu ◽  
Cecilia W. Lo ◽  
...  
Keyword(s):  
Mucociliary Clearance ◽  
Defense Mechanism ◽  
Ex Vivo ◽  
Beat Frequency ◽  
Ciliary Beat Frequency ◽  
Wild Type ◽  
Sulfur Colloid ◽  
Ct System

AbstractRespiratory mucociliary clearance (MCC) is a key defense mechanism that functions to entrap and transport inhaled pollutants, particulates, and pathogens away from the lungs. Previous work has identified a number of anesthetics to have cilia depressive effects in vitro. Wild-type C57BL/6 J mice received intra-tracheal installation of 99mTc-Sulfur colloid, and were imaged using a dual-modality SPECT/CT system at 0 and 6 h to measure baseline MCC (n = 8). Mice were challenged for one hour with inhalational 1.5% isoflurane, or intraperitoneal ketamine (100 mg/kg)/xylazine (20 mg/kg), ketamine (0.5 mg/kg)/dexmedetomidine (50 mg/kg), fentanyl (0.2 mg/kg)/1.5% isoflurane, propofol (120 mg/Kg), or fentanyl/midazolam/dexmedetomidine (0.025 mg/kg/2.5 mg/kg/0.25 mg/kg) prior to MCC assessment. The baseline MCC was 6.4%, and was significantly reduced to 3.7% (p = 0.04) and 3.0% (p = 0.01) by ketamine/xylazine and ketamine/dexmedetomidine challenge respectively. Importantly, combinations of drugs containing fentanyl, and propofol in isolation did not significantly depress MCC. Although no change in cilia length or percent ciliation was expected, we tried to correlate ex-vivo tracheal cilia ciliary beat frequency and cilia-generated flow velocities with MCC and found no correlation. Our results indicate that anesthetics containing ketamine (ketamine/xylazine and ketamine/dexmedetomidine) significantly depress MCC, while combinations containing fentanyl (fentanyl/isoflurane, fentanyl/midazolam/dexmedetomidine) and propofol do not. Our method for assessing MCC is reproducible and has utility for studying the effects of other drug combinations.

scholarly journals Visualisation of interstitial lung disease by molecular imaging of integrin αvβ3 and somatostatin receptor 2

2018 ◽  
Vol 78 (2) ◽  
pp. 218-227 ◽  
Author(s):  
Janine Schniering ◽  
Martina Benešová ◽  
Matthias Brunner ◽  
Stephanie Haller ◽  
Susan Cohrs ◽  
...  
Keyword(s):  
Interstitial Lung Disease ◽  
Lung Disease ◽  
Single Photon ◽  
Ex Vivo ◽  
Somatostatin Receptor ◽  
Photon Emission ◽  
Nuclear Imaging ◽  
Integrin Αvβ3 ◽  
Biodistribution Studies

ObjectiveTo evaluate integrin αvβ3 (alpha-v-beta-3)-targeted and somatostatin receptor 2 (SSTR2)-targeted nuclear imaging for the visualisation of interstitial lung disease (ILD).MethodsThe pulmonary expression of integrin αvβ3 and SSTR2 was analysed in patients with different forms of ILD as well as in bleomycin (BLM)-treated mice and respective controls using immunohistochemistry. Single photon emission CT/CT (SPECT/CT) was performed on days 3, 7 and 14 after BLM instillation using the integrin αvβ3-targeting 177Lu-DOTA-RGD and the SSTR2-targeting 177Lu-DOTA-NOC radiotracer. The specific pulmonary accumulation of the radiotracers over time was assessed by in vivo and ex vivo SPECT/CT scans and by biodistribution studies.ResultsExpression of integrin αvβ3 and SSTR2 was substantially increased in human ILD regardless of the subtype. Similarly, in lungs of BLM-challenged mice, but not of controls, both imaging targets were stage-specifically overexpressed. While integrin αvβ3 was most abundantly upregulated on day 7, the inflammatory stage of BLM-induced lung fibrosis, SSTR2 expression peaked on day 14, the established fibrotic stage. In agreement with the findings on tissue level, targeted nuclear imaging using SPECT/CT specifically detected both imaging targets ex vivo and in vivo, and thus visualised different stages of experimental ILD.ConclusionOur preclinical proof-of-concept study suggests that specific visualisation of molecular processes in ILD by targeted nuclear imaging is feasible. If transferred into clinics, where imaging is considered an integral part of patients’ management, the additional information derived from specific imaging tools could represent a first step towards precision medicine in ILD.

Role of sensory afferents in the myoelectric response to acute enteric inflammation in the rabbit

1997 ◽  
Vol 273 (2) ◽  
pp. G447-G455 ◽  
Author(s):  
T. Shea-Donohue ◽  
J. M. Goldhill ◽  
E. Montcalm-Mazzilli ◽  
C. Colleton ◽  
V. M. Pineiro-Carrero ◽  
...  
Keyword(s):  
Neural Control ◽  
Ex Vivo ◽  
Neutrophil Infiltration ◽  
Mucosal Damage ◽  
Myoelectric Activity ◽  
Sensory Afferents ◽  
Rabbit Small Intestine ◽  
Sustained Inhibition

The role of sensory afferents in inflammation-induced alterations in myoelectric activity in vivo was investigated in the rabbit small intestine. Isolated ileal loops were implanted with serosal electrodes and exposed to ricin or vehicle after pretreatment with 125 mg/kg of subcutaneous (125 mg over 3 days) or intraluminal (640 microM) capsaicin. After 5 h of myoelectric recording, the loops were prepared for histology and for ex vivo generation of eicosanoids. Capsaicin exacerbated mucosal damage after exposure to ricin but did not alter neutrophil infiltration. Subcutaneous capsaicin alone elevated slow-wave frequency and spike events and transiently suppressed the myoelectric response to ricin. In contrast, intraluminal capsaicin alone did not alter myoelectric activity but produced a sustained inhibition of the response to ricin. Eicosanoid production was unchanged by capsaicin alone. Intraluminal capsaicin blocked increases in leukotriene C4 and prostaglandin E2 during inflammation, an effect that paralleled its inhibition of myoelectric activity. Thus the contribution of sensory afferents to altered motility during acute ileitis involves the release of mucosal inflammatory mediators that influence neural control of smooth muscle.

scholarly journals HIV-1 Transcription Inhibitor 1E7-03 Restores LPS-Induced Alteration of Lung Leukocytes’ Infiltration Dynamics and Resolves Inflammation in HIV Transgenic Mice

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 204 ◽  
Author(s):  
Marina Jerebtsova ◽  
Asrar Ahmad ◽  
Xiaomei Niu ◽  
Ornela Rutagarama ◽  
Sergei Nekhai
Keyword(s):  
Lung Disease ◽  
Neutrophil Infiltration ◽  
Lung Macrophage ◽  
Transcription Inhibitor ◽  
Endothelial Migration ◽  
Fast Resolution ◽  
Bleeding Score ◽  
Hiv 1

Human immunodeficiency virus (HIV)-infected individuals treated with anti-retroviral therapy often develop chronic non-infectious lung disease. To determine the mechanism of HIV-1-associated lung disease we evaluated the dynamics of lung leukocytes in HIV-1 transgenic (Tg) mice with integrated HIV-1 provirus. In HIV-Tg mice, lipopolysacharide (LPS) induced significantly higher levels of neutrophil infiltration in the lungs compared to wild-type (WT) mice. In WT mice, the initial neutrophil infiltration was followed by macrophage infiltration and fast resolution of leukocytes infiltration. In HIV-Tg mice, resolution of lung infiltration by both neutrophils and macrophages was significantly delayed, with macrophages accumulating in the lumen of lung capillaries resulting in a 45% higher rate of mortality. Trans-endothelial migration of HIV-Tg macrophages was significantly reduced in vitro and this reduction correlated with lower HIV-1 gene expression. HIV-1 transcription inhibitor, 1E7-03, enhanced trans-endothelial migration of HIV-Tg macrophages in vitro, decreased lung neutrophil infiltration in vivo, and increased lung macrophage levels in HIV-Tg mice. Moreover, 1E7-03 reduced levels of inflammatory IL-6 cytokine, improved bleeding score and decreased lung injury. Together this indicates that inhibitors of HIV-1 transcription can correct abnormal dynamics of leukocyte infiltration in HIV-Tg, pointing to the utility of transcription inhibition in the treatment of HIV-1 associated chronic lung disease.

scholarly journals IMMU-10. ESTABLISHING EFFECTIVE MODELS FOR IMMUNOTHERAPY IN GBM

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi121-vi121
Author(s):  
Daniel Zamler ◽  
Er-Yen Yen ◽  
Takashi Shingu ◽  
Jiangong Ren ◽  
Cynthia Kassab ◽  
...  
Keyword(s):  
Mouse Model ◽  
Cell Function ◽  
Genetically Engineered ◽  
Death Sentence ◽  
Immune Microenvironment ◽  
Knockout Mouse Model ◽  
Arginase 1 ◽  
Genetically Engineered Mouse Model ◽  
Human Gbm

Abstract The introduction of immunotherapies has been paradigm shifting for cancers that were previously a death sentence. However, preclinical/clinical studies on glioblastoma (GBM) have generated mixed outcomes in patients, likely due to its great heterogeneity of immune microenvironment, particularly the myeloid cell populations. Primary patient studies have been limited by a difficulty in performing longitudinal studies, uncontrolled environmental conditions, and genetic variability. There is also, unfortunately, a paucity of mouse models that effectively re-capitulate the immune microenvironment of the human disease. To address these difficulties, we have established the Qk/p53/Pten (QPP) triple knockout mouse model established in our lab. The QPP model uses a cre-lox system to induce Qk deletion on a Pten−/−; p53−/− background which helps NSCs maintain their stemness outside the SVZ in Nes-CreERT2;QkiL/L PtenL/L p53L/L mice, which develops glioblastoma with survival of ~105 days. We have preliminarily assessed the QPP tumors as a faithful model to study the immune response to GBM and found them to recapitulate human GBM with respect to differential response to checkpoint blockade therapy and myeloid and T-cells histopathologically, particularly regarding upregulation of Arginase-1 (Arg1). Arg1 is the canonical marker for tumor-associated macrophages (TAMs), which is a major population of myeloid cells that greatly infiltrate in human GBM, sometimes making up more than ~30% of all GBM cells. Given TAMs’ prevalence in the tumor microenvironment and their upregulation of Arg1 in both human GBM and our QPP model, we are testing whether manipulation of Arg1 will impact TAM function and influence GBM growth. We are also evaluating arginine metabolism in TAMs effect on T cell function in GBM. Lastly, we have developed a genetically engineered mouse model to study the role of Arg1 knockout in a GBM context in-vivo. Our studies suggest that Arg1 plays an important role in GBM immune interaction.

scholarly journals Nme1 and Nme2 genes exert metastasis-suppressor activities in a genetically engineered mouse model of UV-induced melanoma

2020 ◽  
Vol 124 (1) ◽  
pp. 161-165
Author(s):  
Nidhi Pamidimukkala ◽  
Gemma S. Puts ◽  
M. Kathryn Leonard ◽  
Devin Snyder ◽  
Sandrine Dabernat ◽  
...  
Keyword(s):  
Mouse Model ◽  
Suppressor Gene ◽  
Genetically Engineered ◽  
In Vivo Model ◽  
Metastasis Suppressor ◽  
Specific Context ◽  
Genetically Engineered Mouse Model ◽  
Metastatic Activity ◽  
First Time

AbstractNME1 is a metastasis-suppressor gene (MSG), capable of suppressing metastatic activity in cell lines of melanoma, breast carcinoma and other cancer origins without affecting their growth in culture or as primary tumours. Herein, we selectively ablated the tandemly arranged Nme1 and Nme2 genes to assess their individual impacts on metastatic activity in a mouse model (HGF:p16−/−) of ultraviolet radiation (UVR)-induced melanoma. Metastatic activity was strongly enhanced in both genders of Nme1- and Nme2-null mice, with stronger activity in females across all genotypes. The study ascribes MSG activity to Nme2 for the first time in an in vivo model of spontaneous cancer, as well as a novel metastasis-suppressor function to Nme1 in the specific context of UVR-induced melanoma.

scholarly journals Modelling Pancreatic Neuroendocrine Cancer: From Bench Side to Clinic

Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3170
Author(s):  
Alexander Ney ◽  
Gabriele Canciani ◽  
J. Justin Hsuan ◽  
Stephen P. Pereira
Keyword(s):  
Ex Vivo ◽  
Neuroendocrine Differentiation ◽  
Genetically Engineered ◽  
Treatment Modalities ◽  
Ductal Adenocarcinoma ◽  
Disease Models ◽  
Tissue Remodelling ◽  
In Vivo Models ◽  
Original Tumour

Pancreatic neuroendocrine tumours (pNETs) are a heterogeneous group of epithelial tumours with neuroendocrine differentiation. Although rare (incidence of <1 in 100,000), they are the second most common group of pancreatic neoplasms after pancreatic ductal adenocarcinoma (PDAC). pNET incidence is however on the rise and patient outcomes, although variable, have been linked with 5-year survival rates as low as 40%. Improvement of diagnostic and treatment modalities strongly relies on disease models that reconstruct the disease ex vivo. A key constraint in pNET research, however, is the absence of human pNET models that accurately capture the original tumour phenotype. In attempts to more closely mimic the disease in its native environment, three-dimensional culture models as well as in vivo models, such as genetically engineered mouse models (GEMMs), have been developed. Despite adding significant contributions to our understanding of more complex biological processes associated with the development and progression of pNETs, factors such as ethical considerations and low rates of clinical translatability limit their use. Furthermore, a role for the site-specific extracellular matrix (ECM) in disease development and progression has become clear. Advances in tissue engineering have enabled the use of tissue constructs that are designed to establish disease ex vivo within a close to native ECM that can recapitulate tumour-associated tissue remodelling. Yet, such advanced models for studying pNETs remain underdeveloped. This review summarises the most clinically relevant disease models of pNETs currently used, as well as future directions for improved modelling of the disease.

Generation of Dendritic Cells after One-Hit Lentiviral Transduction of Hematopoietic Precursor Cells: Proof of Concept for the Human and Mouse Systems.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3448-3448
Author(s):  
Richard C. Koya ◽  
Nori Kasahara ◽  
Takahiro Kimura ◽  
Antoni Ribas ◽  
Renata Stripecke
Keyword(s):  
Tumor Antigens ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
High Titer ◽  
Lentiviral Vectors ◽  
Genetically Engineered ◽  
Interleukin 4 ◽  
Lentiviral Transduction ◽  
Gm Csf

Abstract Conventional, ex vivo culture of monocytes with recombinant proteins for their differentiation into DCs involves considerable manipulation under “Good Manufacturing Practices” conditions, and is not only more labor intensive but importantly, after ex vivo produced DCs are administered, they lack the stimulatory signals to keep them alive and functional and therefore are short lived. Because of these problems, we have evaluated an one-hit lentiviral transduction approach for genetically modifying monocytes in order to promote autocrine and paracrine production of factors required for their differentiation into immature DCs. High-titer third generation self-inactivating lentiviral vectors expressing granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) efficiently achieved simultaneous and persistent co-delivery of the transgenes into purified human CD14+ monocytes. Co-expression of GM-CSF and IL-4 in monocytes was sufficient to induce their differentiation into lentivirus-modified DCs (“DC/LVs”), as evidenced by their morphology, immunophenotype and immune-function*. Mixed lymphocyte reactions showed that the T-cell stimulating activity of DC/LVs was superior to that of DCs grown by conventional methods. DC/LVs displayed efficient antigen-specific, MHC Class-I restricted stimulation of autologous CD8+ T-cells, as shown by IFN-G production and CTL assays. Importantly, DC/LVs could be maintained metabolically active and viable in culture for 2–3 weeks in the absence of exogenously added growth factors, unlike conventional DCs *. We are now evaluating whether DC/LVs can be re-infused immediately after gene transfer to achieve stable and long-lasting differentiation in vivo. Additionally, the genetic engineering of monocytes is anticipated to generate DCs after one hit of lentiviral transduction, instead of the three consecutive steps for development of DCs (differentiation, maturation, gene delivery of tumor antigens). We have thus established a mouse model for testing DC/LVs in vivo for the treatment of melanoma. Bone marrow cells from C57BL/6 mice transduced with lentiviral vectors expressing GM-CSF and IL-4 recapitulated the same DC/LV morphology and immunophenotype obtained in the human system. Mouse DC/LVs were also more viable in vitro and outperformed conventional mouse DCs in pilot immunization assays as followed by CTL assays and IFN-G ELISPOT. We are currently evaluating the immunotherapeutic efficacy of DC/LVs injected into mice developing B16 melanoma tumors. Co-delivery of a gene for DC maturation (CD40L) and of gene encoding a tumor-associated antigens (MART-1) is being performed. Our goal is to evaluate the implications of simultaneous co-expression of GM-CSF/ IL-4/ CD40L/ MART-1 in DC/LV differentiation and migration to lymph nodes in vivo, immunopotency and safety. Once these pre-clinical considerations are addressed, we foresee a broad clinical application of genetically engineered DCs for vaccination purposes against cancer and chronic infectious diseases.

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