Human birth tissue products as a non-opioid medicine to inhibit post-surgical pain.

Pain after surgery causes significant suffering. Opioid analgesics cause severe side effects and accidental death. Therefore, there is an urgent need to develop non-opioid therapies for managing post-surgical pain. Local application of Clarix Flo (FLO), a human amniotic membrane (AM) product, attenuated established post-surgical pain hypersensitivity without exhibiting known side effects of opioid use in mice. This effect was achieved through direct inhibition of nociceptive dorsal root ganglion (DRG) neurons via CD44-dependent pathways. We further purified the major matrix component, the heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) from human AM that has greater purity and water solubility than FLO. HC-HA/PTX3 replicated FLO-induced neuronal and pain inhibition. Mechanistically, HC-HA/PTX3 induced cytoskeleton rearrangements to inhibit sodium current and high-voltage activated calcium current on nociceptive neurons, suggesting it is a key bioactive component mediating pain relief. Collectively, our findings highlight the potential of naturally derived biologics from human birth tissues as an effective non-opioid treatment for post-surgical pain and unravel the underlying mechanisms.

PACAP activates MRGPRX2 on meningeal mast cells to drive migraine-like pain.

Migraine ranks among the most prevalent disorders worldwide, leading to disability and decreased quality of life in patients. Recently, neurogenic inflammation has been recognized as a potential underlying pathology contributing to the migraine pain pathway. Mast cells reside in the meninges and have been implicated in contributing to the pathophysiology of migraine. Here we report for the first time that the mouse Mas-Related G-protein-coupled Receptor B2 (MrgprB2), is expressed on meningeal connective tissue mast cells and contributes to Pituitary Adenylate Cyclase Activating Peptide (PACAP)-induced migraine-like pain behavior. We also found that PACAP was able to dose-dependently lead to enzyme release from human mast cells via activation of MRGPRX2; the human homolog of MrgprB2. Using a transgenic MRGPRX2 mouse, we observed significant increases in PACAP-induced migraine-like pain behavior in MRGPRX2+ mice vs mice lacking the receptor. These results reveal both MrgprB2 and MRGPRX2 as important contributors to neuropeptide-induced migraine pain.

Biological screening of a unique drug library targeting MRGPRX2.

Background: Allergic drug reaction or drug allergy is an immunologically mediated drug hypersensitivity reaction (DHR). G-protein coupled receptors (GPCRs) are common drug targets and communicate extracellular signals that initiate cellular responses. Recent evidence shows that GPCR MRGPRX2 is of major importance in IgE-independent pseudo-allergic DHRs based on the suspected interactions between many FDA-approved peptidergic compounds and MRGPRX2. Objective: Our aim was to uncover novel MRGPRX2-selective and -potent agonists as drug candidates responsible for clinical features of pseudo-allergic DHRs. Methods: We conducted a primary high-throughput screening (HTS), coupled with mutagenesis targeting the MRGPRX2 N62S mutation, on a panel of 3,456 library compounds. We discovered pharmacologically active hit compounds as agonists of the MRGPRX2 protein according to high degrees of potency evaluated by the calcium response and validated by the degranulation assay. Using the molecular tool Forge, we also characterized the structure-activity relationship shared by identified hit compounds. Results: The alternative allele of single nucleotide polymorphism rs10833049 (N62S) in MRGPRX2 demonstrated loss-of-function property in response to substance P and antineoplastic agent daunorubicin hydrochloride. We applied a unique assay system targeting the N62S mutation to the HTS and identified 84 MRGPRX2-selective active hit compounds representing diverse classes according to primary drug indications. The top five highly represented groups included fluoroquinolone and non-fluoroquinolone antibiotics; antidepressive/antipsychotic; antihistaminic and antineoplastic agents. We classified hit compounds into 14 clusters representing a variety of chemical and drug classes beyond those reported, such as opioids, neuromuscular blocking agents, and fluoroquinolones. We further demonstrated MRGPRX2-dependent degranulation in the human mast cell line LAD2 cells induced by three novel agonists representing the non-fluoroquinolone antibiotics (bacitracin A), anti-allergic agents (brompheniramine maleate) and tyrosine-kinase inhibitors (imatinib mesylate). Conclusion: Our findings could facilitate the development of interventions for personalized prevention and treatment of DHRs, as well as future pharmacogenetic investigations of MRGPRX2 in relevant disease cohorts.

The odorant receptor OR2W3 on airway smooth muscle evokes bronchodilation via a cooperative chemosensory tradeoff between TMEM16A and CFTR.

The recent discovery of sensory (tastant and odorant) G protein-coupled receptors on the smooth muscle of human bronchi suggests unappreciated therapeutic targets in the management of obstructive lung diseases. Here we have characterized the effects of a wide range of volatile odorants on the contractile state of airway smooth muscle (ASM) and uncovered a complex mechanism of odorant-evoked signaling properties that regulate excitation-contraction (E-C) coupling in human ASM cells. Initial studies established multiple odorous molecules capable of increasing intracellular calcium ([Ca2+]i) in ASM cells, some of which were (paradoxically) associated with ASM relaxation. Subsequent studies showed a terpenoid molecule (nerol)-stimulated OR2W3 caused increases in [Ca2+]i and relaxation of ASM cells. Of note, OR2W3-evoked [Ca2+]i mobilization and ASM relaxation required Ca2+ flux through the store-operated calcium entry (SOCE) pathway and accompanied plasma membrane depolarization. This chemosensory odorant receptor response was not mediated by adenylyl cyclase (AC)/cyclic nucleotide-gated (CNG) channels or by protein kinase A (PKA) activity. Instead, ASM olfactory responses to the monoterpene nerol were predominated by the activity of Ca2+-activated chloride channels (TMEM16A), including the cystic fibrosis transmembrane conductance regulator (CFTR) expressed on endo(sarco)plasmic reticulum. These findings demonstrate compartmentalization of Ca2+ signals dictates the odorant receptor OR2W3-induced ASM relaxation and identify a previously unrecognized E-C coupling mechanism that could be exploited in the development of therapeutics to treat obstructive lung diseases.

Osteoclast-derived apoptotic bodies show extended biological effects of parental cell in promoting bone defect healing.

Apoptotic bodies (ABs) traditionally considered as garbage bags that enclose residual components of dead cells are gaining increasing attentions due to their potential roles in intercellular communications. In bone turn over, at the end of bone resorption phase, most osteoclasts undergo apoptosis, generating large amounts of ABs. However, it remains unclear of the role of osteoclast-derived ABs in bone remodeling. Methods: Staurosporine (STS) was used to apoptotic induction and differential centrifugation was used to isolate ABs. Western blotting, flowcytometry and Transmission electron microscopy (TEM) were performed for ABs identification, while whole transcriptome of ABs from osteoclasts at different stages was detected by RNA-seq. VENN analysis and gene set enrichment analysis (GSEA) were performed to compare the profile similarities between ABs and parental cells. In vitro efficacy of ABs on angiogenesis and osteogenesis were evaluated by tube formation assay and ALP staining. In vivo, calvarial defect mice model was used to assess the effects of ABs-modified decalcified bone matrix (DBM) scaffolds on angiogenesis and osteogenesis. Results: Here we mapped the whole transcriptome paralleled with small RNA profiling of osteoclast derived ABs at distinct differentiation stages. Whole transcriptome analysis revealed significant differences in RNA signatures among the ABs generated from osteoclasts at different stages. By comparing with parental osteoclast RNA profiles, we found that the transcriptome of ABs exhibited high similarities with the corresponding parental cells. Functionally, in vitro and in vivo studies showed that similar with the parental cells, pOC-ABs potentiated endothelial progenitor cell proliferation and differentiation, whereas mOC-ABs promoted osteogenic differentiation. The inherited biological effects of ABs were shown mediated by several enriched lncRNAs of which the interference abolished AB functions. Conclusions: Our study revealed the total RNA profiles of osteoclast derived ABs and demonstrated their biological functions. Both gene set and functional analysis indicated that osteoclast derived ABs are biologically similar with the parental cells suggesting their bridging role in osteoclast-osteoblast coupling in bone remodeling.

Progesterone-Based Therapy Protects Against Influenza by Promoting Lung Repair and Recovery in Females.

Over 100 million women use progesterone therapies worldwide. Despite having immunomodulatory and repair properties, their effects on the outcome of viral diseases outside of the reproductive tract have not been evaluated. Administration of exogenous progesterone (at concentrations that mimic the luteal phase) to progesterone-depleted adult female mice conferred protection from both lethal and sublethal influenza A virus (IAV) infection. Progesterone treatment altered the inflammatory environment of the lungs, but had no effects on viral load. Progesterone treatment promoted faster recovery by increasing TGF-ß, IL-6, IL-22, numbers of regulatory Th17 cells expressing CD39, and cellular proliferation, reducing protein leakage into the airway, improving pulmonary function, and upregulating the epidermal growth factor amphiregulin (AREG) in the lungs. Administration of rAREG to progesterone-depleted females promoted pulmonary repair and improved the outcome of IAV infection. Progesterone-treatment of AREG-deficient females could not restore protection, indicating that progesterone-mediated induction of AREG caused repair in the lungs and accelerated recovery from IAV infection. Repair and production of AREG by damaged respiratory epithelial cell cultures in vitro was increased by progesterone. Our results illustrate that progesterone is a critical host factor mediating production of AREG by epithelial cells and pulmonary tissue repair following infection, which has important implications for women's health.

Recruited monocytes modulate malaria-induced lung injury through CD36-mediated clearance of sequestered infected erythrocytes.

Pulmonary complications occur in a significant percentage of adults and children during the course of severe malaria. The cellular and molecular innate immune mechanisms that limit the extent of pulmonary inflammation and preserve lung function during severe Plasmodium infections remain unclear. In particular, the contributions to pulmonary complications by parasitized erythrocyte sequestration and subsequent clearance from the lung microvasculature by immune cells have not been clearly defined. We used the Plasmodium berghei ANKA-C57BL/6 mouse model of severe malaria to investigate the mechanisms governing the nature and extent of malaria-associated lung injury. We have demonstrated that sequestration of infected erythrocytes on postcapillary endothelial surfaces results in acute lung injury and the rapid recruitment of CCR2(+)CD11b(+)Ly6C(hi) monocytes from the circulation. These recruited cells remain in the lungs as monocyte-derived macrophages and are instrumental in the phagocytic clearance of adherent Plasmodium berghei-infected erythrocytes. In contrast, alveolar macrophages do not play a significant role in the clearance of malaria-infected cells. Furthermore, the results obtained from Ccr2(-/-), Cd36(-/-), and CD36 bone marrow chimeric mice showed that sequestration in the absence of CD36-mediated phagocytic clearance by monocytes leads to exaggerated lung pathologic features. In summary, our data indicate that the intensity of malaria-induced lung pathologic features is proportional to the steady-state levels of Plasmodium-infected erythrocytes adhering to the pulmonary vasculature. Moreover, the present work has defined a major role of recruited monocytes in clearing infected erythrocytes from the pulmonary interstitium, thus minimizing lung damage.

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research.

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer research. Although there are a number of methods that have been published showing how to intubate mice for pulmonary function measurements, none are without potential problems for rapid tracheal instillation in large cohorts of mice. In the present paper, a simple and quick method is described that enables an investigator to carry out such instillations in an efficient manner. The method does not require any special tools or lighting and can be learned with very little practice. It involves anesthetizing a mouse, making a small incision in the neck to visualize the trachea, and then inserting an intravenous catheter directly. The small incision is quickly closed with tissue adhesive, and the mice are allowed to recover. A skilled student or technician can do instillations at an average rate of 2 min/mouse. Once the cancer is established, there is frequently a need for quantitative histologic analysis of the lungs. Traditionally pathologists usually do not bother to standardize lung inflation during fixation, and analyses are often based on a scoring system that can be quite subjective. While this may sometime be sufficiently adequate for gross estimates of the size of a lung tumor, any proper stereological quantification of lung structure or cells requires a reproducible fixation procedure and subsequent lung volume measurement. Here we describe simple reliable procedures for both fixing the lungs under pressure and then accurately measuring the fixed lung volume. The only requirement is a laboratory balance that is accurate over a range of 1 mg-300 g. The procedures presented here thus could greatly improve the ability to create, treat, and analyze lung cancers in mice.

Experimental progressive emphysema in BALB/cJ mice as a model for chronic alveolar destruction in humans.

Emphysema, one of the major components of chronic obstructive pulmonary disease (COPD), is characterized by the progressive and irreversible loss of alveolar lung tissue. Even though >80% of COPD cases are associated with cigarette smoking, only a relatively small proportion of smokers develop emphysema, suggesting a potential role for genetic factors in determining individual susceptibility to emphysema. Although strain-dependent effects have been shown in animal models of emphysema, the molecular basis underlying this intrinsic susceptibility is not fully understood. In this present study, we investigated emphysema development using the elastase-induced experimental emphysema model in two commonly used mouse strains, C57BL/6J and BALB/cJ. The results demonstrate that mice with different genetic backgrounds show disparate susceptibility to the development of emphysema. BALB/cJ mice were found to be much more sensitive than C57BL/6J to elastase injury in both a dose-dependent and time-dependent manner, as measured by significantly higher mortality, greater body weight loss, greater decline in lung function, and a greater loss of alveolar tissue. The more susceptible BALB/cJ strain also showed the persistence of inflammatory cells in the lung, especially macrophages and lymphocytes. A comparative gene expression analysis following elastase-induced injury showed BALB/cJ mice had elevated levels of il17A mRNA and a number of classically (M1) and alternatively (M2) activated macrophage genes, whereas the C57BL/6J mice demonstrated augmented levels of interferon-γ. These findings suggest a possible role for these cellular and molecular mediators in modulating the severity of emphysema and highlight the possibility that they might contribute to the heterogeneity observed in clinical emphysema outcomes.

Telomere dysfunction causes alveolar stem cell failure.

Telomere syndromes have their most common manifestation in lung disease that is recognized as idiopathic pulmonary fibrosis and emphysema. In both conditions, there is loss of alveolar integrity, but the underlying mechanisms are not known. We tested the capacity of alveolar epithelial and stromal cells from mice with short telomeres to support alveolar organoid colony formation and found that type 2 alveolar epithelial cells (AEC2s), the stem cell-containing population, were limiting. When telomere dysfunction was induced in adult AEC2s by conditional deletion of the shelterin component telomeric repeat-binding factor 2, cells survived but remained dormant and showed all the hallmarks of cellular senescence. Telomere dysfunction in AEC2s triggered an immune response, and this was associated with AEC2-derived up-regulation of cytokine signaling pathways that are known to provoke inflammation in the lung. Mice uniformly died after challenge with bleomycin, underscoring an essential role for telomere function in AEC2s for alveolar repair. Our data show that alveoloar progenitor senescence is sufficient to recapitulate the regenerative defects, inflammatory responses, and susceptibility to injury that are characteristic of telomere-mediated lung disease. They suggest alveolar stem cell failure is a driver of telomere-mediated lung disease and that efforts to reverse it may be clinically beneficial.

Puerarin exhibits weak estrogenic activity in female rats.

A weak estrogenicity of puerarin on reproductive organs was addressed in female rats. In short-term treatment, immature ovariectomized rats were injected with 0.7 mg/kg BW/day of puerarin, for 14 days. Puerarin did not increase uterus weights, endometrium and myometrium areas, and the percent of cornified cells (%Co), but it increased the number of uterine glands. In long-term treatment, mature rats were injected with 7.0mg/kg BW/day of puerarin for 140 days. Puerarin did not increase uterus weights, endometrium and myometrium areas, and the number of uterine glands, but a significant increase in the %Co was observed from day 98 onwards.