Upregulation Of Inflammatory Markers Research Articles

#897 Peritoneal inflammation in PD-related peritonitis induces systemic eryptosis: in vitro and in vivo assessments

Abstract Background and Aims Erythrocytes (RBCs) have a highly specialized and organized membrane structure and undergo programmed cell death, known as eryptosis ( = stress-induced RBC death mechanism). Triggers of eryptosis include oxidative stress, inflammation and several uremic toxins. Our preliminary data show a significant increase of eryptosis during peritoneal dialysis (PD) -associated peritonitis in PD patients. The aims of this study were: assessment of incrementation of eryptosis in PD patients with peritonitis, evaluation of the relationship between systemic eryptosis in peritonitis and specific peritonitis biomarkers in PD effluent (PDE), and confirmation of the induction of eryptosis by peritonitis in a vitro setting. Method We enrolled 34 PD patients in stable condition (without systemic inflammation nor PD-related peritonitis in the last 3 months), 31 PD patients with acute peritonitis, and 17 healthy subjects (CTR). For PD stable patients, blood samples for eryptosis evaluation were drawn during the regular outpatient. For PD patients with peritonitis, blood samples and PDE samples were collected at the time of peritonitis diagnosis. PDE samples were used for peritoneal White Blood Cell count (pWBC), peritoneal Neutrophil Gelatinase-associated Lipocalin (pNGAL) and peritoneal cytokines levels (IL-1β and IL-6) measurements. For in vitro study, healthy RBCs were exposed to plasma of 31 PD patients with peritonitis and plasma of CTR group for 2, 4 and 24 hours. Morphological markers of eryptosis (cell membrane scrambling, cell shrinkage) and eryptosis percentage (based on PS exposure at RBC surface and AnnexinV-binding) were evaluated by flow cytometric analyses in vivo and in vitro. Results Totally, 65 chronic PD patients were included in this study. 57% of patients were treated with continuous ambulatory PD (CAPD) and 43% with automated PD (APD). The median PD vintage was 38 months (IQR 3.6–124.9 months). Table 1 reports clinical data for our PD population (Table 1). The percentage of AnnexinV-binding RBCs was significantly higher in PD patients than in CTR (2.7%; IQR 1.6–3.9 vs CTR: 0.8; IQR 0.7–1.3 P < .05). Eryptosis levels did not differ significantly between PD pts with and without diabetes, with and without hypertension, with and without cardiovascular disease. Eryptosis showed no significant differences between patients treated by CAPD/APD, and with Kt/Vurea values ≤ 1.7 and > 1.7. Generally, RBCs of all 65 PD patients were dramatically deranged in their morphology. In particular, RBCs from PD patients with peritonitis were characterized by dramatically deranged morphology and increased median cell volume in comparison with PD stable patients (P < .001). The percentages of AnnexinV-binding RBCs were significantly increased in the PD-associated peritonitis group (9.6%; IQR 4.2–16.7 versus 2.7%; IQR 1.6–3.9) (P<.0001) (Fig. 1). The percentage of AnnexinV-binding RBCs was significantly higher in PD patients with peritonitis than in healthy CTR (P < .001). We confirmed these in vivo data by in vitro results: healthy RBCs incubated with plasma from PD patients with peritonitis demonstrated a significant increase of eryptosis compared to healthy RBCs exposed to plasma from control group at all times (Fig. 2). Furthermore, significant positive strong correlations were observed between eryptosis level and all analysed peritoneal biomarkers of peritonitis (Spearman's rho pWBC = 0.77, P<.001, Spearman's rho pNGAL = 0.64, P = .001, Spearman's rho IL-6 = 0.61, P = .003 and Spearman's rho IL-1β = 0.64, P = .001,) (Fig. 3). Conclusion We investigated a potential connection between systemic eryptosis and peritoneal biomarkers of peritonitis. In particular, we theorized that the eryptosis enhancement is directly connected with peritonitis, and, based on the results, we hypothesized that the peritoneal membrane injury could be related to eryptosis. Upregulation of inflammatory markers could explain the increased rate of systemic eryptosis during PD-related peritonitis. In particular, we corroborated this point with our observations about in vitro induction of eryptosis by plasma from PD patients on the first day of peritonitis.

Long-term haplodeficency of DSPP causes temporomandibular joint osteoarthritis in mice

BackgroundExtracellular matrix (ECM) protein malfunction or defect may lead to temporomandibular joint osteoarthritis (TMJ OA). Dentin sialophophoprotein (DSPP) is a mandibular condylar cartilage ECM protein, and its deletion impacted cell proliferation and other extracellular matrix alterations of postnatal condylar cartilage. However, it remains unclear if long-term loss of function of DSPP leads to TMJ OA. The study aimed to test the hypothesis that long-term haploinsufficiency of DSPP causes TMJ OA.Materials and methodsTo determine whether Dspp+/– mice exhibit TMJ OA but no severe tooth defects, mandibles of wild-type (WT), Dspp+/–, and Dspp homozygous (Dspp−/−) mice were analyzed by Micro-computed tomography (micro-CT). To characterize the progression and possible mechanisms of osteoarthritic degeneration over time in Dspp+/– mice over time, condyles of Dspp+/– and WT mice were analyzed radiologically, histologically, and immunohistochemically.ResultsMicro-CT and histomorphometric analyses revealed that Dspp+/– and Dspp−/− mice had significantly lower subchondral bone mass, bone volume fraction, bone mineral density, and trabecular thickness compared to WT mice at 12 months. Interestingly, in contrast to Dspp−/− mice which exhibited tooth loss, Dspp+/– mice had minor tooth defects. RNA sequencing data showed that haplodeficency of DSPP affects the biological process of ossification and osteoclast differentiation. Additionally, histological analysis showed that Dspp+/– mice had condylar cartilage fissures, reduced cartilage thickness, decreased articular cell numbers and severe subchondral bone cavities, and with signs that were exaggerated with age. Radiographic data showed an increase in subchondral osteoporosis up to 18 months and osteophyte formation at 21 months. Moreover, Dspp+/– mice showed increased distribution of osteoclasts in the subchondral bone and increased expression of MMP2, IL-6, FN-1, and TLR4 in the mandibular condylar cartilage.ConclusionsDspp+/– mice exhibit TMJ OA in a time-dependent manner, with lesions in the mandibular condyle attributed to hypomineralization of subchondral bone and breakdown of the mandibular condylar cartilage, accompanied by upregulation of inflammatory markers.

Peritoneal Inflammation in PD-Related Peritonitis Induces Systemic Eryptosis: In Vitro and In Vivo Assessments.

Erythrocytes (RBCs) have a highly specialized and organized membrane structure and undergo programmed cell death, known as eryptosis. Our preliminary data show a significant increase in the eryptosis during peritoneal dialysis (PD)-associated peritonitis. The objectives of the present study were assessment of the incrementation of eryptosis in PD patients with peritonitis, evaluation of the relationship between systemic eryptosis in peritonitis and specific peritonitis biomarkers in PD effluent (PDE), and confirmation of the induction of eryptosis by peritonitis in a vitro setting. We enrolled 22 PD patients with peritonitis and 17 healthy subjects (control group, CTR). For the in vivo study, eryptosis was measured in freshly isolated RBCs. For the in vitro study, healthy RBCs were exposed to the plasma of 22 PD patients with peritonitis and the plasma of the CTR group for 2, 4, and 24 h. Eryptosis was evaluated by flow cytometric analyses in vivo and in vitro. PDE samples were collected for biomarkers analysis.The percentage of eryptotic RBCs was significantly higher in PD patients with peritonitis than in CTR (PD patients with peritonitis: 7.7; IQR 4.3-14.2, versus CTR: 0.8; IQR 0.7-1.3; p < 0.001). We confirmed these in vivo results by in vitro experiments: healthy RBCs incubated with plasma from PD patients with peritonitis demonstrated a significant increase in eryptosis compared to healthy RBCs exposed to plasma from the control group at all times. Furthermore, significant positive correlations were observed between eryptosis level and all analyzed peritoneal biomarkers of peritonitis. We investigated a potential connection between systemic eryptosis and peritoneal biomarkers of peritonitis. Up-regulation of inflammatory markers could explain the increased rate of systemic eryptosis during PD-related peritonitis.

Microglial Transforming Growth Factor-β Signaling in Alzheimer's Disease.

Novel technologies such as single-cell RNA and single-nucleus RNA sequencing have shed new light on the complexity of different microglia populations in physiological and pathological states. The transcriptomic profiling of these populations has led to the subclassification of specific disease-associated microglia and microglia clusters in neurodegenerative diseases. A common profile includes the downregulation of homeostasis and the upregulation of inflammatory markers. Furthermore, there is concordance in few clusters between murine and human samples. Apolipoprotein E, which has long been considered a high-risk factor for late-onset Alzheimer's disease, is strongly regulated in both these murine and human clusters. Transforming growth factor-β plays an essential role during the development and maturation of microglia. In a pathological state, it attenuates their activation and is involved in numerous cell regulatory processes. Transforming growth factor-β also has an influence on the deposition of amyloid-beta, as it is involved in the regulation of key proteins and molecules. Taken together, this review highlights the complex interaction of apolipoprotein E, the triggering receptor on myeloid cells 2, and transforming growth factor-β as part of a regulatory axis in microglia at the onset and over the course of Alzheimer's disease.

Differential Outcomes of Infection by Wild-Type SARS-CoV-2 and the B.1.617.2 and B.1.1.529 Variants of Concern in K18-hACE2 Transgenic Mice.

SARS-CoV-2 is a respiratory virus with neurological complications including the loss of smell and taste, headache, and confusion that can persist for months or longer. Severe neuronal cell damage has also been reported in some cases. The objective of this study was to compare the infectivity of the wild-type virus, Delta (B.1.617.2) and Omicron (B.1.1.529) variants in transgenic mice that express the human angiotensin-converting enzyme 2 (hACE2) receptor under the control of the keratin 18 promoter (K18) and characterize the progression of infection and inflammatory response in the lungs, brain, medulla oblongata, and olfactory bulbs of these animals. We hypothesized that wild type, Delta and Omicron differentially infect K18-hACE2 mice, thereby inducing distinct cellular responses. K18-hACE2 female mice were intranasally infected with wild-type, Delta, or Omicron variants and euthanized either at 3 days post-infection (dpi) or at the humane endpoint. None of the animals infected with the Omicron variant reached the humane endpoint and were euthanized at day 8 dpi. Virological and immunological analyses were performed in the lungs, brains, medulla oblongata and olfactory bulbs isolated from infected mice. At 3 dpi, mice infected with wild type and Delta displayed significantly higher levels of viral RNA in the lungs than mice infected with Omicron, while in the brain, Delta and Omicron resulted in higher levels of viral RNA than with the wild type. Viral RNA was also detected in the medulla oblongata of mice infected by all these virus strains. At this time point, the mice infected with wild type and Delta displayed a marked upregulation of many inflammatory markers in the lungs. On the other hand, the upregulation of inflammatory markers was observed only in the brains of mice infected with Delta and Omicron. At the humane endpoint, we observed a significant increase in the levels of viral RNA in the lungs and brains of mice infected with wild type and Delta, which was accompanied by the elevated expression of many inflammatory markers. In contrast, mice which survived infection with the Omicron variant showed high levels of viral RNA and the upregulation of cytokine and chemokine expression only in the lungs at 8 dpi, suggesting that infection and inflammatory response by this variant is attenuated in the brain. Reduced RNA levels and the downregulation of inflammatory markers was also observed in the medulla oblongata and olfactory bulbs of mice infected with Omicron at 8 dpi as compared with mice infected with wild-type and Delta at the humane end point. Collectively, these data demonstrate that wild-type, Delta, and Omicron SARS-CoV-2 induce distinct levels of infection and inflammatory responses in K18-hACE2 mice. Notably, sustained brain infection accompanied by the upregulation of inflammatory markers is a critical outcome in mice infected with wild type and Delta but not Omicron.

Abstract 11641: The Role of Inflammation in a Humanized Mouse Model of Transthyretin Cardiac Amyloidosis

Transthyretin cardiac amyloidosis (ATTR) is an under-diagnosed, progressive form of cardiomyopathy, leading to heart failure and death. It is caused by the deposition of amyloid fibrils in the myocardium derived from the plasma protein transthyretin (TTR), but its pathogenesis is poorly understood. Informative in vivo models have proven elusive. Our research identifies inflammation as a potential exacerbating factor in ATTR, suggested by gene expression elevations in inflammatory markers from cardiac biopsies of ATTR-CM patients. To explore this, we created a murine TTR knockout model expressing the human V122I TTR variant. Significant elevations of human TTR were observed in the plasma post-injection (human TTR level (ng/ml) ATTR: 110 ± 10; Control: 28 ± 12; p=0.0008, N=3-5) with preserved cardiac function (FS% ATTR: 20 ± 6; Control: 23± 5; p=0.8162, N=3-5). Notably, the mouse model exhibited amyloid deposits in the heart, confirmed by immunofluorescence and Congo Red staining. Transmission electron microscopy revealed both immature and mature amyloid fibrils in the extracellular matrix (Figure1). RNA-sequencing of the ATTR mouse heart identified distinct transcriptomic patterns and conserved inflammation pathways similar to those seen in a cohort of human ATTR heart samples, such as TNF, T-cell, and B-cell receptor signaling. Up-regulation of inflammatory markers, including CXCL1/2/3 and CCL20, was also observed, and an increased level of CCL5 (MFI ATTR: 801 ± 105; Control: 426± 64; p=0.0061, N=3) was confirmed via luminance-based immunoassay. These chemokines are critical for directing leukocytes to inflammation sites. This study suggests that inflammation may play a role in ATTR pathogenesis. Further in vivo studies are needed to elucidate the mechanisms by which inflammation modulates ATTR amyloidosis.

Sigmar1 ablation leads to lung pathological changes associated with pulmonary fibrosis, inflammation, and altered surfactant proteins levels.

Sigma1 receptor protein (Sigmar1) is a small, multifunctional molecular chaperone protein ubiquitously expressed in almost all body tissues. This protein has previously shown its cardioprotective roles in rodent models of cardiac hypertrophy, heart failure, and ischemia-reperfusion injury. Extensive literature also suggested its protective functions in several central nervous system disorders. Sigmar1's molecular functions in the pulmonary system remained unknown. Therefore, we aimed to determine the expression of Sigmar1 in the lungs. We also examined whether Sigmar1 ablation results in histological, ultrastructural, and biochemical changes associated with lung pathology over aging in mice. In the current study, we first confirmed the presence of Sigmar1 protein in human and mouse lungs using immunohistochemistry and immunostaining. We used the Sigmar1 global knockout mouse (Sigmar1-/-) to determine the pathophysiological role of Sigmar1 in lungs over aging. The histological staining of lung sections showed altered alveolar structures, higher immune cells infiltration, and upregulation of inflammatory markers (such as pNFκB) in Sigmar1-/- mice compared to wildtype (Wt) littermate control mice (Wt). This indicates higher pulmonary inflammation resulting from Sigmar1 deficiency in mice, which was associated with increased pulmonary fibrosis. The protein levels of some fibrotic markers, fibronectin, and pSMAD2 Ser 245/250/255 and Ser 465/467, were also elevated in mice lungs in the absence of Sigmar1 compared to Wt. The ultrastructural analysis of lungs in Wt mice showed numerous multilamellar bodies of different sizes with densely packed lipid lamellae and mitochondria with a dark matrix and dense cristae. In contrast, the Sigmar1-/- mice lung tissues showed altered multilamellar body structures in alveolar epithelial type-II pneumocytes with partial loss of lipid lamellae structures in the lamellar bodies. This was further associated with higher protein levels of all four surfactant proteins, SFTP-A, SFTP-B, SFTP-C, and SFTP-D, in the Sigmar1-/- mice lungs. This is the first study showing Sigmar1's expression pattern in human and mouse lungs and its association with lung pathophysiology. Our findings suggest that Sigmar1 deficiency leads to increased pulmonary inflammation, higher pulmonary fibrosis, alterations of the multilamellar body stuructures, and elevated levels of lung surfactant proteins.

Relationship between Severity of Lumbar Spinal Stenosis and Ligamentum Flavum Hypertrophy and Serum Inflammatory Factors.

Objective This study is aimed at investigating the correlation between lumbar spinal stenosis (LSS) severity, ligamentum flavum hypertrophy, and the upregulation of inflammatory markers. Methods From March 2019 and May 2022, eighty-five inpatients with LSS were enlisted as the study's research group, while sixty-five patients hospitalized for lumbar intervertebral disc herniation over the same time period served as the study's control group. Moreover, mild, moderate, and severe subgroups of patients were created within the research population based on their LSS severity. The ligamentum flavum thickness and the positive expression rates of TNF-α, TGF-β1, and IL-1α were compared between the study group and the control group. The levels of TNF-α, TGF-β1, and IL-1α that were found to be positively expressed were compared between the mild, moderate, and severe groups. Patients with LSS had their ligamentum flavum thickness and their positive expression rates of TNF-α, TGF-β1, and IL-1α analyzed using Spearman correlation analysis. We evaluated the diagnostic utility of the positive expression rates of IL-α1, TGF-β1, and TNF-α and ligamentum flavum thickness in distinguishing the severity of LSS using a receiver operating characteristic (ROC) curve. Results The rates of both lower limb pain (40.00%) and intermittent claudication (80.00%) in the LSS group were higher than those in the lumbar disc herniation group (15.38%, 12.31%), with statistical significance (P < 0.05). However, no substantial disparity was observed in left lower limb pain, right lower limb pain, low back pain, lower limb sensation, muscle strength, and reflex abnormalities between the two groups (P > 0.05). Positive expressions of TGF-β1, TNF-α, and IL-1α and thicker ligamentum flavum were more prevalent in the LSS group than in the lumbar intervertebral disc herniation group. All indexes were significantly (P < 0.05) higher in the moderate stenosis group than in the severe stenosis group. Additionally, the thickness of the ligamentum flavum and the positive expression rates of TNF-α, TGF-β1, and IL-1α were higher in the mild and moderate stenosis groups than in the severe stenosis group. The expression levels of TNF-α, TGF-β1, and IL-1α were favorably linked with ligamentum flavum thickness (P < 0.05). ROC curve analysis showed that the thickness of ligamentum flavum, the expression of IL-1α, the expression of TGF-β1, and the expression of TNF-α could effectively diagnose mild, moderate, and severe LSS (P < 0.05). Conclusion Ligamentum flavum hypertrophy and positive expression rates of IL-1α, TGF-β1, and TNF-α are closely linked to LSS, which can effectively identify mild, moderate, and severe LSS.

Amelioration of ethanol-induced gastric ulcer in rats by quercetin: implication of Nrf2/HO1 and HMGB1/TLR4/NF-κB pathways

Gastric ulcer is a serious medical condition that can be developed due to an imbalance in the protective and destructive factors of the gastric system. Available therapies do not provide definite cure, thus, there is an urge to seek for alternative treatments. Quercetin is a natural flavonoid that possesses antioxidant and anti-inflammatory properties. In the current study, the antiulcerogenic effect of quercetin in ethanol-induced gastric ulcer (EI-GU) rat model was compared to Antodine® (a reference drug), to elucidate the potential underlying mechanisms. Quercetin (50 mg/kg) and Antodine® (20 mg/kg) were given orally for one week post ulcer induction by ethanol. EI-GU was associated with downregulation of SOD, CAT, Nrf2 and HO1, and accompanied by upregulation of inflammatory markers (i.e., HMGB1, NF-κB and TNFα) and an increase in Bax/Bcl2 ratio. Administration of quercetin resulted in a significant reduction in gastric volume in the stomach of ulcerative rats by 86% and a significant decrease in gastric lesion count by 3.5- folds, as compared with the ulcerative rats. Moreover, rats treated with quercetin showed upregulation of Nrf2 by 3.3-fold change and in HO1 by 3.5-fold change when compared to ulcerated rats, and decreased HMGB1, TLR4, NF-κB p65 and TNF-α by 50%, 53%, 52.9% and 54.9%, respectively. Treatment of rats with quercetin reduced Bax and Bax/Bcl2 ratio and increased Bcl2 relative to ulcerated rats. Thus, it can be concluded that the ulcerogenic curative properties of quercetin were mediated by antioxidant, anti-inflammatory and antiapoptotic activities.

Neurovascular dysfunction and neuroinflammation in a Cockayne syndrome mouse model.

Cockayne syndrome (CS) is a rare, autosomal genetic disorder characterized by premature aging-like features, such as cachectic dwarfism, retinal atrophy, and progressive neurodegeneration. The molecular defect in CS lies in genes associated with the transcription-coupled branch of the nucleotide excision DNA repair (NER) pathway, though it is not yet clear how DNA repair deficiency leads to the multiorgan dysfunction symptoms of CS. In this work, we used a mouse model of severe CS with complete loss of NER (Csa−/−/Xpa−/−), which recapitulates several CS-related phenotypes, resulting in premature death of these mice at approximately 20 weeks of age. Although this CS model exhibits a severe progeroid phenotype, we found no evidence of in vitro endothelial cell dysfunction, as assessed by measuring population doubling time, migration capacity, and ICAM-1 expression. Furthermore, aortas from CX mice did not exhibit early senescence nor reduced angiogenesis capacity. Despite these observations, CX mice presented blood brain barrier disruption and increased senescence of brain endothelial cells. This was accompanied by an upregulation of inflammatory markers in the brains of CX mice, such as ICAM-1, TNFα, p-p65, and glial cell activation. Inhibition of neovascularization did not exacerbate neither astro- nor microgliosis, suggesting that the pro-inflammatory phenotype is independent of the neurovascular dysfunction present in CX mice. These findings have implications for the etiology of this disease and could contribute to the study of novel therapeutic targets for treating Cockayne syndrome patients.

Pharmacological Inhibition of STAT3 by Stattic Ameliorates Clinical Symptoms and Reduces Autoinflammation in Myeloid, Lymphoid, and Neuronal Tissue Compartments in Relapsing-Remitting Model of Experimental Autoimmune Encephalomyelitis in SJL/J Mice.

Multiple sclerosis (MS) is an immune-mediated inflammatory disease that leads to demyelination and neuronal loss in the central nervous system. Immune cells of lymphoid and myeloid origin play a significant role in the initiation and amplification of neuronal inflammation in MS. STAT3 signaling plays a pivotal role in both myeloid and lymphoid immune cells, such as neutrophils and CD4+ T cells, through regulation of their inflammatory potential. Dysregulation in STAT3 signaling in myeloid and lymphoid cell compartments has been reported in MS. In this report, we attempted to investigate the effect of a small molecular inhibitor of STAT3, i.e., Stattic, in a relapsing–remitting (RR) model of experimental autoimmune encephalomyelitis (EAE). The effect of Stattic was investigated for clinical features, oxidative stress parameters, and Th17-related signaling in both the periphery and brain of SJL/J mice. Our data report that p-STAT3 expression is elevated in granulocytes, CD4+ T cells, and brain tissue in myelin proteolipid protein (PLP)-immunized SJL/J mice, which is associated with the presence of clinical symptoms and upregulation of inflammatory markers in these cells/tissues. Treatment with Stattic leads to the amelioration of disease symptoms and attenuation of inflammatory markers in neutrophils (iNOS/nitrotyrosine/IL-1β), CD4+ T cells (IL-17A/IL-23R), and brain tissue (IL-17A/iNOS/IL-1β/MPO activity/lipid peroxides) in mice with EAE. These data suggest that the blockade of STAT3 signaling in cells of lymphoid and myeloid origin may cause the attenuation of systemic and neuronal inflammation, which could be responsible for the amelioration of disease symptoms in an RR model of EAE. Therefore, pharmacological inhibition of STAT3 in RRMS could be a potential therapeutic strategy.

Inhibition of HDAC6 Attenuates Diabetes-Induced Retinal Redox Imbalance and Microangiopathy.

We investigated the contributing role of the histone deacetylase 6 (HDAC6) to the early stages of diabetic retinopathy (DR). Furthermore, we examined the mechanism of action of HDAC6 in human retinal endothelial cells (HuREC) exposed to glucidic stress. Streptozotocin-induced diabetic rats (STZ-rats), a rat model of type 1 diabetes, were used as model of DR. HDAC6 expression and activity were increased in human diabetic postmortem donors and STZ-rat retinas and were augmented in HuREC exposed to glucidic stress (25 mM glucose). Administration of the HDAC6 specific inhibitor Tubastatin A (TS) (10 mg/kg) prevented retinal microvascular hyperpermeability and up-regulation of inflammatory markers. Furthermore, in STZ-rats, TS decreased the levels of senescence markers and rescued the expression and activity of the histone deacetylase sirtuin 1 (SIRT1), while downregulating the levels of free radicals and of the redox stress markers 4-hydroxynonenal (4-HNE) and nitrotyrosine (NT). The antioxidant effects of TS, consequent to HDAC6 inhibition, were associated with preservation of Nrf2-dependent gene expression and up-regulation of thioredoxin-1 activity. In vitro data, obtained from HuREC, exposed to glucidic stress, largely replicated the in vivo results further confirming the antioxidant effects of HDAC6 inhibition by TS in the diabetic rat retina. In summary, our data implicate HDAC6 activation in mediating hyperglycemia-induced retinal oxidative/nitrative stress leading to retinal microangiopathy and, potentially, DR.

MP80-01 RADIOPROTEOMIC ANALYSIS AS A POTENTIAL PREDICTOR OF RENAL TUMOR HISTOPATHOLOGY

You have accessJournal of UrologyKidney Cancer: Epidemiology & Evaluation/Staging/Surveillance III (MP80)1 Apr 2020MP80-01 RADIOPROTEOMIC ANALYSIS AS A POTENTIAL PREDICTOR OF RENAL TUMOR HISTOPATHOLOGY Jorge Daza*, Berengere Salome, Octavia Bane, Sara Lewis, John Sfakianos, Andrew Charap, Kirolos N. Meilika, Bheesham Dayal, Kennedy Okhawere, Ketan Badani, and Amir Horowitz Jorge Daza*Jorge Daza* More articles by this author , Berengere SalomeBerengere Salome More articles by this author , Octavia BaneOctavia Bane More articles by this author , Sara LewisSara Lewis More articles by this author , John SfakianosJohn Sfakianos More articles by this author , Andrew CharapAndrew Charap More articles by this author , Kirolos N. MeilikaKirolos N. Meilika More articles by this author , Bheesham DayalBheesham Dayal More articles by this author , Kennedy OkhawereKennedy Okhawere More articles by this author , Ketan BadaniKetan Badani More articles by this author , and Amir HorowitzAmir Horowitz More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000972.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Renal biopsy is the available option to evaluate tumor characteristics in small renal masses (SMRs). However, this technique has some drawbacks including potential complications and false negative results due to tumor heterogeneity. We aim to show a novel and comprehensive approach composed by multiparametric magnetic resonance imaging (mpMRI) and high throughput proteome analysis in urine and blood from patients with SMRs that can potentially be a novel diagnostic tool to identify benign or indolent tumors. METHODS: A cohort of 16 patients who underwent robotic partial nephrectomy (RPN) had a mpMRI. Histogram measures of diffusion-weighted MRI parameters in the tumor were compared between tumor subtypes by Mann-Whitney tests and one-way ANOVA. A high throughput analysis of the plasma and urine proteome using a panel of 92 inflammation related markers from Olink proteomics was obtained in 14 out 16 patients with previous mpMRI. Samples were collected at the day of surgery. NPX values for the biomarkers were compared across patients with benign, clear cell renal cell carcinoma (ccRCC) and non-ccRCC. T-test, p-value< 0.05 was considered statistically significant). RESULTS: Mean (p=0.048) and median (p=0.034) apparent diffusion coefficient (ADC), and mean (p=0.048) and median (p=0.048) diffusion parameter D were significantly elevated in ccRCC versus other subtypes. Protein expression clustering analysis of the markers with significant differential expression across the groups showed a predominant upregulation of the inflammation markers in ccRCC cases in urine (Fig 1). On the other hand, protein expression in plasma showed clustering of benign cases with a predominant downregulation of the analytes from the panel. Interestingly, when comparing ccRCC vs non-ccRCC, the latter cluster in a subset of cases with upregulation of inflammatory markers (Fig 2). CONCLUSIONS: Radioproteomic analysis showed to be a promising approach to differentiate across ccRCC, non-ccRCC and benign tumors and guide therapeutic approaches to different subtypes of RCC. Source of Funding: None © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e1224-e1224 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jorge Daza* More articles by this author Berengere Salome More articles by this author Octavia Bane More articles by this author Sara Lewis More articles by this author John Sfakianos More articles by this author Andrew Charap More articles by this author Kirolos N. Meilika More articles by this author Bheesham Dayal More articles by this author Kennedy Okhawere More articles by this author Ketan Badani More articles by this author Amir Horowitz More articles by this author Expand All Advertisement PDF downloadLoading ...

P146 OXPHOS INDUCTION IN HEALING: A CRITICAL MISSING STEP IN INFLAMMATORY BOWEL DISEASE?

Abstract Background In Inflammatory Bowel Disease (IBD), mucosal healing represents a key outcome in clinical remission. Mitochondrial dysfunction is one of the major features of IBD, which is hallmarked by increased oxidative stress and impaired ATP production. However, partial wound healing continues even with repressed mitochondrial respiration in an inflammatory environment. In this study, we demonstrate that intestinal epithelial cells (IEC) responding to colitis produce a gene signature with downregulated mitochondrial expression associated with oxidative phosphorylation (OxPHOS) and upregulate epithelial-to-mesenchymal transition (EMT). These data were consistent with the notion that EMT induced in IEC is fueled by glycolysis (repressed OxPHOS). We also wish to determine if this OxPHOS repression is present in normal healing. Methods We are actively enrolling patients with Ulcerative Colitis (UC) and normal patients into a prospective study evaluating biopsy site healing. Biopsy samples from UC patients were collected in Allprotect® or PBS. Biopsy samples collected in PBS were processed for IEC isolation using enzymatic digestion followed by flow sorting of EpCAM+ cells. To investigate the healing process in colitis and normal patients, the sigmoid colon is tattooed at initial endoscopy and 10–12 biopsies are collected in Allprotect®. Patients are brought back a week later for a flexible sigmoidoscopy. The tattooed area is examined, and biopsies are obtained from the previous biopsy sites (“biopsy of the biopsy”) and collected in Allprotect®. RNA is extracted from Allprotect® preserved tissue or isolated IECs and analyzed with RT-PCR. Results Genes corresponding to subunits of NADH dehydrogenase (Ubiquinone) of complex I (ND1, ND2, ND3, ND4, ND5 and ND6) were consistently downregulated in ulcer sites of colitis patients as compared to “biopsy of the biopsy” sites near the tattoo in normal patients. As expected, samples from colitis patients exhibited significant upregulation of inflammatory markers (iNOS, NLRP3, CCL2, RANTES, TGF-b1, CCL20 and CXCL10) compared to “biopsy of the biopsy” sites in normal patients undergoing healing. Intriguingly, epithelial marker E-cadherin was noted to be decreased in samples obtained from colitis patients with concomitant increase in mesenchymal markers (Vimentin, Snail, Twist-1 and Zeb-1) compared to previously biopsied normal controls. Discussion Our data suggests that repressed OxPHOS observed in inflamed IEC from colitis patients may serves as an inducer of EMT governing delayed but partial healing in IBD. We found this to be in stark opposition to healing in normal patients, who exhibit enhanced OxPHOS associated with rapid healing. These important findings indicate OxPHOS metabolism may be an important target for therapeutic agents to improve healing in refractory colitis.

Intraperitoneal administration of mesenchymal stem cells is effective at mitigating detrusor deterioration after pBOO.

Partial bladder outlet obstruction (pBOO) results in bladder fibrosis that is initiated by an inflammatory cascade and the decompensation after smooth muscle hypertrophy. We have been using an animal model to develop the hypothesis that mesenchymal stem cells (MSCs) are able to mitigate this cytokine cascade and prevent bladder deterioration. We hypothesized that intraperitoneal administration of MSCs can produce the same effects as intravenously administered cells but may require higher dosing. Intraperitoneal treatment will provide insights into the mechanisms of action and may offer advantages over intravenous administration, as it will permit allow higher doses and potentially reduce systemic exposure. Rats underwent a surgical induction of pBOO and instillation of either 1 × 106 or 5 × 106 commercially acquired MSCs into the peritoneum. RT-PCR, immunohistochemistry, and urodynamics were used to compare treatment groups with controls. pBOO resulted in a marked, statistically significant, upregulation of inflammatory markers in the bladder, including transforming growth factor-β, hypoxia-inducible factor-1α, hypoxia-inducible factor-3α, mammalian target of rapamycin, and collagen types I and III. Moderate but inconsistent levels of downregulation were seen with 1 × 106 MSCs, but excellent and reliable downregulation was seen with 5 × 106 MSCs (P < 0.05). Immunohistochemistry confirmed that protein levels were affected in accordance with mRNA upregulation. Urodynamics demonstrated MSC treatment resulted in whole organ physiological benefits, as they prevented elevations in detrusor pressure. In conclusion, intraperitoneal administration of MSCs resulted in a similar effect as intravenous administration; however, this required a higher dose. This has significant implications for determining the mechanism of action and potential clinical application for human therapy.

Myricetin Abrogates Cisplatin-Induced Oxidative Stress, Inflammatory Response, and Goblet Cell Disintegration in Colon of Wistar Rats.

Cisplatin [cis-diamminedichloroplatinum II] is an extensively prescribed drug in cancer chemotherapy; it is also useful for the treatment of diverse types of malignancies. Conversely, cisplatin is associated with a range of side effects such as nephrotoxicity, hepatotoxicity, gastrointestinal toxicity, and so on. Myricetin (3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-4chromenone) is a very common natural flavonoid found in fruits, tea, and plants. It has been found to have high-value pharmacological properties and strong health benefits. To examine the role of myricetin in colon toxicity induced by cisplatin, we conducted a concurrent prophylactic study in experimental animals that were treated orally with myricetin for 14 days at two doses—25 and 50 mg/kg of body weight. On the 14th day, a single intraperitoneal injection of cisplatin (7.5 mg/kg body weight) was administered in all groups except control. The effects of myricetin in cisplatin-induced toxicity in the colon were assessed in terms of antioxidant status, phase-II detoxification enzymes, the level of inflammatory markers, and goblet cell disintegration. Myricetin was found to restore the level of all the antioxidant enzymes analyzed in the study. In addition, the compound ameliorated cisplatin-induced lipid peroxidation, increase in xanthine oxidase activity, and phase-II detoxifying enzyme activity. Myricetin also attenuated deteriorative effects induced by cisplatin by regulating the level of molecular markers of inflammation (NF-κB, Nrf-2, IL-6, and TNF-α), restoring Nrf-2 levels, and controlling goblet cell disintegration. The current study reinforces the conclusion that myricetin exerts protection in colon toxicity via up-regulation of inflammatory markers, improving anti-oxidant status, and protecting tissue damage.

PRAS40 suppresses atherogenesis through inhibition of mTORC1-dependent pro-inflammatory signaling in endothelial cells

Endothelial pro-inflammatory activation plays a pivotal role in atherosclerosis, and many pro-inflammatory and atherogenic signals converge upon mechanistic target of rapamycin (mTOR). Inhibitors of mTOR complex 1 (mTORC1) reduced atherosclerosis in preclinical studies, but side effects including insulin resistance and dyslipidemia limit their clinical use in this context. Therefore, we investigated PRAS40, a cell type-specific endogenous modulator of mTORC1, as alternative target. Indeed, we previously found PRAS40 gene therapy to improve metabolic profile; however, its function in endothelial cells and its role in atherosclerosis remain unknown. Here we show that PRAS40 negatively regulates endothelial mTORC1 and pro-inflammatory signaling. Knockdown of PRAS40 in endothelial cells promoted TNFα-induced mTORC1 signaling, proliferation, upregulation of inflammatory markers and monocyte recruitment. In contrast, PRAS40-overexpression blocked mTORC1 and all measures of pro-inflammatory signaling. These effects were mimicked by pharmacological mTORC1-inhibition with torin1. In an in vivo model of atherogenic remodeling, mice with induced endothelium-specific PRAS40 deficiency showed enhanced endothelial pro-inflammatory activation as well as increased neointimal hyperplasia and atherosclerotic lesion formation. These data indicate that PRAS40 suppresses atherosclerosis via inhibition of endothelial mTORC1-mediated pro-inflammatory signaling. In conjunction with its favourable effects on metabolic homeostasis, this renders PRAS40 a potential target for the treatment of atherosclerosis.

072 The Inflammatory Response and FL2 (an inhibitor of axonal growth) is Deferentially Modulated in Animal Models of Nerve Sparing Prostatectomy and Laparotomy

Radical prostatectomy (RP), including nerve sparing RP (NSRP), carries high risk of erectile dysfunction (ED) through a mechanism primarily thought to involve neuropraxia of the cavernous nerve (CN). Activation of the inflammatory response has been proposed to contribute to neuropraxia. In preliminary studies, using a rat model of NSRP, we observed significant ED 3-days postsurgery accompanied by upregulation of inflammatory markers in the CN. We have also observed that using siRNA to downregulate an inhibitor of axonal growth called Fidgetin-like 2 protein (FL2) in a rat model of bilateral CN transection, promoted a more rapid regeneration of the CN and recovery of erectile function. Given the proposed association between inflammation, FL2 and CN repair, the objective of the present study was to determine if even in the absence of direct damage to the CN, trauma associated with NSRP triggers an inflammatory response or modulation of FL2 expression which contributes to neuropraxia and ED.

The changes in immune cell concentration during the progression of pre-diabetes to type 2 diabetes in a high-fat high-carbohydrate diet-induced pre-diabetic rat model

Pre-diabetes is a long-lasting condition that precedes type 2 diabetes (T2D). T2D has been shown to suppress the immune response. However, it remains unclear if immune activation occurs before the onset of T2D during the progression of the pre-diabetic state. This study sought to characterize the changes in general immunity occurring during the progression from pre-diabetes to T2D. Male rats were fed a high-fat high-carbohydrate diet for 20 weeks (pre-diabetes induction period) and kept on the same diet being monitored for a further 12 weeks (experimental period). Blood was collected for haemocytometer analysis on week 0, 4, 8, and 12 of the experimental period after which the animals were sacrificed. Plasma was collected from centrifuged blood for ELISA (TNF-α, CRP, P-selectin, CD40 L, fibrinogen, and IL-6). Blood neutrophils percentage significantly decreased at week 12 possibly due to recruited neutrophils migrating to an inflamed area such as visceral adipose tissue as further observed. Due to hyperglycaemia, there was significant increase in blood lymphocytes percentage at week 12. Blood monocytes percentage significantly increased at week 12. Monocytes recruited and circulated in blood due to hyperglycaemia for glucose uptake to decrease it from circulation. Blood eosinophils percentage significantly decreased at week 12. Eosinophils migrated to inflamed areas such as visceral adipose tissue as further observed. Blood basophils percentage significantly increased due to their recruitment and activation. TNF-α, CRP, and IL-6 increased significantly after 12 weeks. There was also upregulation of fibrinogen, P-selectin, and CD40L. The results of this study show that there are changes in immune cells concentration and that immune cells such as neutrophils and eosinophils migrate to inflamed areas such as adipose tissue. There is also upregulation of various inflammatory cytokines. Based on these findings, immune activation begins during the pre-diabetic state as there is upregulation of inflammatory markers.

Pathological Clotting and Deep Vein Thrombosis in Patients with HIV.

The number of people infected with human immunodeficiency virus (HIV) is rapidly increasing and the majority of those infected are living in sub-Saharan Africa. Some hallmarks of HIV are inflammation and upregulation of inflammatory markers. A pathological coagulation system may accompany these inflammatory changes and potentially result in venous thromboembolism such as a deep vein thrombosis (DVT). In this review, the authors describe the inflammatory profile in HIV, the treatment regimens currently in place in South Africa, and in particular how HIV affects the hematological system, with specific focus on platelets, red blood cells (RBCs; erythrocytes), and fibrin(ogen). They also discuss the presence of DVT in HIV, focus on screening tests, and suggest a more proactive approach to track the inflammatory profile of HIV patients, by specifically using parameters that might point to pathological coagulation; these should involve platelet, RBC, and fibrin(ogen) analysis. They conclude by suggesting that including coagulation function tests to study the effect of treatment interventions would improve outcomes in these individuals, as it could help in the diagnosis of thromboembolic disease. Furthermore, this approach could streamline treatment strategies due to improved monitoring. A better understanding of hypercoagulability of HIV-infected patients is therefore urgently needed. In conclusion, the authors suggest a panel of pathology tests that should be considered as standard procedures when HIV is present.