scholarly journals Optical Detection of Distal Lung Enzyme Activity in Human Inflammatory Lung Disease

BME Frontiers ◽  
2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Alicia Megia-Fernandez ◽  
Adam Marshall ◽  
Ahsan R. Akram ◽  
Bethany Mills ◽  
Sunay V. Chankeshwara ◽  
...  

Objective and Impact Statement. There is a need to develop platforms delineating inflammatory biology of the distal human lung. We describe a platform technology approach to detect in situ enzyme activity and observe drug inhibition in the distal human lung using a combination of matrix metalloproteinase (MMP) optical reporters, fibered confocal fluorescence microscopy (FCFM), and a bespoke delivery device. Introduction. The development of new therapeutic agents is hindered by the lack of in vivo in situ experimental methodologies that can rapidly evaluate the biological activity or drug-target engagement in patients. Methods. We optimised a novel highly quenched optical molecular reporter of enzyme activity (FIB One) and developed a translational pathway for in-human assessment. Results. We demonstrate the specificity for matrix metalloproteases (MMPs) 2, 9, and 13 and probe dequenching within physiological levels of MMPs and feasibility of imaging within whole lung models in preclinical settings. Subsequently, in a first-in-human exploratory experimental medicine study of patients with fibroproliferative lung disease, we demonstrate, through FCFM, the MMP activity in the alveolar space measured through FIB One fluorescence increase (with pharmacological inhibition). Conclusion. This translational in situ approach enables a new methodology to demonstrate active drug target effects of the distal lung and consequently may inform therapeutic drug development pathways.

Author(s):  
Ameen A. Salahudeen ◽  
Shannon S. Choi ◽  
Arjun Rustagi ◽  
Junjie Zhu ◽  
Sean M. de la O ◽  
...  

ABSTRACTThe distal lung contains terminal bronchioles and alveoli that facilitate gas exchange and is affected by disorders including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. Investigations of these localized pathologies have been hindered by a lack of 3D in vitro human distal lung culture systems. Further, human distal lung stem cell identification has been impaired by quiescence, anatomic divergence from mouse and lack of lineage tracing and clonogenic culture. Here, we developed robust feeder-free, chemically-defined culture of distal human lung progenitors as organoids derived clonally from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids exhibited AT1 transdifferentiation potential, while basal cell organoids progressively developed lumens lined by differentiated club and ciliated cells. Organoids consisting solely of club cells were not observed. Upon single cell RNA-sequencing (scRNA-seq), alveolar organoids were composed of proliferative AT2 cells; however, basal organoid KRT5+ cells contained a distinct ITGA6+ITGB4+ mitotic population whose proliferation segregated to a TNFRSF12Ahi subfraction. Clonogenic organoid growth was markedly enriched within the TNFRSF12Ahi subset of FACS-purified ITGA6+ITGB4+ basal cells from human lung or derivative organoids. In vivo, TNFRSF12A+ cells comprised ~10% of KRT5+ basal cells and resided in clusters within terminal bronchioles. To model COVID-19 distal lung disease, we everted the polarity of basal and alveolar organoids to rapidly relocate differentiated club and ciliated cells from the organoid lumen to the exterior surface, thus displaying the SARS-CoV-2 receptor ACE2 on the outwardly-facing apical aspect. Accordingly, basal and AT2 “apical-out” organoids were infected by SARS-CoV-2, identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung alveolar and basal stem cells, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and exemplifies progenitor identification within a slowly proliferating human tissue. Further, our studies establish a facile in vitro organoid model for human distal lung infectious diseases including COVID-19-associated pneumonia.


2008 ◽  
Vol 5 (2) ◽  
pp. 139-147 ◽  
Author(s):  
D. Hüttenberger ◽  
T. Gabrecht ◽  
G. Wagnières ◽  
B. Weber ◽  
A. Linder ◽  
...  
Keyword(s):  

Author(s):  
John J. Godleski ◽  
Rebecca C. Stearns ◽  
Marshall Katler ◽  
Robyn Rufner ◽  
Theresa D. Sweeney ◽  
...  

We have previously reported methods to cryofix the extracellular lining layers of the large airways and trachea. We seek to study the extracellular lining layers of both airways and alveoli in situ because it is the interaction of these lining materials with inhaled particles that determines the subsequent responses of phagocytic cells to the particles. Cryopreservation of the lining fluids of lung parenchyma is difficult because of the complexity of distal lung structure, and the need to fix the lung in its natural state, i.e., filled with air. Any method used must be able to obtain the frozen specimen from the inflated lung. This requirement precludes the possibility of having a cryogen come into primary contact with the internal surface of an alveolus. However, if optimal fixation through the pleura could be attained, then the lining layers of subjacent alveoli could be studied. In this report, we describe a new method to cryopreserve distal lung tissue for optimal study of the extracellular lining layers of the alveolus. Alveolar tissue is sufficiently preserved in the inflated state to maintain its in vivo characteristics.


2021 ◽  
Author(s):  
Moataz Dowaidar

As many medications are administered jointly, they often give larger benefits, counteract disadvantages, and enhance treatment results compared to monotherapy. Whether natural or synthetic, injectable biomaterials can form degradable networks in situ, decreasing patient pain and cost while presenting new and promising possibilities for minimally invasive surgery. Biomaterials' ability to create and manufacture injectable systems is strongly impacted by their physicochemical and mechanical properties. The design and manufacture of injectable systems containing cells, therapeutic molecules, particles, and biomolecules that can be injected into geometrically complex body tissue regions poses a significant challenge as they must ensure drug/biomolecule/material bioactivity, cell survival and retention. Hydrogels are a promising choice in this case given their amazing ability to manipulate, encapsulate and co-deliver pharmaceutical chemicals, cells, biomolecules, and nanomaterials. Hydrogels can alter their mechanical and deteriorating qualities by adjusting the cross-linking technique and chemical composition. The ability to modify IH's mechanical strength permits co-encapsulation of medicinal compounds, cells, nanomaterials, and growth factors in the matrix in situ, allowing for multimodal synergistic therapies.To boost the prospects of translating IHs into normal clinics, various barriers and outstanding scientific issues must be tackled in the future. Future investigations, including the application of IHs in multimodal synergistic treatment, should start with large animal models such as monkeys and dogs or even ex vivo human tissue models. In addition, the period of in vivo evaluations should be prolonged from weeks to months for trustworthy and accurate data to be translated to clinical trials. On the one hand, the toxicity of certain crosslinking agents used in IH synthesis must be considered, as the residues will cause unwanted in vivo reactions.Toxic crosslinkers, on the other hand, may interact with therapeutic molecules/biomolecules or nanomaterials trapped in the hydrogel matrix, causing loss of bioactivity. Similarly, IHs' sol–gel transition is a vital issue requiring much investigation. A quick sol–gel transition of precursor solutions might cause the fluid to be caught in the needle, whereas high-viscosity precursor solutions need high injection force, resulting in physician hand fatigue and patient annoyance. Other concerns for clinical IH translation include fast release and rate of degradation. Degradation rate is critical in controlling therapeutic drug release and tissue regeneration. Fast hydrogel breakdown may trigger early inflammatory reaction due to breakdown products, whereas delayed degradation may result in insufficient release of therapeutic drugs. Changing the composition, structure, and crystallinity of polymers must be employed to customize the breakdown rate. Expert researchers will be better equipped to tackle these challenges if they have a deeper knowledge of polymers' physiochemical features. Overall, future IH design should focus on building simple, well-defined 3D networks with low toxicity, high biodegradation rate, and acceptable functionality.


2020 ◽  
Vol 319 (4) ◽  
pp. C675-C693
Author(s):  
Kalpaj R. Parekh ◽  
Janna Nawroth ◽  
Albert Pai ◽  
Shana M. Busch ◽  
Christiana N. Senger ◽  
...  

The ability to replace defective cells in an airway with cells that can engraft, integrate, and restore a functional epithelium could potentially cure a number of lung diseases. Progress toward the development of strategies to regenerate the adult lung by either in vivo or ex vivo targeting of endogenous stem cells or pluripotent stem cell derivatives is limited by our fundamental lack of understanding of the mechanisms controlling human lung development, the precise identity and function of human lung stem and progenitor cell types, and the genetic and epigenetic control of human lung fate. In this review, we intend to discuss the known stem/progenitor cell populations, their relative differences between rodents and humans, their roles in chronic lung disease, and their therapeutic prospects. Additionally, we highlight the recent breakthroughs that have increased our understanding of these cell types. These advancements include novel lineage-traced animal models and single-cell RNA sequencing of human airway cells, which have provided critical information on the stem cell subtypes, transition states, identifying cell markers, and intricate pathways that commit a stem cell to differentiate or to maintain plasticity. As our capacity to model the human lung evolves, so will our understanding of lung regeneration and our ability to target endogenous stem cells as a therapeutic approach for lung disease.


2007 ◽  
Vol 102 (5) ◽  
pp. 1969-1975 ◽  
Author(s):  
Emma H. Baker ◽  
Nicholas Clark ◽  
Amanda L. Brennan ◽  
Donald A. Fisher ◽  
Khin M. Gyi ◽  
...  

In animals, glucose concentrations are 3–20 times lower in lung lining fluid than in plasma. In humans, glucose concentrations are normally low (<1 mmol/l) in nasal and bronchial fluid, but they are elevated by inflammation or hyperglycemia. Furthermore, elevated bronchial glucose is associated with increased respiratory infection in intensive care patients. Our aims were to estimate normal glucose concentrations in fluid from distal human lung sampled noninvasively and to determine effects of hyperglycemia and lung disease on lung glucose concentrations. Respiratory fluid was sampled as exhaled breath condensate, and glucose was measured by chromatography with pulsed amperometric detection. Dilution corrections, based on conductivity, were applied to estimate respiratory fluid glucose concentrations (breath glucose). We found that breath glucose in healthy volunteers was 0.40 mmol/l (SD 0.24), reproducible, and unaffected by changes in salivary glucose. Breath-to-blood glucose ratio (BBGR) was 0.08 (SD 0.05). Breath glucose increased during experimental hyperglycemia ( P < 0.05) and was elevated in diabetic patients without lung disease [1.20 mmol/l (SD 0.69)] in proportion to hyperglycemia [BBGR 0.09 (SD 0.06)]. Breath glucose was elevated more than expected for blood glucose in cystic fibrosis patients [breath 2.04 mmol/l (SD 1.14), BBGR 0.29 (SD 0.17)] and in cystic fibrosis-related diabetes [breath 4.00 mmol/l (SD 2.07), BBGR 0.54 (0.28); P < 0.0001]. These data indicate that 1) this method makes a biologically plausible estimate of respiratory fluid glucose concentration, 2) respiratory fluid glucose concentrations are elevated by hyperglycemia and lung disease, and 3) effects of hyperglycemia and lung disease can be distinguished using the BBGR. This method will support future in vivo investigation of the cause and effect of elevated respiratory fluid glucose in human lung disease.


Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.


Author(s):  
Greg V. Martin ◽  
Ann L. Hubbard

The microtubule (MT) cytoskeleton is necessary for many of the polarized functions of hepatocytes. Among the functions dependent on the MT-based cytoskeleton are polarized secretion of proteins, delivery of endocytosed material to lysosomes, and transcytosis of integral plasma membrane (PM) proteins. Although microtubules have been shown to be crucial to the establishment and maintenance of functional and structural polarization in the hepatocyte, little is known about the architecture of the hepatocyte MT cytoskeleton in vivo, particularly with regard to its relationship to PM domains and membranous organelles. Using an in situ extraction technique that preserves both microtubules and cellular membranes, we have developed a protocol for immunofluorescent co-localization of cytoskeletal elements and integral membrane proteins within 20 µm cryosections of fixed rat liver. Computer-aided 3D reconstruction of multi-spectral confocal microscope images was used to visualize the spatial relationships among the MT cytoskeleton, PM domains and intracellular organelles.


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