Early Inflammatory Phase Research Articles

#4976 DIFFERENTIAL ROLES OF REGNASE3 IN RESIDENT MACROPHAGE VERSUS TUBULAR EPITHELIAL CELLS IN THE HEALTHY OR POSTISCHEMIC KIDNEY

Abstract Background and Aims Acute kidney injury (AKI) is a prevalent yet severe condition that occurs in clinical settings. The prognosis for AKI is poor, with many patients progressing to AKD, CKD, and even ESKD especially in those who were admitted to the ICU. RNA-binding proteins (RBPs) are a class of proteins that play a vital role in regulating gene expression and have been implicated in a wide range of diseases. Regnase3 is a member of the Regnase RBPs family and has been shown to promote inflammation by increasing TNF in macrophages. Despite this knowledge, the full extent of the role played by Regnase3 in AKI remains unknown. Therefore, we hypothesized that Regnase3 plays a role in both macrophages and tubular epithelial cells, influencing inflammation and tubular repair in AKI. Method In order to investigate the role of Regnase3 in kidney injury, a series of genetically-modified mice were developed on the C57BL/6J genetic background. These mice included the paired box gene 8 (Pax8)-reverse tetracycline transactivator (rTtA, Pax8rTtA), tetracycline resistance protein (TetO)-Cre (TetOCre), transgenic receptor activator of nuclear factor kappa-Β (Rank)-Cre (RankCre), and Regnase3 floxed (Regnase-3fl/fl) mice. Using these genetically modified mice, we applied a range of experimental animal models to study the effects of Regnase3 on kidney injury, including unilateral kidney ischemia-reperfusion surgery with or without nephrectomy and nephrocalcinosis-related kidney injury. Additionally, we employed in vitro models utilizing primary cells to study Regnase3 on the cellular level. Furthermore, we utilized scRNA-seq and RNA-seq to investigate the underlying mechanisms of Regnase3’s function. Results We conducted a thorough investigation and discovered that the Regnase3 is highly expressed in macrophages located within the kidney after AKI. The scRNA-seq showed the expression Regnase3 positively correlated with the phagocytosis, chemokines, and monocyte maturation. Furthermore, Rank-Regnase3 mice suffered from more inflammation and kidney injury after AKI, characterized by an increase in CCR2 positive leukocyte infiltration. Additionally, in vitro experiments revealed that Regnase3 is involved in modulating macrophage behavior, acting as a regulator of macrophage polarization towards an M1 and M2 phenotype and influencing the capacity of cell migration. These findings indicate that Regnase3 is essential in the migration of macrophages after AKI, which contribute to both early phase inflammation and progression towards AKI- CKD. Next, we aimed to examine the role of Regnase3 in the context of renal tubule injury. We observed that Regnase3 is highly expressed in healthy renal tubules, but its expression is reduced following injury. Through in silico, in vivo and in vitro experiments, we found that Regnase3 controls early apoptosis, cell death, proliferation, and wound healing capability of tubular epithelial cells. These effects contribute to kidney injury in postischemic kidneys. Additionally, we discovered that the deletion of Regnase3 provides protection against ischemic AKI (Figure 1). Mechanistically, Regnase3 targets pre-RNA and modulates alternative splicing by increasing the proportion of targeted skipped exon events and decreasing the probability of targeted retained intron events. Overall, our findings indicate that Regnase3 plays a vital role in tubular epithelial cells in the response to kidney injury by regulating alternative splicing. Conclusion In summary, our findings indicate that Regnase3 contributes to kidney injury in postischemic kidneys. However, the impact of Regnase3 is contingent upon the specific cell lineage in question. The Rank-Regnase3 leads to an exacerbation of kidney injury by increasing macrophage recruitment, whereas the Pax8-Regnase3 leads to an improvement in kidney injury through its effects on cell death and wound healing capability of tubular epithelial cells. Therefore, the role of Regnase3 in kidney injury is dependent on the cell lineage, which ultimately determines the detrimental or beneficial impact on kidney injury.

Spatiotemporal and microstructural characterization of heterotopic ossification in healing rat Achilles tendons.

Achilles tendon rupture is a common debilitating medical condition. The healing process is slow and can be affected by heterotopic ossification (HO), which occurs when pathologic bone-like tissue is deposited instead of the soft collagenous tendon tissue. Little is known about the temporal and spatial progression of HO during Achilles tendon healing. In this study we characterize HO deposition, microstructure, and location at different stages of healing in a rat model. We use phase contrast-enhanced synchrotron microtomography, a state-of-the-art technique that allows 3D imaging at high-resolution of soft biological tissues without invasive or time-consuming sample preparation. The results increase our understanding of HO deposition, from the early inflammatory phase of tendon healing, by showing that the deposition is initiated as early as one week after injury in the distal stump and mostly growing on preinjury HO deposits. Later, more deposits form first in the stumps and then all over the tendon callus, merging into large, calcified structures, which occupy up to 10% of the tendon volume. The HOs were characterized by a looser connective trabecular-like structure and a proteoglycan-rich matrix containing chondrocyte-like cells with lacunae. The study shows the potential of 3D imaging at high-resolution by phase-contrast tomography to better understand ossification in healing tendons.

IL-6 and cfDNA monitoring throughout COVID-19 hospitalization are accurate markers of its outcomes

BackgroundSevere COVID-19 entails a dysregulated immune response, most likely inflammation related to a lack of virus control. A better understanding of immune toxicity, immunosuppression balance, and COVID-19 assessments could help determine whether different clinical presentations are driven by specific types of immune responses. The progression of the immune response and tissular damage could predict outcomes and may help in the management of patients.MethodsWe collected 201 serum samples from 93 hospitalised patients classified as moderately, severely, and critically ill. We differentiated the viral, early inflammatory, and late inflammatory phases and included 72 patients with 180 samples in separate stages for longitudinal study and 55 controls. We studied selected cytokines, P-selectin, and the tissue damage markers lactate dehydrogenase (LDH) and cell-free DNA (cfDNA).ResultsTNF-α, IL-6, IL-8, and G-CSF were associated with severity and mortality, but only IL-6 increased since admission in the critical patients and non-survivors, correlating with damage markers. The lack of a significant decrease in IL-6 levels in the critical patients and non-survivors in the early inflammatory phase (a decreased presence in the other patients) suggests that these patients did not achieve viral control on days 10–16. For all patients, lactate dehydrogenase and cfDNA levels increased with severity, and cfDNA levels increased in the non-survivors from the first sample (p = 0.002) to the late inflammatory phase (p = 0.031). In the multivariate study, cfDNA was an independent risk factor for mortality and ICU admission.ConclusionsThe distinct progression of IL-6 levels in the course of the disease, especially on days 10–16, was a good marker of progression to critical status and mortality and could guide the start of IL-6 blockade. cfDNA was an accurate marker of severity and mortality from admission and throughout COVID-19 progression.

Temporal changes in glucose metabolism reflect polarization in resident and monocyte-derived macrophages after myocardial infarction.

Metabolic reprogramming from glycolysis to the mitochondrial tricarboxylic acid (TCA) cycle and oxidative phosphorylation may mediate macrophage polarization from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype. We hypothesized that changes in cardiac macrophage glucose metabolism would reflect polarization status after myocardial infarction (MI), ranging from the early inflammatory phase to the later wound healing phase. MI was induced by permanent ligation of the left coronary artery in adult male C57BL/6J mice for 1 (D1), 3 (D3), or 7 (D7) days. Infarct macrophages were subjected to metabolic flux analysis or gene expression analysis. Monocyte versus resident cardiac macrophage metabolism was assessed using mice lacking the Ccr2 gene (CCR2 KO). By flow cytometry and RT-PCR, D1 macrophages exhibited an M1 phenotype while D7 macrophages exhibited an M2 phenotype. Macrophage glycolysis (extracellular acidification rate) was increased at D1 and D3, returning to basal levels at D7. Glucose oxidation (oxygen consumption rate) was decreased at D3, returning to basal levels at D7. At D1, glycolytic genes were elevated (Gapdh, Ldha, Pkm2), while TCA cycle genes were elevated at D3 (Idh1 and Idh2) and D7 (Pdha1, Idh1/2, Sdha/b). Surprisingly, Slc2a1 and Hk1/2 were increased at D7, as well as pentose phosphate pathway (PPP) genes (G6pdx, G6pd2, Pgd, Rpia, Taldo1), indicating increased PPP activity. Macrophages from CCR2 KO mice showed decreased glycolysis and increased glucose oxidation at D3, and decreases in Ldha and Pkm2 expression. Administration of dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, robustly decreased pyruvate dehydrogenase phosphorylation in the non-infarcted remote zone, but did not affect macrophage phenotype or metabolism in the infarct zone. Our results indicate that changes in glucose metabolism and the PPP underlie macrophage polarization following MI, and that metabolic reprogramming is a key feature of monocyte-derived but not resident macrophages.

Characterization of acute lung injury in the bleomycin rat model.

The aim of this study was to describe and characterize the pathophysiological changes occurring during the early inflammatory phase (first 3 days) in the rat bleomycin model of lung injury preceding the development of fibrosis. Further, we wanted to understand the kinetics and factors contributing to bleomycin-induced acute lung injury (ALI) and provide a robust, reliable and reproducible framework of features of ALI readouts to assess effects of therapeutics on bleomycin-induced ALI in rats. We induced ALI in rats with intratracheal (i.t.) installation of bleomycin. The animals were sacrificed on predetermined time points, that is, Day 0, 1, 2, and 3 post the bleomycin challenge. We analyzed bronchoalveolar lavage fluid (BALF) and lung tissue to establish and assess relevant experimental features of ALI. We demonstrated that bleomycin induced key features of experimental ALI including a profound increase in neutrophils in BALF (50-60%), pulmonary edema, and lung pathology on Day 3 after challenge. Furthermore, we showed that TGF-β1, IL-1β, TNF-α, IL-6, CINC-1, TIMP-1, and WISP-1 were induced by studying their kinetic profile during the first 3 days after bleomycin injury consistent with their known role ALI. We also confirmed that detectable fibrogenesis occurs at the earliest on Day 3 after injury based on collagen content, along with changes in the TGF-β/Smad signaling pathway and increased expression of Galectin-3, Vimentin, and Fibronectin in lung homogenate. Our report presents robust features and contributing mediators/factors to the pathology of bleomycin-induced ALI in rats on Day 3. The kinetic data provide insights on the progression of ALI and a detailed understanding of early events before actual fibrosis development. This set of experimental endpoints is very appropriate and invaluable for efficacy testing of potential novel therapeutic treatments (single or combined) in ALI and understanding their mechanism of action.

Altered early immune response after fracture and traumatic brain injury.

Clinical and preclinical data suggest accelerated bone fracture healing in subjects with an additional traumatic brain injury (TBI). Mechanistically, altered metabolism and neuro-endocrine regulations have been shown to influence bone formation after combined fracture and TBI, thereby increasing the bone content in the fracture callus. However, the early inflammatory response towards fracture and TBI has not been investigated in detail so far. This is of great importance, since the early inflammatory phase of fracture healing is known to be essential for the initiation of downstream regenerative processes for adequate fracture repair. Therefore, we analyzed systemic and local inflammatory mediators and immune cells in mice which were exposed to fracture only or fracture + TBI 6h and 24h after injury. We found a dysregulated systemic immune response and significantly fewer neutrophils and mast cells locally in the fracture hematoma. Further, local CXCL10 expression was significantly decreased in the animals with combined trauma, which correlated significantly with the reduced mast cell numbers. Since mast cells and mast cell-derived CXCL10 have been shown to increase osteoclastogenesis, the reduced mast cell numbers might contribute to higher bone content in the fracture callus of fracture + TBI mice due to decreased callus remodeling.

Loss of Protein Kinase D2 Activity Protects Against Bleomycin-Induced Dermal Fibrosis in Mice

Protein kinase D (PKD) has been linked to inflammatory responses in various pathologic conditions; however, its role in inflammation-induced dermal fibrosis has not been evaluated. In this study, we aimed to investigate the roles and mechanisms of protein kinase D2 (PKD2) in inflammation-induced dermal fibrosis and evaluate the therapeutic potential of PKD inhibitors in this disease. Using homozygous kinase-dead PKD2 knock-in (KI) mice, we examined whether genetic ablation or pharmacologic inhibition of PKD2 activity affected dermal inflammation and fibrosis in a bleomycin (BLM)-induced skin fibrosis model. Our data showed that dermal thickness and collagen fibers were significantly reduced in BLM-treated PKD2 KI mice compared with that in wild-type mice, and so was the expression of α-smooth muscle actin and collagens and the mRNA levels of transforming growth factor-β1 and interleukin-6 in the KI mice. Corroboratively, pharmacologic inhibition of PKD by CRT0066101 also significantly blocked BLM-induced dermal fibrosis and reduced α-smooth muscle actin, collagen, and interleukin-6 expression. Further analyses indicated that loss of PKD2 activity significantly blocked BLM-induced infiltration of monocytes/macrophages and neutrophils in the dermis. Moreover, using bone marrow–derived macrophages, we demonstrated that PKD activity was required for cytokine production and migration of macrophages. We have further identified Akt as a major downstream target of PKD2 in the early inflammatory phase of the fibrotic process. Taken together, our findings indicate that PKD2 promotes dermal fibrosis via regulating immune cell infiltration, cytokine production, and downstream activation of Akt in lesional skin, and targeted inhibition of PKD2 may benefit the treatment of this condition.

Epigenetic regulation of macrophage polarization in wound healing.

The immune microenvironment plays a critical role in regulating skin wound healing. Macrophages, the main component of infiltrating inflammatory cells, play a pivotal role in shaping the immune microenvironment in the process of skin wound healing. Macrophages comprise the classic proinflammatory M1 subtype and anti-inflammatory M2 population. In the early inflammatory phase of skin wound closure, M1-like macrophages initiate and amplify the local inflammatory response to disinfect the injured tissue. In the late tissue-repairing phase, M2 macrophages are predominant in wound tissue and limit local inflammation to promote tissue repair. The biological function of macrophages is tightly linked with epigenomic organization. Transcription factors are essential for macrophage polarization. Epigenetic modification of transcription factors determines the heterogeneity of macrophages. In contrast, transcription factors also regulate the expression of epigenetic enzymes. Both transcription factors and epigenetic enzymes form a complex network that regulates the plasticity of macrophages. Here, we describe the latest knowledge concerning the potential epigenetic mechanisms that precisely regulate the biological function of macrophages and their effects on skin wound healing.

The biological role of IL-1, IL-6 and CRP and their application in the diagnosis of the inflammatory process

Activation of inflammation is a type of innate, non-specific defence of the body against harmful factors of external or internal origin. During this process, various types of cytokines are released, including interleukin-1 (IL-1) and interleukin-6 (IL-6), responsible for the intensification of inflammatory reactions and the activation of hepatic acute phase proteins synthesis. IL-1 exists in two isoforms (IL-1α and IL-1β), showing similar, pro-inflammatory biological properties, but differing in origin, place of release and method of activation. The use of IL-1 in the diagnosis of the inflammatory process is limited, mainly due to the short half-life of this cytokine. IL-6 has a pleiotropic nature of action by using different types of receptors signaling. IL-6 is responsible for activating the synthesis of a wide range of acute phase proteins, also is involved in hematopoiesis and the immune response. IL-6 is a good biomarker of the early phase of inflammation because it has a longer half-life than other cytokines, and its concentration in the blood may increase several thousand times during the first hours after the initiation of inflammation. Due to its high sensitivity in detecting inflammation, C-reactive protein is the most commonly determined highly positive acute phase protein. During the inflammatory process, the pentameric CRP isoform (pCRP) dissociates into monomeric CRP (mCRP), which changes the anti-inflammatory properties of CRP into highly pro-inflammatory. The currently used laboratory tests detect only the pCRP isoform, because it is well soluble in blood and accumulates in it when conversion to mCRP is no longer effective.

Recurrent acute myocarditis: An under-recognized clinical entity associated with the later diagnosis of a genetic arrhythmogenic cardiomyopathy.

Myocardial inflammation has been consistently associated with genetic arrhythmogenic cardiomyopathy (ACM) and it has been hypothesized that episodes mimicking acute myocarditis (AM) could represent early inflammatory phases of the disease. We evaluated the temporal association between recurrent acute myocarditis (RAM) episodes and the later diagnosis of a genetic ACM. Between January 2012 and December 2021, patients with RAM and no previous cardiomyopathy were included (Recurrent Acute Myocarditis Registry, NCT04589156). A follow-up visit including clinical evaluation, resting and stress electrocardiogram, cardiac magnetic resonance imaging, and genetic testing was carried out. Endpoints of the study was the incidence of both ACM diagnosis criteria and ACM genetic mutation at the end of follow-up. Twenty-one patients with RAM were included and follow-up was completed in 19/21 patients (90%). At the end of follow-up, 3.3 ± 2.9 years after the last AM episode, 14/21 (67%) patients with an ACM phenotype (biventricular: 10/14, 71%; left ventricular: 4/14, 29%) underwent genetic testing. A pathogenic or likely pathogenic mutation was found in 8/14 patients (57%), 5/8 in the Desmoplakin gene, 2/8 in the Plakophillin-2 gene, and 1/8 in the Titin gene. Family history of cardiomyopathy or early sudden cardiac death had a positive predictive value of 88% for the presence of an underlying genetic mutation in patients with RAM. RAM is a rare entity associated with the latter diagnosis of an ACM genetic mutation in more than a third of the cases. In those patients, RAM episodes represent early inflammatory phases of the disease. Including RAM episodes in ACM diagnosis criteria might allow early diagnosis and potential therapeutic interventions.

TNFα and IFNγ cooperate for efficient pro- to anti-inflammatory transition of macrophages during muscle regeneration

IFNγ is traditionally known as a proinflammatory cytokine with diverse roles in antimicrobial and antitumor immunity. Yet, findings regarding its sources and functions during the regeneration process following a sterile injury are conflicting. Here, we show that natural killer (NK) cells are the main source of IFNγ in regenerating muscle. Beyond this cell population, IFNγ production is limited to a small population of T cells. We further show that NK cells do not play a major role in muscle regeneration following an acute injury or in dystrophic mice. Surprisingly, the absence of IFNγ per se also has no effect on muscle regeneration following an acute injury. However, the role of IFNγ is partially unmasked when TNFα is also neutralized, suggesting a compensatory mechanism. Using transgenic mice, we showed that conditional inhibition of IFNGR1 signaling in muscle stem cells or fibro-adipogenic progenitors does not play a major role in muscle regeneration. In contrast to common belief, we found that IFNγ is not present in the early inflammatory phase of the regeneration process but rather peaks when macrophages are acquiring an anti-inflammatory phenotype. Further transcriptomic analysis suggests that IFNγ cooperates with TNFα to regulate the transition of macrophages from pro- to anti-inflammatory states. The absence of the cooperative effect of these cytokines on macrophages, however, does not result in significant regeneration impairment likely due to the presence of other compensatory mechanisms. Our findings support the arising view of IFNγ as a pleiotropic inflammatory regulator rather than an inducer of the inflammatory response.

A TOUCH OF THE HEART AND THE TOOLS FOR DIAGNOSIS

A TOUCH OF THE HEART AND THE TOOLS FOR DIAGNOSIS

Chemerin as a Potential Marker of Resolution of Inflammation in COVID-19 Infection.

Chemerin is one of the specialized pro-resolving mediators that participate in the early phase of inflammation and contribute to the initiation of the pro-resolving response. There is a paucity of data regarding the time course of chemerin during acute infections. We aimed to evaluate the sequence of inflammatory responses in the acute COVID-19 phase throughout onset and resolution of inflammation. We evaluated changes in selected biomarkers in COVID-19 survivors on the 7-day and 28-day follow up. Chemerin was lower in patients with baseline moderate/severe disease at day 7 compared with asymptomatic patients and individuals with mild illness (7265 [5526–9448] vs. 8730 [6888–11,058] pg/mL; p = 0.03). Only in patients with moderate/severe disease, but not in those with mild symptoms, were chemerin concentrations decreased one week after infection onset compared with baseline (7265 [5526–9448] vs. 8866 [6383–10,690] pg/mL; p < 0.05) with a subsequent increase on the 28-day follow up (9313 [7353–11,033] pg/mL; p < 0.05). Resolution of inflammation in the group of moderate/severe SARS-CoV2 infection was associated with increasing serum concentrations of chemerin, contrary to pro-inflammatory cytokines and adipokines (pentraxin 3, TNFα, resistin, leptin). A similar pattern of angiopoietin-2 dynamics may suggest signs of enhanced vascularization as a consequence of acute SARS-CoV2 infection.

57-Year-Old Man With Headache, Vomiting, and Gait Instability

57-Year-Old Man With Headache, Vomiting, and Gait Instability

Lymphatic endothelial sphingosine 1-phosphate receptor 1 enhances macrophage clearance via lymphatic system following myocardial infarction.

Lymphatic endothelial cell homeostasis plays important roles in normal physiological cardiac functions, and its dysfunction significantly influences pathological cardiac remodeling after myocardial infarction (MI). Our results revealed that sphingosine 1-phosphate receptor 1 (S1pr1) expression in cardiac lymphatic endothelial cells (LECs) was sharply changed after MI. It has been shown that S1pr1 tightly controlled LEC functions and homeostasis. We thus hypothesized that lymphatic endothelial S1pr1 might be involved in post-MI cardiac remodeling. We generated LEC-conditional S1pr1 transgenic mice, in which S1pr1 expression was reduced in cardiac LECs. We performed the left anterior descending coronary artery (LAD) ligation operation to induce MI in these mice. Cardiac functions and remodeling were examined by echocardiography analysis and serial histological analysis. Meanwhile, we performed adoptive cell transfer experiments to monitor macrophage trafficking in post-MI myocardium and their draining lymphatic system. Furthermore, in vitro cell culture experiments and mechanism studies were undertaken to uncover the molecular mechanism by which LEC-S1pr1 regulated cardiac inflammation and remodeling after MI. Our results showed that S1pr1 expression significantly decreased in cardiac LECs after MI. Our in vivo experiments showed that the reduced expression of LEC-S1pr1 deteriorated cardiac function and worsened pathological cardiac remodeling after MI. Our further results demonstrated that the reduced expression of LEC-S1pr1 did not influence macrophage infiltration in an early inflammatory phase of MI, but significantly affected macrophages clearance in the later phase of MI via afferent cardiac lymphatics, and thus influenced inflammatory responses and cardiac outcome after MI. Further study showed that S1P/S1pr1 activated ERK signaling pathway and enhanced CCL2 expression, which promoted macrophage trafficking in a paracrine manner. This study reveals that cardiac lymphatic endothelial cells tightly control macrophage trafficking via lymphatic vessels in injured hearts via S1P/S1pr1/ERK/CCL2 pathway and thus regulate post-MI immune modulation and heart repair. This study highlights the importance of cardiac lymphatic vessel system in orchestrating post-MI immune responses and cardiac remodeling by regulating macrophage transit in injured hearts. Our finding implies that a feasible modulation of S1pr1 signaling in LECs might provide a promising target to resolve excessive inflammation and to ameliorate adverse cardiac remodeling after MI.

In vivo efficacy & phantom imaging connote the theranostic potential of a drug-loaded lipid nanobubble

The current American College of Rheumatology (ACR) guidelines emphasize on targeting rheumatoid arthritis in its early inflammatory phase for diagnosis and intervention to be effective. Ultrasound has been implicated as an affordable non-invasive modality to assess early pathology in synovitis. Additionally, therapeutic ultrasound can be used as an effective trigger for drug delivery. Commercial ultrasound contrast agents are microbubbles which have limitations as ‘theranostic’ agents with both diagnostic and therapeutic functions. In the present study, we have reported the development of a dexamethasone loaded nanobubble (DNB) which could have dual function as a theranostic contrast agent. In-vitro echogenicity studies were carried out using phantom imaging which suggested that these nanobubbles were quite stable up to 6 months at 2–8 °C in lyophilized form and displayed echogenic signal comparable to commercial Sonovue® up to 10mins. A significant anti-inflammatory activity was observed in presence of ultrasound on Raw 264.7 and THP1 cells. In vivo efficacy studies on adjuvant induced arthritic rodent model indicated significant reduction in visible and inflammatory markers in group treated with DNBs when compared to diseased control. Overall, these DNBs when subjected to therapeutic or diagnostic ultrasound provide a triggered drug release and contrast enhancement respectively highlighting theranostic potential and thus, can be explored in future for a precision therapy in RA.

Effect of low-intensity pulsed ultrasound on mast cell degranulation and fibroblast expression on type 2 diabetes mellitus rats wound healing process

Impaired wound healing is one of the Diabetes mellitus complications. Low-intensity Pulsed Ultrasound (LIPUS) therapy may accelerate the impaired wound healing. The use of LIPUS therapy in the early inflammatory phase can induce mast cell degranulation, and in the proliferative phase it can increase collagen synthesis by fibroblasts. The purpose of this study was to determine the effects of LIPUS therapy on mast cell degranulation and fibroblastexpression in the healing process of punch biopsy wound in rats with type 2 diabetes mellitus. Twenty-four Sprague dawley (n=24) were designed into type 2 diabetes mellitus by injecting Nicotinamide and Streptozotocin, then divided into six groups: diabetes mellitus without LIPUS (DM3, DM7, DM14) and diabetes mellitus with LIPUS (DML3, DML7, DML14), 4 each, and punch biopsy wounds were made on the dorsal skin. The DML group received LIPUS therapy in the wound area (frequency 3 MHz, intensity 0.5 W/cm2, duty cycle 20%, duration 3 minutes every day for 3 days (DML3), 7 days (DML7), and 14 days (DML14). The wounded tissue area was stained with toluidine blue to observe mast cell degranulation and immunohistochemical type HSP-47 to observe fibroblast expression. Two-Way ANOVA and Post Hoc LSD tests were used to determine the differences in mast cell degranulation and fibroblast expression. The results showed that mast cell degranulation and fibroblast expression in the DML group were higher than in the DM group (table 1). Pearson test showed a correlation between mast cell degranulation and fibroblast expression (p=0.00; r= 0.839). LIPUS therapy increases mast cell degranulation and fibroblast expression in type 2 diabetes mellitus rat model. The higher the mast cell degranulation, the higher fibroblast expressions.

Inhalation of tetrandrine liposomes for the treatment of bleomycin induced idiopathic pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease of unknown cause, with limited therapeutic options and an urgent need to develop new treatment methods. However, there is still a lack of suitable inhaled drugs for the treatment of IPF due to non-specific side effects, inability to cross the pulmonary barrier, and inadequate management of heterogeneity. In this study, we hypothesized that tetrandrine liposomes (TET-LIPs) delivered by inhalation might have a direct targeting effect on lung tissue, effectively treat IPF and limit its adverse side effects on other organs. MethodsMorphology, particle size, zeta potential, and in vitro dissolution studies of the prepared TET-LIPs were performed. Blood aspartate aminotransferase (AST), alanine aminotransferase (ALT), Urea nitrogen (BUN), creatinine (Cr) were measured to determine the liver and renal function. Bleomycin-induced mice model of IPF was used to evaluate the in vivo pharmacodynamics of TET-LIPs. The levels of hydroxyproline, tumor necrosis factor (TNF-α), and transforming growth factor (TGF-β1) were examined by enzyme-linked immunosorbent assay (ELISA). Finally, alveolar lavage fluid lipid profiles were obtained by using ultra-performance liquid chromatography combined with quadrupole-Exactive Orbitrap mass spectrometry (UPLC-Q-Exactive Orbitrap MS). ResultsInhaled TET-LIPs significantly inhibited the progression of bleomycin-induced acute inflammation and pulmonary fibrosis in lung tissue. Furthermore, delivering the drug locally to the lungs showed little accumulation in the liver and kidneys after long-term inhalation and had a favorable safety profile. Interestingly, we observed a relationship between inhaled TET-LIPs and increased levels of triglycerides (TG), fatty acids (FA) while decreased levels of lysophosphatidylcholine (LPC) in BALF. ConclusionsOverall, as TET-LIPs were safe and well-tolerated in the BLM-induced mice model, tetrandrine encapsulation in liposomes could be an effective method for locally delivering to patients with IPF. Simultaneously, our findings revealed that various lipid metabolic disorders were linked to the early inflammatory phase of the fibrosis process, and the regulation of lipid metabolism may be one of the mechanisms to improve pulmonary fibrosis using TET-LIPs.

Risk factors and clinical impact of COVID-19-associated pulmonary aspergillosis: Multicenter retrospective cohort study.

Background/AimsThe risk factors and clinical impacts of coronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) remain controversial, and no data have been reported in Korea. This study aimed to investigate the epidemiology and importance of CAPA diagnostic efforts and to identify the predictors of CAPA and the impacts on clinical outcomes.MethodsBetween January 2020 and May 2021, data of severely to critically ill COVID-19 patients were extracted from seven hospitals of the Catholic Medical Center through a clinical data warehouse. Corticosteroid use was subcategorized into total cumulative dose, early 7-day dose, mean daily dose, and duration of use.ResultsA total of 2,427 patients were screened, and 218 patients were included. CAPA was diagnosed in 4.6% (10/218) of all hospitalized and 11.2% (10/89) of intensive care unit patients. Total cumulative dose (over 1,000 mg as methylprednisolone) and daily high-dose corticosteroid use (over 60 mg/day) were independent predictors but not early 7-day high-dose corticosteroid use (over 420 mg/week) (odds ratio [OR], 1.731; 95% confidence interval [CI], 0.350 to 8.571) nor prolonged use (OR, 2.794; 95% CI, 0.635 to 13.928). In-hospital overall mortality was 11.9% (26 of 218). CAPA itself did not affect the outcome; rather, daily high-dose steroid use significantly increased the 30-day mortality (hazard ratio, 5.645; 95% CI, 1.225 to 26.091).ConclusionsCAPA was not uncommon, especially in critically ill patients. Daily high-dose corticosteroid use was the predictor of CAPA and associated with high mortality rates. High-dose corticosteroids use after early inflammatory phase should be avoided, and active surveillance methods for CAPA are essential for those high-risk patients.

Helminth TGF-β mimic, TGM, increases leukocyte migration and activation while also enhancing cutaneous wound healing and tissue regeneration.

Abstract Intestinal parasites express excretory/secretory (ES) molecules, which modulate the type-2 immune response including anti-inflammatory and tissue repair mechanisms. TGF-β mimic (TGM), an ES molecule secreted by Heligmosomoides polygyrus (Hp), binds TGF-β receptors yet lacks structural homology to TGF-β and distinct receptor interactions. We now demonstrate that TGM enhanced wound healing using an in vivo wound biopsy model in which TGM, in a 1.5% carboxymethylcellulose solution, was topically administered beneath a Tegaderm layer. Through histological analysis, greater restoration of normal tissue structure in the wound beds of TGM treated mice was observed during mid to late stage wound healing. These observations included accelerated re-epithelialization as well as hair follicle regeneration, in the absence of increased scarring. Using flow cytometric, histological and gene expression analyses, differential expansion of myeloid populations at different stages of wound healing was observed. This included enhanced early accumulation and persistence of migratory Ly6C+ macrophages in TGM-treated wounds during the early inflammatory phase. Additionally, at mid-stage wound healing, we observed increased CD301b+ myeloid populations, a subset that is critical to efficient tissue restoration. In summary, TGM can enhance skin wound healing and associated recruitment of specific macrophage subsets and elevation of type 2 cytokines. This study indicates a role for TGM as a potential novel therapeutic option for enhanced wound healing.