Influx Of Inflammatory Cells Research Articles

The Role of Fibrinolytic Factors, a Subset of Angiocrine Factors in Cytokine Storm-Associated Diseases

Cytokine storm syndrome is a general term applied to maladaptive cytokine release in response to infection and other stimuli. It occurs during graft versus host disease after hematopoietic stem cell transplantation.Recent evidence suggested that, during the coronavirus disease 2019 (COVID-19) epidemic, the severe deterioration of some patient's health with Coronavirus Disease 2019 (COVID-19) was compatible with symptoms as they are known for the cytokine storm syndrome. The cytokine storm syndrome in COVID-19 is associated with the development and progression of macrophage activation syndrome (MAS), vascular endotheliitis like Kawasaki disease, and acute respiratory distress syndrome. Abnormalities of coagulation and fibrinolysis are known clinical features of COVID-19 or MAS. We reported previously that plasmin inhibition reduced GVHD associated lethality and prevented increases in inflammatory cytokines in mice. But the role of the fibrinolytic system and its key player, plasmin, in the development of COVID-19 is not well defined. Toll-like receptors (TLRs) might contribute to the pathogenesis of the disease.We established a murine model of fulminant MAS by repeated injections of TLR-9 agonist and D-galactosamine in immunocompetent mice. We found increases in circulating urokinase and the angiocrine factor tissue-type plasminogen activator (tPA) levels during the progression of fulminant MAS in mice, which causes the enhanced conversion of the proenzyme plasminogen into plasmin. Genetic and pharmacological inhibition of plasmin counteracted MAS-associated lethality and other related symptoms. We show that plasmin regulates the influx of inflammatory cells and the production of inflammatory cytokines, chemokines, and proteases like soluble forms or membrane forms of matrix metalloproteases generating an amplification loop. Based on these data, we hypothesize that COVID-19-induced vascular endothelial dysfunction causes the aggravation of COVID-19 leading up to cytokine storm or MAS syndrome, endotheliitis, and hypercoagulability with the induction of disseminated intravascular coagulation syndrome(Fig.1.). In summary, we propose that plasmin and potentially MMPs inhibitors might offer a novel treatment to control the deadly cytokine storm syndrome in patients with MAS or COVID-19, thereby preventing multiple organ failure.FigureDisclosuresNo relevant conflicts of interest to declare.

Effect of TNFα blockade on UVB-induced inflammatory cell migration and collagen loss in mice

UVB irradiation induces pro-inflammatory cytokines including interleukin-1 (IL-1) and tumor necrosis factor-α (TNFα) in the skin. TNFα stimulates the chemotaxis of inflammatory cells to the skin. These cells secrete metalloproteinases (MMPs) and other enzymes that damage the cutaneous matrix. Therefore, blocking TNFα activity could be effective in preventing the influx of inflammatory cells and subsequent collagen degradation in the skin. In addition, TNFα downregulates procollagen mRNA, and thus blockade may be beneficial to production of type I collagen. Female C57BL/6 J mice were treated with etanercept (TNFα blocker, 4 mg/kg/day) for 4 days 1 h prior to UVB irradiation (100 mJ/cm2/day for 5 days). On the 5th day mice were sacrificed 3 h after UVB exposure. Blocking TNFα significantly inhibited UVB-induced recruitment of macrophages, mast cells, and neutrophils. UVB-irradiated mice skin contained more mature collagen compared to etanercept and UVB + etanercept-treated mice. Skin from UVB + etanercept-treated mice had more collagen fragments relative to UVB-irradiated mice. Procollagen protein was lower in UVB-irradiated and UVB + etanercept-treated mice. TNFα blockade decreased decorin and TGF-β1 in UVB-irradiated mice compared to UVB alone. MMP13 was inhibited by etanercept in UVB-irradiated mice (p < 0.01). In conclusion, blockade of TNFα significantly decreased mature collagen in UVB-irradiated mice, while increasing collagen fragmentation and decreasing procollagen.

Callicarpa japonica Thunb. ameliorates allergic airway inflammation by suppressing NF-κB activation and upregulating HO-1 expression

Ethnopharmacological relevanceCallicarpa japonica Thunb., as an herbal medicine has been used for the treatment of inflammatory diseases in China and Korea. Materials and methodsUltra performance liquid chromatography–photodiode array–quadrupole time–of–flight mass spectrometer (UPLC–PDA–QTof MS) was used to detect the major phenylethanoid glycosides in the C. japonica extract. BALB/c mice were intraperitoneally sensitized by ovalbumin (OVA) (on days 0 and 7) and challenged by OVA aerosol (on days 11–13) to induce airway inflammatory response. The mice were also administered with C. japonica Thunb. (CJT) (20 and 40 mg/kg Per oral) on days 9–13. CJT pretreatment was conducted in lipopolysaccharide (LPS)-stimulated RAW264.7 or phorbol 12-myristate 13-acetate (PMA)-stimulated A549 cells. ResultsCJT administration significantly reduced the secretion of Th2 cytokines, TNF-α, IL-6, immunoglobulin E (IgE) and histamine, and the recruitment of eosinophils in an OVA-exposed mice. In histological analyses, the amelioration of inflammatory cell influx and mucus secretion were observed with CJT. The OVA-induced airway hyperresponsiveness (AHR), iNOS expression and NF-κB activation were effectively suppressed by CJT administration. In addition, CJT led to the upregulation of HO-1 expression. In an in vitro study, CJT pretreatment suppressed the LPS-induced TNF-α secretion in RAW264.7 cells and attenuated the PMA-induced IL-6, IL-8 and MCP-1 secretion in A549 cells. These effects were accompanied by downregulated NF-κB phosphorylation and by upregulated HO-1 expression. ConclusionThese results suggested that CJT has protective activity against OVA-induced airway inflammation via downregulation of NF-κB activation and upregulation of HO-1, suggesting that CJT has preventive potential for the development of allergic asthma.

Human recombinant interleukin-38 suppresses inflammation in mouse models of local and systemic disease

Interleukin (IL)–38 belongs to the IL–1 family and is part of the IL–36 subfamily due to its binding to the IL–36 Receptor (IL–1R6). In the current study, we assessed the anti-inflammatory properties of IL–38 in murine models of arthritis and systemic inflammation. First, the anti-inflammatory properties of mouse and human IL–38 precursors were compared to forms with a truncated N–terminus. In mouse bone marrow derived dendritic cells (BMDC), human and mouse IL–38 precursors with a truncation of the two N-terminal amino acids (3–152) suppressed LPS-induced IL–6. Recombinant human IL–38 (3–152) was further investigated for its immunomodulatory potential using four murine models of inflammatory disease: streptococcal cell wall (SCW)-induced arthritis, monosodium urate (MSU) crystal-induced arthritis, MSU crystal-induced peritonitis, and systemic endotoxemia. In each of these models IL–38 significantly reduced inflammation. In SCW and MSU crystal-induced arthritis, joint swelling, inflammatory cell influx, and synovial levels of IL–1β, IL–6, and KC were reduced by 50% or greater. These suppressive properties of IL–38 in SCW-induced arthritis were independent of the anti-inflammatory co-receptor IL–1R8, as IL–38 reduced arthritis equally in IL–1R8 deficient and WT mice. In MSU crystal-induced peritonitis, IL–38 reduced hypothermia, while plasma IL–6 and KC and peritoneal KC levels were reduced by 65–70%. In the LPS endotoxemia model, IL–38 pretreatment reduced systemic IL–6, TNFα and KC. Furthermore, in ex vivo cultured bone marrow, LPS-induced IL–6, TNFα and KC were reduced by 75–90%. Overall, IL–38 exhibits broad anti-inflammatory properties in models of systemic and local inflammation and therefore may be an effective cytokine therapy.

COVID-19: Targeting Proteases in Viral Invasion and Host Immune Response.

An acute respiratory disorder (COVID-19) that accelerated across the globe has been found to be caused by a novel strain of coronaviruses (SARS-CoV-2). The absence of a specific antiviral drug or vaccination has promoted the development of immediate therapeutic responses against SARS-CoV-2. As increased levels of plasma chemokines and, cytokines and an uncontrolled influx of inflammatory cells were observed in lethal cases, it was concluded that the severity of the infection corresponded with the imbalanced host immunity against the virus. Tracing back the knowledge acquired from SERS and MERS infections, clinical evidence suggested similar host immune reactions and host ACE2 receptor-derived invasion by SARS-CoV-2. Further studies revealed the integral role of proteases (TMPRSS2, cathepsins, plasmin, etc.) in viral entry and the immune system. This review aims to provide a brief review on the latest research progress in identifying the potential role of proteases in SARS-CoV-2 viral spread and infection and combines it with already known information on the role of different proteases in providing an immune response. It further proposes a multidisciplinary clinical approach to target proteases specifically, through a combinatorial administration of protease inhibitors. This predictive review may help in providing a perspective to gain deeper insights of the proteolytic web involved in SARS-CoV-2 viral invasion and host immune response.

Immunomodulatory effect of different extracts from Angiostrongylus cantonensis on airway inflammation in an allergic asthma model.

This study aimed to evaluate the effects of early-life exposure to different extracts of Angiostrongylus cantonensis (A. cantonensis) on airway inflammation in an allergic asthma model. The total soluble extract (TE) and the soluble extracts of the digestive (AcD), reproductive (AcR), and cuticle (AcC) systems of A. cantonensis were used for immunisation before ovalbumin (OVA)-sensitisation/challenge in an OVA-induced allergic asthma model. The initial hypothesis of the study was that some soluble extract of the systems (AcD, AcR, or AcC) could be more potent to the modulation of inflammation than the TE. Our data, however, shows that immunisation with the TE is more promising because it decreased the high influx of inflammatory cells on airways and promoted an increase of interferon-γ (IFN-ɣ) and interleukin-10 (IL-10) levels. Besides this, the immunisation with the TE also led to a reduction of goblet cells and mucus overproduction in the lung tissue of asthmatic mice. We believe that the extracts have a distinct capacity to modulate the immune system, due to the TE possessing a greater variability of molecules, which together leads to control of airway inflammation. In conclusion, this is the first study to reveal that the TE of A. cantonensis adult worms has a greater potential for developing a novel therapeutic for allergic asthma.

Biflavonoid-rich fraction from Daphne pseudomezereum var. koreana Hamaya exerts anti-inflammatory effect in an experimental animal model of allergic asthma

Ethnopharmacological relevanceDaphne pseudomezereum var. koreana Hamaya is distributed in the Gangwon-do of South Korea and is traditionally used to treat chronic inflammatory diseases, including rheumatoid arthritis.Aim of the study: We investigated the anti-inflammatory effect of biflavonoid-rich fraction (BF) obtained from an extract of D. pseudomezereum leaves on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and mouse model of ovalbumin (OVA)-induced allergic asthma. Materials and methodsNeochamaejasmin B (NB) and chamaejasmin D (CD) were spectroscopically characterized as major components of BF obtained from the leaves of D. pseudomezereum. RAW264.7 cells pretreated with NB, CD and BF and activated by LPS (500 ng/ml) were used to assess the anti-inflammatory effects of these materials in vitro. To evaluate the protective effect of BF on allergic asthma, female BALB/c mice were sensitized to OVA by intraperitoneal (i.p.) injection and treated with BF by oral administration (15 or 30 mg/kg). ResultsPretreatment with BF inhibited LPS-stimulated nitric oxide (NO), TNF-α and IL-6, and led to upregulation of heme oxygenase-1 (HO-1) in RAW264.7 macrophages. Orally administered BF significantly inhibited the recruitment of eosinophils and the production of IL-5, IL-6, IL-13 and MCP-1 as judged by the analysis of BALF from OVA-induced asthma animal model. BF also decreased the levels of IgE in the serum of asthmatic mice. BF suppressed the influx of inflammatory cells into nearby airways and the hypersecretion of mucus by the airway epithelium of asthmatic mice. In addition, the increase in Penh in asthmatic mice was reduced by BF administration. Furthermore, BF led to Nrf2 activation and HO-1 induction in the lungs of mice. ConclusionsThese data have shown the anti-asthmatic effects of BF, and therefore we expect that BF may be a potential candidate as a natural drug/nutraceutical for the prevention and treatment of allergic asthma.

Lack of myeloid cell infiltration as an acquired resistance strategy to immunotherapy

BackgroundImmunotherapy of cancer is successful but tumor regression often is incomplete and followed by escape. Understanding the mechanisms underlying this acquired resistance will aid the development of more effective treatments.MethodsWe...

A critical role for CARD9 in pneumocystis pneumonia host defence.

Caspase recruitment domains-containing protein 9 (CARD9) is an adaptor molecule critical for key signalling pathways initiated through C-type lectin receptors (CLRs). Previous studies demonstrated that Pneumocystis organisms are recognised through a variety of CLRs. However, the role of the downstream CARD9 adaptor signalling protein in host defence against Pneumocystis infection remains to be elucidated. Herein, we analysed the role of CARD9 in host defence against Pneumocystis both in CD4-depleted CARD9-/- and immunocompetent hosts. Card9 gene-disrupted (CARD9-/- ) mice were more susceptible to Pneumocystis, as evidenced by reduced fungal clearance in infected lungs compared to wild-type (WT) infected mice. Our data suggests that this defect was due to impaired proinflammatory responses. Furthermore, CARD9-/- macrophages were severely compromised in their ability to differentiate and express M1 and M2 macrophage polarisation markers, to enhanced mRNA expression for Dectin-1 and Mincle, and most importantly, to kill Pneumocystis in vitro. Remarkably, compared to WT mice, and despite markedly increased organism burdens, CARD9-/- animals did not exhibit worsened survival during pneumocystis pneumonia (PCP), perhaps related to decreased lung injury due to altered influx of inflammatory cells and decreased levels of proinflammatory cytokines in response to the organism. Finally, although innate phase cytokines were impaired in the CARD9-/- animals during PCP, T-helper cell cytokines were normal in immunocompetent CARD9-/- animals infected with Pneumocystis. Taken together, our data demonstrate that CARD9 has a critical function in innate immune responses against Pneumocystis.

Anakinra for patients with COVID-19.

Anakinra for patients with COVID-19.

Preliminary Trichinella spiralis Infection Ameliorates Subsequent RSV Infection-Induced Inflammatory Response.

Respiratory syncytial virus (RSV) infection affects the lives of neonates throughout the globe, causing a high rate of mortality upon hospital admission. Yet, therapeutic options to deal with this pulmonary pathogen are currently limited. Helminth therapy has been well received for its immunomodulatory role in hosts, which are crucial for mitigating a multitude of diseases. Therefore, in this study, we used the helminth Trichinella spiralis and assessed its capabilities for modulating RSV infection as well as the inflammatory response induced by it in mice. Our results revealed that RSV-specific antibody responses were enhanced by pre-existing T. spiralis infection, which also limited pulmonary viral replication. Diminished lung inflammation, indicated by reduced pro-inflammatory cytokines and inflammatory cell influx was confirmed, as well as through histopathological assessment. We observed that inflammation-associated nuclear factor kappa-light-chain enhancement of activated B cells (NF-κB) and its phosphorylated forms were down-regulated, whereas antioxidant-associated nuclear factor erythroid 2-related factor 2 (Nrf2) protein expression was upregulated in mice co-infected with T. spiralis and RSV. Upregulated Nrf2 expression contributed to increased antioxidant enzyme expression, particularly NQO1 which relieved the host of oxidative stress-induced pulmonary inflammation caused by RSV infection. These findings indicate that T. spiralis can mitigate RSV-induced inflammation by upregulating the expression of antioxidant enzymes.

Ocular exposure to infectious laryngotracheitis virus alters leukocyte subsets in the head-associated lymphoid tissues and trachea of 6-week-old White Leghorn chickens

ABSTRACT Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes acute respiratory disease primarily infecting the upper respiratory tract and conjunctiva. Administration of live attenuated ILTV vaccines via eye drop, drinking water, or by coarse spray elicits protective mucosal immunity in the head-associated lymphoid tissues (HALT), of which conjunctiva-associated lymphoid tissue (CALT) and the Harderian gland (HG) are important tissue components. The trachea, a non-lymphoid tissue, also receives significant influx of inflammatory cells that dictate the outcome of ILTV infection. The objective of this study was to evaluate leukocyte cellular and phenotypic changes in the CALT, HG and trachea following ocular infection with a virulent ILTV strain. At 1, 3, 5, 7 and 9 days post-infection, CALT, HG, and trachea of 6-week-old specific pathogen free (SPF) chickens ocularly-exposed to vehicle or virulent ILTV strain 63140 were dissociated, the cells enumerated and then phenotyped using flow cytometry. The CALT had the highest viral genomic load, which peaked on day 3. In ILTV-infected birds, the CALT had a decreased percentage of leukocytes. This was reflected by decreased numbers of MHCI+MHCII–, MHCI+MHCIIlow+, and CD4+ cells, while IgM+ and MHCI+MHCIIHigh+ expressing cell populations increased. In the HG, the most notable change in cells from ILTV-infected birds was a decrease in IgM expressing cells and histologically, an increase in Mott cells. In summary, an acute, ocular exposure to ILTV strain 63140 in young birds shifts subsets of lymphocyte populations in the CALT and HG with minimal impact on the trachea.

COVID-19 in solid organ transplant recipients: Initial report from the US epicenter.

Solid organ transplant recipients may be at a high risk for SARS‐CoV‐2 infection and poor associated outcomes. We herein report our initial experience with solid organ transplant recipients with SARS‐CoV‐2 infection at two centers during the first 3 weeks of the outbreak in New York City. Baseline characteristics, clinical presentation, antiviral and immunosuppressive management were compared between patients with mild/moderate and severe disease (defined as ICU admission, intubation or death). Ninety patients were analyzed with a median age of 57 years. Forty‐six were kidney recipients, 17 lung, 13 liver, 9 heart, and 5 dual‐organ transplants. The most common presenting symptoms were fever (70%), cough (59%), and dyspnea (43%). Twenty‐two (24%) had mild, 41 (46%) moderate, and 27 (30%) severe disease. Among the 68 hospitalized patients, 12% required non‐rebreather and 35% required intubation. 91% received hydroxychloroquine, 66% azithromycin, 3% remdesivir, 21% tocilizumab, and 24% bolus steroids. Sixteen patients died (18% overall, 24% of hospitalized, 52% of ICU) and 37 (54%) were discharged. In this initial cohort, transplant recipients with COVID‐19 appear to have more severe outcomes, although testing limitations likely led to undercounting of mild/asymptomatic cases. As this outbreak unfolds, COVID‐19 has the potential to severely impact solid organ transplant recipients.

Essential role of CCL2-CCR2 axis in the pathogenesis of chronic irritant contact dermatitis

Abstract Chronic irritant contact dermatitis (ICD) including hand eczema is a common skin disorder caused by the repetitive exposure to irritants such as chemical detergents and organic solvents. Products including sodium dodecyl sulfate (SDS), such as soap or shampoo, are the leading cause of occupational ICD. Previous studies have shown that irritants cause epidermal damage which induce the production of proinflammatory mediators, followed by a dermal influx of inflammatory cells. However, the detailed immunological cascades occurring in the chronic ICD remains unclear. Repeated application of SDS, onto murine ear skin induced ear swelling, accompanied by epidermal hyperplasia and barrier dysfunction. Dermal infiltration of neutrophils, eosinophils and monocytes were remarkable in the SDS-applied skin. Our observation described above was consistent with the features of human chronic ICD. Here, we found that CCR2 deficiency or blockade of CCL2, a major ligand for CCR2, significantly attenuated SDS-induced ear swelling as well as inflammatory cell accumulation. Among the differentially expressed genes, which were up-regulated by SDS application, genes related to cell interaction at the vascular wall were significantly affected by CCR2 deficiency. Further, we investigated whether CCR2 on radiosensitive cells or radioresistant cells is critical. Lethally irradiated CCR2−/− mice transplanted with CCR2+/+ bone marrow cells (BMC) exhibited impaired SDS-induced ICD, while CCR2+/+ mice transplanted with CCR2−/− did not. Collectively, our data suggest that CCR2 expression by radioresistant cells plays an essential role in the pathogenesis of chronic ICD.

Modeling Immune Effector Responses in Alveolar Epithelial Cell Driven Lung Injury and Fibrosis

Abstract Mutations in the Surfactant Protein-C gene [SP-C] have been linked to pulmonary fibrosis (PF), with the missense isoleucine to threonine substitution at position 73 [I73T] as the most common. Clinically, PF is characterized by unrelenting tissue scarring and respiratory failure, intermitted by episodic exacerbations, bouts of inflammatory cell influx of unknown cause, which drastically reduce patient prognosis. Current murine models of PF rely on exposure to toxic agents or overexpression of pro-fibrotic signaling molecules in order to generate fibrotic endpoints. This reduces the overall relevance to human disease. To address these shortcomings, we developed a murine model of fibrosis designed to expressed mutant SP-CI73T. The resulting phenotype is accompanied by elevations in bronchoalveolar lavage cell counts, diffuse alveolar damage, and mortality. RNA and protein analysis demonstrate SP-CI73T epithelial cells initiate immune cell recruitment via CCL-2, IL-5, CCL-11, and CCL-17. Flow cytometric analysis highlighted temporally definite waves of inflammatory cells: an initial Ly6C+ inflammatory monocytes influx (3d), transient neutrophilic influx, and subsequent alveolar eosinophilia (2wk). RNA-seq analysis of sorted Ly6C+ monocytes revealed both inflammatory (inos, Il-6) and pro-fibrotic (col1a1, col1a2) phenotype. Pharmacologic and genetic monocyte ablation reduced inflammatory burden and improved survival of SP-CI73T mice. Taken together, we show that our mutant SP-CI73T model recapitulates the clinical features of PF. These findings support the concept that epithelial dysfunction is a key component of fibrogenesis and that inflammatory monocytes is responsible for disease exacerbation.

The involvement of annexin A1 in human placental response to maternal Zika virus infection

The association of Zika virus infection (ZIKV) with congenital malformation and neurological sequelae brought a significant global concern. Recent studies have shown that maternal viral infection leads to inflammation in the placental tissue. In this context, the antiinflammatory protein annexin 1 (ANXA1) has a major determination of the resolution of inflammation and it has been positively associated with antiparasitic activity in infected placental explants. Although these effects have been explored to some degree, ANXA1 expression and potential properties have not yet been fully elucidated in placentas infected with ZIKV. This study was conducted to evaluate the histopathology, inflammatory process and elucidate if ANXA1 were differently expressed in placentas of ZIKV-infected mothers. Three classification groups were used in this study: Neg/Neg (mother and placenta negative for the virus), Pos/Neg (infected mother, but no virus detected in placenta) and Pos/Pos (mother and placenta infected with ZIKV). ANXA1 was expressed in syncytiotrophoblast cells of all studied groups, and its expression was decreased in Pos/Neg group, which displayed also an increase of the inflammatory response, as evinced from the recruitment of inflammatory cells, increased levels of placenta cytokines, and evidence of impaired tissue repair. The presence of ZIKV in placentas of Pos/Pos group shows structural alterations, including detachment and disorganization of the trophoblastic epithelium. In summary, our results suggest that maternal infection with ZIKV, even without direct tissue infection, leads to a placental inflammatory response probably related to the modulation of ANXA1. After placental infection, structural changes - including inflammatory cells influx - are observed leading to placental dysfunction and reduced fetal weight. Our study sheds additional light on the outcomes of ZIKV infection in trophoblast and reveals a potential involvement of ANXA1 in the placental biology.

Obese Preeclamptic‐like BPH/5 Female Mice Have Evidence of Hepatic Steatosis with Progression to Non‐Alcoholic Fatty Liver Disease and Steatohepatitis with Pregnancy That Is Partially Reversed by Maternal Weight Loss

Maternal obesity before and during early pregnancy has been associated with adverse outcomes, including preeclampsia (PE). Recently, pre‐conception fatty liver in women has been identified as a significant risk factor for the development of PE. Non‐alcoholic fatty liver disease (NAFLD) is the most common cause of liver dysfunction with a prevalence of 30% worldwide and 10% in women of childbearing age. To better understand the relationship of maternal obesity and liver function in PE pregnancies, we sought to characterize the hepatic phenotype before and during early pregnancy in the obese preeclamptic‐like BPH/5 mouse. We hypothesized that BPH/5 female mice would have pre‐existing hepatic steatosis that is exacerbated by pregnancy to NAFLD and non‐alcoholic steatohepatitis (NASH), which could be reversed by weight loss to improve pregnancy outcomes.We have previously shown that BPH/5 female mice have an adverse metabolic phenotype in adulthood with obesity, increased pro‐inflammatory white adipose tissue (WAT), and hyperleptinemia as well as dyslipidemia that is exaggerated by pregnancy. To test our hypothesize, BPH/5 females were examined for signs of liver dysfunction before and at early pregnancy, embryonic day (e) 7.5, as well as after calorie restriction beginning at the time of conception, e0.5, by pair‐feeding to age‐ and gestation‐matched ad libitum fed control C57 female mice. This pair‐feeding paradigm has been shown to reduce BPH/5 female body weight significantly compared to ad libitum fed BPH/5 by e3.5 (n=6; p&lt;0.05) and WAT by e10.5 (n=6; p&lt;0.05).BPH/5 female mice have a 25% increase in liver mass before pregnancy versus C57, while their body weight is increased by only 15% (p&lt;0.05, n=3–9). Moreover, non‐pregnant BPH/5 female mice have a trend for increased serum alanine aminotransferase, a circulating hepatic leakage enzyme, versus C57 (45.7±19.5 vs. 30.3±2.3U/L; n=3–4, p=0.1). Hepatic expression of pro‐inflammatory genes, tumor necrosis factor (TNF)α, pan interferon (IFNa), interleukin (IL)‐1β and ‐6, increased by 25, 50, 20, and 100‐fold, respectively with pregnancy at e7.5 as compared to non‐pregnant BPH/5 female livers (n=3–4, p&lt;0.05), but this was not attenuated by pair‐feeding (p&gt;0.05). C57 females do not have increased hepatic expression of TNFα, IFNa, IL‐1 β nor IL‐6 mRNA with pregnancy (n=3–4, p&gt;0.05). Histologic examination of non‐pregnant BPH/5 female livers show excessive hepatic lipid accumulation with microvesicular steatosis. While pregnant (e7.5) C57 livers are free of inflammatory cells and lipid accumulation (n=3), e7.5 BPH/5 livers have an influx of inflammatory cells (1, scale 0–3) along with excess lipid accumulation (1.5, scale 0–3) that is attenuated by pair‐feeding (0.8, n=3). Further investigations are necessary to determine if maternal obesity and liver disease underlie the heightened inflammatory state observed in BPH/5 preeclamptic‐like mice. Furthermore, weight loss in obese BPH/5 may reverse pre‐existing metabolic derangements, including NAFLD to prevent PE associated adverse pregnancy outcomes.

Nitric Oxide Inhalation is Associated with Decreased Incidence of Acute Rejection after Cardiac Transplantation

Inhaled nitric oxide (iNO) has been shown to decrease ischemia-reperfusion injury (IRI) by several putative mechanisms, while preclinical studies have shown it to reduce acute rejections. In heart transplantation (HTx), iNO is routinely used to decrease pulmonary vascular resistance. However, iNO's effect on IRI after HTx and later outcomes on acute rejections remains unknown. We performed post hoc analysis on clinical data and plasma samples of 84 donor-recipient pairs collected in a prospective, double-blinded single-center trial. Recipient plasma was collected prior to HTx, and 1, 6, 12, and 24h after transplantation. In addition cardiac biopsies were collected before and 1h after reperfusion, and 2 weeks after HTx. We measured TnT, TnI, CKMBm, and proBNP as biomarkers for early organ-specific injury and function. Follow-up was conducted until one year for rejections and mortality. Of the 84 patients, 58.3% received iNO after HTx based on perioperative pulmonary vascular resistance. Preoperatively, iNO-treated patients had higher bilirubin and proBNP (P<0.01 and P<0.05), and a trend for higher transpulmonary gradient (P=0.058). Postoperatively, iNO-treated patients had higher TnT, TnI, and proBNP plasma levels (P<0.01-0.05), and an increased need for renal replacement therapy (P<0.001) compared to patients not receiving iNO. iNO-treated patients received more likely ATG, and therefore patients were divided to subgroups based on ATG and iNO treatment. In a 1-year follow-up, mortality between groups was similar. In groups without ATG treatment, iNO-treated patients had fewer biopsy-proven acute rejection episodes, and their 1-y freedom from rejection was higher (20% vs 52%) (both P<0.01). There was a similar, albeit statistically non-significant, trend for groups receiving ATG. iNO-treated patients were sicker pre- and postoperatively compared to patients not receiving iNO. Despite iNO-patients being overall sicker, iNO-treatment associated with less rejections. Our findings suggest that iNO may have some long-term cardioprotective effects after HTx. In ISHLT 2020 meeting we aim to present also results on the effect of iNO on early inflammatory cell influx to the graft, and on changes in cardiac transcriptome using IHC and next-generation sequencing from cardiac biopsies.

Modifying the Non-muscle Invasive Bladder Cancer Immune Microenvironment for Optimal Therapeutic Response.

It is now well-recognized that the tumor microenvironment (TME) is not only a key regulator of cancer progression but also plays a crucial role in cancer treatment responses. Recently, several high-profile publications have demonstrated the importance of particular immune parameters and cell types that dictate responsiveness to immunotherapies. With this increased understanding of TME-mediated therapy, approaches that increase therapeutic efficacy by remodeling the TME are actively being pursued. A classic example of this, in practice by urologists for over 40 years, is the manipulation of the bladder microenvironment for the treatment of non-muscle invasive bladder cancer (NMIBC) by instillation of intravesical bacillus Calmette-Guerin (BCG). The success of BCG treatment is thought to be due to its ability to induce a massive influx of Th1-polarized inflammatory cells, production of Th1 inflammatory cytokines and the generation of tumor-targeted Th1-mediated cytotoxic responses. Whilst BCG immunotherapy is currently the best treatment for NMIBC, ~30% of patients show no response to this treatment. Here we present a review highlighting a variety of promising alternative immunotherapies being developed that remodel the bladder tumor microenvironment. These include (1) the use of oncolytic viruses which selectively replicate within cancer cells whilst also modifying the immunological components of the TME, (2) manipulation of the bladder microbiome to augment the response to BCG or other immunotherapies (3) utilizing Toll-like Receptor agonists as anti-tumor agents due to their potent stimulation of innate and adaptive immunity and (4) the growing recognition that immunotherapeutic strategies that will have the largest impact on patients may require multiple therapeutic approaches combined together. The accumulating knowledge on TME remodeling holds promise for providing an alternative therapy for patients with BCG-unresponsive NMIBC.

Aluminum hydroxide nebulization-induced redox imbalance and acute lung inflammation in mice

Purpose: Aluminum is the third most abundant metal in the earth’s crust and is widely used in industry. Chronic contact with aluminum results in a reduction in the activity of electron transport chain complexes, leading to excessive production of reactive oxygen species (ROS) and oxidative stress. This study aimed to evaluate the effects of short-term exposure of aluminum hydroxide on oxidative stress and pulmonary inflammatory response.Materials and methods: Male BALB/c mice were divided into three groups: control group (CG); phosphate buffered saline group (PBSG) and aluminum hydroxide group (AHG). CG was exposed to ambient air, while PBSG and AHG were exposed to PBS or aluminum hydroxide solutions via nebulization, three times per day for five consecutive days. Twenty-four hours after the last exposure, all animals were euthanized for subsequent analysis.Results: Exposure to aluminum hydroxide in the blood resulted in lower platelet levels, higher neutrophils, and lower monocytes compared to CG and PBSG. Aluminum hydroxide promoted the recruitment of inflammatory cells to the lung. Macrophage, neutrophil and lymphocyte counts were higher in AHG compared to CG and PBSG. Protein oxidation and superoxide dismutase activity were higher, while catalase activity and reduced and oxidizes glutathione ratio in AHG were lower compared to CG and PBSG. Furthermore, there was an increase in the inflammatory markers CCL2 and IFN-γ in AHG compared to CG and PBSG.Conclusion: In conclusion, short-term nebulization with aluminum hydroxide induces the influx of inflammatory cells and oxidative stress in adult BALB/c mice.