Evaluation of patients with organizing pneumonia in the post-COVID-19 period

Objective: This study aimed to retrospectively evaluate the cases of healthy adults, diagnosed with histologically confirmed organizing pneumonia and had shortness of breath and fatigue persist for at least three months in the post-COVID-19 period. Materials and Methods: Seventeen patients admitted to our hospital with shortness of breath and fatigue complaints between September 2020 and May 2021 were diagnosed with SARS-CoV-2 infection at least three months before the last admission and diagnosed with organizing pneumonia confirmed both radiologically and pathologically were included. Results: All of the patients were previously hospitalized due to COVID-19. The median time elapsed between the diagnosis of COVID-19 and recurrent hospitalization due to organizing pneumonia was four months. Our patients had bilateral lobular;subpleural densities vary from ground glass to the middle/lower lung consolidation, which is the typical radiological feature of organizing pneumonia. Partial lobular excision of the lung was performed in all patients. Intra-alveolar exudate, characterized by granulation tissue and fibroblast proliferation in the lung parenchyma, was detected in all patients. Conclusion: We believe that secondary organizing pneumonia should be considered among patients with COVID-19 pneumonia, especially in the case of persistent or recurrent respiratory symptoms and ongoing lung infiltrates. Thus, it will be possible to provide the proper medical approaches instead of unnecessary surgical intervention.

A review of fibroblast-like synoviocytes in the pathogenesis of Rheumatoid arthritis: Their activation and the inhibition of their apoptosis

Rheumatoid Arthritis (RA) is a systemic, autoimmune, inflammatory disease characterized by synovial hyperplasia, inflammatory cell infiltration in the synovial tissues, and progressive destruction of cartilage and bones. This disease often leads to chronic disability. More recently, activation of synovial fibroblasts (SFs) has been linked to innate immune responses and several cellular signalingpathways that ultimately result in the aggressive and invasive stages of RA. SFs are the major sources of pro-inflammatory cytokines in RA synovium. They participate in maintaining the inflammatory state that leads to synovial hyperplasia and angiogenesis in the inflamed synovium. The altered apoptotic response of synovial and inflammatory cells has been connected to these alterations of inflamed synovium. RA synovial fibroblasts (RASFs) have the ability to inhibit several apoptotic proteins that cause their abnormal proliferation. This proliferation leads to synovial hyperplasia. Apoptotic pathway proteins have thus been identified as possible targets for modifying the pathophysiology of RA. This review summarizes current knowledge of SF activation and its roles in the inhibition of apoptosis in the synovium, which is involved in joint damage during the effector phase of RA development.Copyright © 2022 Biomedpress.

The mast cell exosome-fibroblast connection: A novel pro-fibrotic pathway.

Introduction: In addition to the traditional activation of resident receptors by release of local mediators, new evidence favors the existence of exosomes in cell-to-cell communication that mediates delivery of specific cargo to modulate recipient cell function. We report that mast cell exosomes are an additional source of pro-fibrotic substances and constitute a unique pathway for the generation of excess collagen. Methods: We use primary human lung fibroblasts (HLFs) to demonstrate the uptake of labeled exosomes isolated from the human mast cell line HMC-1 (MC-EXOs), previously shown to contain protein cargo in common with human mast cell exosomes. Results: The MC-EXO uptake by HLF is to the cytosol and increases both proline hydroxylation in HLF lysate and secreted collagen, within 24 h, which is sustained over 72 h, the same time required for transforming growth factor-ß (TGF-ß) to activate collagen synthesis in the HLFs. Unlike TGF-ß, MC-EXO uptake does not induce fibrillar gene activation or invoke the Smad-nuclear transcription pathway. We show that MC-EXO uptake and TGF-ß have an additive effect on collagen synthesis in HLF and postulate that MC-EXO uptake by HLFs is a contributing factor to excess collagen synthesis and represents a unique paradigm for understanding fibrosis. Discussion: It is known that, in the lungs, mast cells are more activated and increase in number with inflammation, injury and viral infection associated with fibrosis. With the reported increased incidence of post-COVID-pulmonary fibrosis (PCPF), data from patients with severe COVID-19 are presented that show an increase in the mast cell number in lung parenchyma, the site of PCPF. Our findings provide a rationale for targeting multiple fibrogenic pathways in the management of lung fibrosis and the use of mast cell exosomes as a biomarker for the prognostic and diagnostic management of evolving fibrotic lung disease.

Optimization of Primary Human Bronchial Epithelial 3D Cell Culture with Donor-Matched Fibroblasts and Comparison of Two Different Culture Media.

In vitro airway models are increasingly important for pathomechanistic analyses of respiratory diseases. Existing models are limited in their validity by their incomplete cellular complexity. We therefore aimed to generate a more complex and meaningful three-dimensional (3D) airway model. Primary human bronchial epithelial cells (hbEC) were propagated in airway epithelial cell growth (AECG) or PneumaCult ExPlus medium. Generating 3D models, hbEC were airlifted and cultured on a collagen matrix with donor-matched bronchial fibroblasts for 21 days comparing two media (AECG or PneumaCult ALI (PC ALI)). 3D models were characterized by histology and immunofluorescence staining. The epithelial barrier function was quantified by transepithelial electrical resistance (TEER) measurements. The presence and function of ciliated epithelium were determined by Western blot and microscopy with high-speed camera. In 2D cultures, an increased number of cytokeratin 14-positive hbEC was present with AECG medium. In 3D models, AECG medium accounted for high proliferation, resulting in hypertrophic epithelium and fluctuating TEER values. Models cultured with PC ALI medium developed a functional ciliated epithelium with a stable epithelial barrier. Here, we established a 3D model with high in vivo-in vitro correlation, which has the potential to close the translational gap for investigations of the human respiratory epithelium in pharmacological, infectiological, and inflammatory research.

Safety Assessment of the Modified Lactoperoxidase System-In Vitro Studies on Human Gingival Fibroblasts.

One strategy in caries prevention is to inhibit the formation of cariogenic biofilms. Attempts are being made to develop oral hygiene products enriched with various antimicrobial agents. One of them is lactoperoxidase-an enzyme that can oxidise (pseudo)halide ions to reactive products with antimicrobial activity. Currently, commercially available products utilise thiocyanate as a substrate; however, several alternatives that are oxidised to products with greater antimicrobial potential have been found. In this study, toxicity against human gingival fibroblasts of the lactoperoxidase system was evaluated using four different (pseudo)halide substrate systems-thiocyanate, iodide, selenocyanate, and a mixture of thiocyanate and iodide. For this purpose, cells were treated with the systems and then apoptosis, cell cycle, intracellular glutathione concentration, and mitochondrial superoxide production were assessed. The results showed that each system, after generating 250 µM of the product, inhibited cell divisions, increased apoptosis, and increased the percentage of dead cells. It was concluded that the mechanism of the observed phenomena was not related to increased superoxide production or the depletion of glutathione concentration. These findings emphasised the need for the further in vitro and in vivo toxicity investigation of the modified lactoperoxidase system to assess its safety and the possibility of use in oral hygiene products.

Interleukin-17 and the COVID-19 cytokine storm: Cases reports

We report two cases of patients with COVID-19. Clinical and biological features of the two patients confirm severe form of COVID-19 associated with cytokine storm. High levels of IL-6 and IL-17 were found. Unfortunately the patients died because of the multi-organ failure secondary to the cytokine storm. Cytokine storm is a systemic inflammatory syndrome which leads to aberrant release of cytokines. IL-6 is the most frequently reported cytokine to be increased in COVID-19 patients. Naive T CD4+ cells in the presence of TGF ß and IL-6 will differentiate into T helper 17 cells responsible for secreting IL-17A and 17F, target macrophages, dendritic cells, endothelial cells, and fibroblasts to increase the production of cytokines. IL-6 and IL-17 have been shown to play a role in increasing risk of airway disease. They synergistically promote viral persistence by protecting virus-infected cells from apoptosis. Immune hyperactivation in cytokine storm amplified levels of cytokines that will have systemic effects and cause collateral damage to vital organ systems. Immunotherapy can play a crucial role in COVID-19 managing. Tocilizumab an anti-IL6 receptor antibody was used with clinical improvement. The possibility of inhibiting IL17 as therapy for COVID-19 should be also considered.

A review on post-COVID-19 pulmonary fibrosis and the role of nintedanib in its management

[...]the current evidence is limited and more evidence is awaited from the ongoing randomized clinical trials evaluating the safety and efficacy of nintedanib in the management of post-COVID-19 pulmonary fibrosis. Angiotensin II (1-10 amino acid long) has a number of profibrotic effects on lung parenchymal cells such as the induction of growth factors like TGF-ß1 for mesenchymal cells, ECM molecules, cytokines like IL-1, and increased motility of lung fibroblasts. [22] Antifibrotic Agent-Nintedanib Nintedanib, an indolinone derivative, also known by its developmental code BIBF1120, is a small molecule inhibitor of receptor tyrosine kinase of fibroblast growth factor receptor (FGFR)-1, platelet derived growth factor receptor (PDGFR)-a and ß, and vascular endothelial growth factor receptor (VEGFR-2). [29] Therefore, it appears that nintedanib by inhibiting the receptor tyrosine kinase of all the above mentioned growth factors is shown to have an effect in the treatment of idiopathic pulmonary fibrosis.

Complement Regulation in Immortalized Fibroblast-like Synoviocytes and Primary Human Endothelial Cells in Response to SARS-CoV-2 Nucleocapsid Protein and Pro-Inflammatory Cytokine TNFα.

Background: Case reports are available showing that patients develop symptoms of acute arthritis during or after recovery from SARS-CoV-2 infection. Since the interrelation is still unknown, our aim was to study the impact of the SARS-CoV-2 nucleocapsid protein (NP) on human fibroblast-like synoviocytes and human endothelial cells (hEC) in terms of complement and cytokine regulation. Methods: Non-arthritic (K4IM) synoviocyte, arthritic (HSE) synoviocyte cell lines and primary hEC were stimulated with recombinant NP and/or TNFα. Analyses of cell viability, proliferation, gene and protein expression of cytokines and complement factors were performed. Results: NP suppressed significantly the vitality of hEC and proliferation of HSE. NP alone did not induce any significant changes in the examined gene expressions. However, NP combined with TNFα induced significantly higher TNFα in HSE and K4IM as well as higher IL-6 and CD55 gene expression in HSE and suppressed C3aR1 gene expression in hEC. HSE proliferated twice as fast as K4IM, but showed significantly lesser gene expressions of CD46, CD55, CD59 and TNFα with significantly higher IL-6 gene expression. CD35 gene expression was undetectable in K4IM, HSE and hEC. Conclusions: NP might contribute in combination with other inflammatory factors to complement regulation in arthritis.

Sirt1 overexpression improves senescence-associated pulmonary fibrosis induced by vitamin D deficiency through downregulating IL-11 transcription.

Determining the mechanism of senescence-associated pulmonary fibrosis is crucial for designing more effective treatments for chronic lung diseases. This study aimed to determine the following: whether Sirt1 and serum vitamin D decreased with physiological aging, promoting senescence-associated pulmonary fibrosis by activating TGF-ß1/IL-11/MEK/ERK signaling, whether Sirt1 overexpression prevented TGF-ß1/IL-11/MEK/ERK signaling-mediated senescence-associated pulmonary fibrosis in vitamin D-deficient (Cyp27b1-/- ) mice, and whether Sirt1 downregulated IL-11 expression transcribed by TGF-ß1/Smad2 signaling through deacetylating histone at the IL-11 promoter in pulmonary fibroblasts. Bioinformatics analysis with RNA sequencing data from pulmonary fibroblasts of physiologically aged mice was conducted for correlation analysis. Lungs from young and physiologically aged wild-type (WT) mice were examined for cell senescence, fibrosis markers, and TGF-ß1/IL-11/MEK/ERK signaling proteins, and 1,25(OH)2 D3 and IL-11 levels were detected in serum. Nine-week-old WT, Sirt1 mesenchymal transgene (Sirt1Tg ), Cyp27b1-/- , and Sirt1Tg Cyp27b1-/- mice were observed the pulmonary function, aging, and senescence-associated secretory phenotype and TGF-ß1/IL-11/MEK/ERK signaling. We found that pulmonary Sirt1 and serum vitamin D decreased with physiological aging, activating TGF-ß1/IL-11/MEK/ERK signaling, and promoting senescence-associated pulmonary fibrosis. Sirt1 overexpression improved pulmonary dysfunction, aging, DNA damage, senescence-associated secretory phenotype, and fibrosis through downregulating TGF-ß1/IL-11/MEK/ERK signaling in Cyp27b1-/- mice. Sirt1 negatively regulated IL-11 expression through deacetylating H3K9/14ac mainly at the region from -871 to -724 of IL-11 promoter, also the major binding region of Smad2 which regulated IL-11 expression at the transcriptional level, and subsequently inhibiting TGF-ß1/IL-11/MEK/ERK signaling in pulmonary fibroblasts. This signaling in aging fibroblasts could be a therapeutic target for preventing senescence-associated pulmonary fibrosis induced by vitamin D deficiency.

Human Lung Fibroblasts Exhibit Induced Inflammation Memory via Increased IL6 Gene Expression and Release.

Fibroblasts of different origins are known to possess stromal memory after inflammatory episodes. However, there are no studies exploring human lung fibroblast memory which may predict a subsequent inflammatory response in chronic respiratory diseases and COVID-19. MRC-5 and HF19 human lung fibroblast cell lines were treated using different primary and secondary stimulus combinations: TNFα-WD-TNFα, Poly (I:C)-WD-TNFα, TNFα-WD-Poly (I:C), or LPS-WD-TNFα with a 24-h rest period (withdrawal period; WD) between the two 24-h stimulations. TLR3 and NF-κB inhibitors were used to determine pathways involved. The effect of SARS-Cov-2 spike protein to inflammatory response of lung fibroblasts was also investigated. mRNA expressions of genes and IL6 release were measured using qRT-PCR and ELISA, respectively. Statistical significance was determined by using one- or two-way ANOVA, followed by Bonferroni's post hoc analysis for comparison of multiple groups. Preexposure with Poly (I:C) significantly increased TNFα-induced IL6 gene expression and IL6 release in both cell lines, while it affected neither gene expressions of IL1B, IL2, IL8, and MMP8 nor fibrosis-related genes: ACTA2, COL1A1, POSTN, and TGFB1. Inhibition of TLR3 or NF-κB during primary stimulation significantly downregulated IL6 release. Simultaneous treatment of MRC-5 cells with SARS-CoV-2 spike protein further increased TNFα-induced IL6 release; however, preexposure to Poly (I:C) did not affect it. Human lung fibroblasts are capable of retaining inflammatory memory and showed an augmented response upon secondary exposure. These results may contribute to the possibility of training human lung fibroblasts to respond suitably on inflammatory episodes after viral infection.

The second decade of anti-TNF-a therapy in clinical practice: new lessons and future directions in the COVID-19 era.

Since the late 1990s, tumor necrosis factor alpha (TNF-α) inhibitors (anti-TNFs) have revolutionized the therapy of immune-mediated inflammatory diseases (IMIDs) affecting the gut, joints, skin and eyes. Although the therapeutic armamentarium in IMIDs is being constantly expanded, anti-TNFs remain the cornerstone of their treatment. During the second decade of their application in clinical practice, a large body of additional knowledge has accumulated regarding various aspects of anti-TNF-α therapy, whereas new indications have been added. Recent experimental studies have shown that anti-TNFs exert their beneficial effects not only by restoring aberrant TNF-mediated immune mechanisms, but also by de-activating pathogenic fibroblast-like mesenchymal cells. Real-world data on millions of patients further confirmed the remarkable efficacy of anti-TNFs. It is now clear that anti-TNFs alter the physical course of inflammatory arthritis and inflammatory bowel disease, leading to inhibition of local and systemic bone loss and to a decline in the number of surgeries for disease-related complications, while anti-TNFs improve morbidity and mortality, acting beneficially also on cardiovascular comorbidities. On the other hand, no new safety signals emerged, whereas anti-TNF-α safety in pregnancy and amid the COVID-19 pandemic was confirmed. The use of biosimilars was associated with cost reductions making anti-TNFs more widely available. Moreover, the current implementation of the "treat-to-target" approach and treatment de-escalation strategies of IMIDs were based on anti-TNFs. An intensive search to discover biomarkers to optimize response to anti-TNF-α treatment is currently ongoing. Finally, selective targeting of TNF-α receptors, new forms of anti-TNFs and combinations with other agents, are being tested in clinical trials and will probably expand the spectrum of TNF-α inhibition as a therapeutic strategy for IMIDs.

The “End Life” of the Grape Pomace Waste Become the New Beginning: The Development of a Virtuous Cycle for the Green Synthesis of Gold Nanoparticles and Removal of Emerging Contaminants from Water

During the last decades, the demand for processes developed according to the Circular Economy Principles has increased, searching for an alternative life for wastes. For this purpose, a one-pot green approach is exploited during this work to synthesize gold nanoparticles (AuNPs) by using grape pomace waste from Vitis vinifera. A raw aqueous extract of grape seeds, skin, and stems is used for AuNPs synthesis. UV-Vis, XPS, SEM, and ATR-FTIR spectroscopies demonstrate the main role of the extract’s polyphenolic components in stabilizing nanoparticles. XRD, DLS, and Zeta Potential analyses were used to characterize AuNPs. Moreover, the ionic strength, pH, and temperature role was investigated through the Surface Plasmon Resonance (SPR) band observation to assess AuNPs’ stability and photostability. For foreseeing the as-synthesized AuNPs’ potential use in cosmetic and biomedical fields as multifunctional platforms, their antioxidant, and skin-lightening properties were tested, together with their sunscreen ability. A preliminary in-vitro evaluation is reported about the AuNPs’ cytoprotective effects against H2O2 oxidative stress-induced in normal human dermal fibroblasts. Briefly, the possibility of reusing the grape pomace waste after the AuNPs synthesis as an adsorbent for the efficient removal of emergent contaminants is preliminarily discussed in the paper, further valorizing the use of waste according to a bio circular approach.

Disulfiram Exerts Antifibrotic and Anti-Inflammatory Therapeutic Effects on Perimysial Orbital Fibroblasts in Graves' Orbitopathy.

Fibrosis of extraocular muscles (EOMs) is a marker of end-stage in Graves' orbitopathy (GO). To determine the antifibrotic and anti-inflammatory therapeutic effects and the underlying molecular mechanisms of disulfiram (DSF) on perimysial orbital fibroblasts (pOFs) in a GO model in vitro, primary cultures of pOFs from eight patients with GO and six subjects without GO (NG) were established. CCK-8 and EdU assays, IF, qPCR, WB, three-dimensional collagen gel contraction assays, cell scratch experiments, and ELISAs were performed. After TGF-ß1 stimulation of pOFs, the proliferation rate of the GO group but not the NG group increased significantly. DSF dose-dependently inhibited the proliferation, contraction, and migration of pOFs in the GO group. Additionally, DSF dose-dependently inhibited fibrosis and extracellular matrix production markers (FN1, COL1A1, α-SMA, CTGF) at the mRNA and protein levels. Furthermore, DSF mediates antifibrotic effects on GO pOFs partially through the ERK-Snail signaling pathway. In addition, DSF attenuated HA production and suppressed inflammatory chemokine molecule expression induced by TGF-ß1 in GO pOFs. In this in vitro study, we demonstrate the inhibitory effect of DSF on pOFs fibrosis in GO, HA production, and inflammation. DSF may be a potential drug candidate for preventing and treating tissue fibrosis in GO.

Can Extracts from the Leaves and Fruits of the Cotoneaster Species Be Considered Promising Anti-Acne Agents?

This study aimed to evaluate the phenolic profile and biological activity of the extracts from the leaves and fruits of Cotoneaster nebrodensis and Cotoneaster roseus. Considering that miscellaneous species of Cotoneaster are thought to be healing in traditional Asian medicine, we assumed that this uninvestigated species may reveal significant therapeutic properties. Here, we report the simultaneous assessment of chemical composition as well as biological activities (antioxidant, anti-inflammatory, antibacterial, and cytotoxic properties) of tested species. Complementary LC-MS analysis revealed that polyphenols (especially flavonoids and proanthocyanidins) are the overriding phytochemicals with the greatest significance in tested biological activities. In vitro chemical tests considering biological activities revealed that obtained results showed different values depending on concentration, extraction solvent as well as phenolic content. Biological assays demonstrated that the investigated extracts possessed antibacterial properties and were not cytotoxic toward normal skin fibroblasts. Given the obtained results, we concluded that knowledge of the chemical composition and biological activities of investigated species are important to achieve a better understanding of the utilization of these plants in traditional medicine and be useful for further research in their application to treat various diseases, such as skin disorders.

Potential of Ferritin-Based Platforms for Tumor Immunotherapy.

Ferritin is an iron storage protein that plays a key role in iron homeostasis and cellular antioxidant activity. Ferritin has many advantages as a tumor immunotherapy platform, including a small particle size that allows for penetration into tumor-draining lymph nodes or tumor tissue, a unique structure consisting of 24 self-assembled subunits, cavities that can encapsulate drugs, natural targeting functions, and a modifiable outer surface. In this review, we summarize related research applying ferritin as a tumor immune vaccine or a nanocarrier for immunomodulator drugs based on different targeting mechanisms (including dendritic cells, tumor-associated macrophages, tumor-associated fibroblasts, and tumor cells). In addition, a ferritin-based tumor vaccine expected to protect against a wide range of coronaviruses by targeting multiple variants of SARS-CoV-2 has entered phase I clinical trials, and its efficacy is described in this review. Although ferritin is already on the road to transformation, there are still many difficulties to overcome. Therefore, three barriers (drug loading, modification sites, and animal models) are also discussed in this paper. Notwithstanding, the ferritin-based nanoplatform has great potential for tumor immunotherapy, with greater possibility of clinical transformation.

Combined administration of inhaled DNase, baricitinib and tocilizumab as rescue treatment in COVID-19 patients with severe respiratory failure.

Aiming to reduce mortality in COVID-19 with severe respiratory failure we administered a combined rescue treatment (COMBI) on top of standard-of-care (SOC: dexamethasone/heparin) consisted of inhaled DNase to dissolve thrombogenic neutrophil extracellular traps, plus agents against cytokine-mediated hyperinflammation, namely anti-IL-6-receptor tocilizumab and JAK1/2 inhibitor baricitinib. Patients with PaO2/FiO2 < 100 mmHg were analysed. COMBI group (n = 22) was compared with similar groups that had received SOC alone (n = 26) or SOC plus monotherapy with either IL-1-receptor antagonist anakinra (n = 19) or tocilizumab (n = 11). COMBI was significantly associated with lower in-hospital mortality and intubation rate, shorter duration of hospitalization, and prolonged overall survival after a median follow-up of 110 days. In vitro, COVID-19 plasma induced tissue factor/thrombin pathway in primary lung fibroblasts. This effect was inhibited by the immunomodulatory agents of COMBI providing a mechanistic explanation for the clinical observations. These results support the conduct of randomized trials using combined immunomodulation in COVID-19 to target multiple interconnected pathways of immunothrombosis.

Patomorphology of the liver in pregnant with Coronavirus infection

Researchers have shown that SARS-CoV and MERS-CoV can also cause liver damage in an infected organism, but the mechanisms of injury are poorly understood. In this study, pathomorphological changes in the liver during coronavirus infection in pregnant women were studied. As material, the liver was studied at autopsy of maternal mortality from 33 coronaviruses conducted at RCPA (Republican Center for Pathological Anatomy) between 2020 and 2021. Morphological examinations of liver tissue showed that the development of various pathomorphological changes in the liver was also observed depending on the periods of coronavirus infection. In the exudative period of the coronavirus is observed a strong process of circulating in the liver, swelling, destruction and bleeding of interstitial tissue, the development of protein and hydropic dystrophy in the liver parenchyma, i.e. hepatocytes. In the second proliferative inflammatory period of the disease, there is an increase in lymphoid infiltration along the portal pathways of the liver, myxomatous metaplasia of Kupffer cells, proliferation and proliferation of fibroblasts, growth of connective tissue, portal pathways of fibrous structures, periphery and even sinusoidal wall.

Targeting Fibroblast Growth Factor Receptors in Preclinical Patient-Derived Xenograft Models of CRPC

In this reporting period, the didactic coursework and training opportunities that were proposed for this project were completed. However, significant disruptions to research activities were encountered due to the ongoing COVID-19 pandemic response and ACURO approval delays. These factors led to the application and approval of a no cost extension to complete all proposed work. Nevertheless, all study approvals (IACUC, ACURO, HRPO) have been obtained and experiments using fibroblast growth factor receptor and androgen receptor inhibitors inpatient derived xenograft models are underway. The approved project objectives and scope will not change and the work to be completed in the extension period will include all experiments and analyses related to Specific Aim 1.

Evaluation of in vitro human skin models for studying effects of external stressors and stimuli and developing treatment modalities

Skin is exposed to a variety of potential stressors and stimulators that may impact homeostasis, healing, tumor development, inflammation, and irritation. As such it is important to understand the impact that these stimuli have on skin health and function, and to develop therapeutic interventions. Animal experiments have been the gold standard for testing the safety and efficacy of therapeutics and observing disease pathology for centuries. However, complex ethics, costs, time consumption, and interspecies variation limit the transferability of results to humans and reduce their repeatability and reliability. Furthermore, traditional 2D cell studies are not representative of human tissue. Skin tissue is a dynamic environment, and when cells are isolated in unphysiologically stiff, static petri dishes their behavior, and phenotypic expression is altered. Increasingly complex in vitro models of human skin, including organoids, 3D bioprinting, and skin‐on‐a‐chip platforms, present the opportunity to gain insight into how stressors affect tissue at a cellular level in a controlled and repeatable environment. This insight can be leveraged to further understand pathological skin conditions and better formulate and validate drugs and therapeutics. Here, we will discuss the application of in vitro skin modeling to investigating the effects of mechanical, electromagnetic, and chemical stressors on skin.

Countermeasures against COVID-19 from the viewpoints of dental diseases: strengthening innate immunity with the toothpick method of brushing. (Special Issue: COVID-19 and historical pandemics part 2.)

Coronavirus disease (COVID-19) patients with periodontal disease have an 8.8-fold higher mortality rate than those in the patients without periodontal diseases. This was higher than the odds ratio for patients with diabetes. Periodontal disease is associated with ulcers in the periodontal pocket, and gram-negative bacteria called periodontal pathogens invade the tissue through ulcers. Bacteria in the ulcer site are phagocytosed and sterilized by leukocytes. Following the autolysis of leukocytes, lipopolysaccharides (LPS) on the bacterial cell wall spread throughout the body, which is a major cause of multiple organ failure. Thus, periodontal disease is considered to contribute to the mortality rate of COVID-19. Ulcers in the periodontal pocket can be repaired using by a new developed brushing method called the toothpick method. The toothpick method can significantly improve gingival bleeding in one week, which is quicker than conventional periodontal treatment methods. Mechanical stimulation by the toothbrush causes gingival basal cells, fibroblasts, vascular endothelial cells and osteoblasts to proliferate and repair the tissue. However, these cell proliferations cease to occur 0.5 mm away from where the toothbrush bristles make contact with the gingiva. The toothpick method of brushing is characterized by its ability to stimulate the interdental gingiva, which is the initial site of periodontitis. As the toothpick method can repair periodontal ulcers, it will strengthen biological defense mechanisms against chronic degenerative and infectious diseases.