ACE Levels Research Articles

MO287: A Recombinant BIO-HDL (CER-001) Can Prevent SARS-COV2-Induced Renal Dysfunction by Restoring SR-BI Signalling

BACKGROUND AND AIMSReplication of the enveloped SARS-COV2 virus can alter lipidomic composition and metabolism of infected cells [1]. These alterations commonly result in a decline in HDL, total cholesterol and LDL, and an increase in triglyceride levels in COVID-19 patients. Furthermore, the ‘cytokine storm’ subsequent to release of inflammatory cytokines can severely impair lipid homeostasis. Importantly, decreased HDL-cholesterol correlates with severity of COVID-19 infection and represents a significant prognostic factor in predicting poor clinical outcomes [2]. Similarly, it has been observed that COVID-19 patients’ recovery is accompanied by a rise in serum HDL levels. Pharmacological intervention that aims to restore ApoA-1 or functional HDL particles may have beneficial roles for clinical outcome of COVID-19 patients and has recently been approved for compassionate use [3].SARS-CoV 2 spike proteins S1 and S2 can bind free cholesterol and HDL-bound cholesterol, facilitating virus entry by binding the ACE2 co-receptor Scavenger Receptor-BI (SR-BI) [4]. When activated at the trans-membrane level, SR-BI signalling culminates in Ser1173-eNOS phosphorylation with both anti-inflammatory and anti-apoptotic effect. We hypothesized that SARS-COV2 binding promoted SR-BI internalization, so that it could not exert its essential protective function. Therefore, the aim of this study is to evaluate the effects of CER-001, a mimetic HDL, in antagonizing this process.METHODEndothelial and tubular (RPTEC) cells were exposed to S1, S2 and S1 + S2 (50–250 nM) with or without CER-001 (CER-001 50–500 ug/mL) and cholesterol (10–50 uM). Apoptosis tests (MTT and AnnV/PI) were performed. Internalization of SR-BI, ACE2 with S1 and activation of eNOS was evaluated by FACS analysis. SR-BI and ACE2 expression were evaluated on kidney biopsies from COVID-19 patients.RESULTSAt concentrations used, the exposition of S1, S2 and S1 + S2 in the presence of CER-001 and cholesterol did not induce apoptosis of endothelial cells and RPTEC. Endothelial and tubular cells stimulated by S1, in presence of cholesterol, showed an increased intracellular level of SR-BI and ACE-2, with significantly reduced eNOS phosphorylation compared to baseline (P < 0.05). The treatment with CER-001 reversed trans-membrane SR-BI levels and eNOS phosphorylation to baseline values. The detection of S1 spike protein by endothelial cells immunohistochemistry revealed an increased level in S1-exposed cells with cholesterol and reduced S1 intracellular positive staining in CER-001-exposed cells (P < 0.05). Interestingly, S1-exposed cells without cholesterol appeared not to be capable of mediating S1 spike protein internalization.Consistent with in vitro results, analysis of renal biopsies from COVID-19 patients with proteinuria showed increased SR-BI and ACE-2 cytoplasmic signals and reduced expression at the apical domain of injured tubules.CONCLUSIONOur data confirmed the key role of lipid profile in SARS-COV2 infection, evaluating the molecular signalling involved in HDL metabolism and inflammatory processes, and could offer new therapeutic strategies for COVID-19 patients.

MO041: Impaired ACE2 glycosylation and protease activity lowers susceptibility to SARS-COV-2 infection in Gitelman/Bartter syndromes

BACKGROUND AND AIMSACE2, part of the counter-regulatory arm of the renin–angiotensin system, serves both as protective toward oxidative stress and cardiovascular remodeling and as a key entry for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ACE2 has two isoforms, non-glycosylated and glycosylated, the latter being accountable for the binding with SARS-CoV-2. After the binding, viruses use proteases as cathepsin-L (Cat-L) to entry the cells. Both ACE2 glycosylation and Cat-L activity are pH-dependent. Gitelman and Bartter syndromes (GS/BS), rare genetic tubulopathies, are characterized by electrolytic alterations, activation of the renin–angiotensin system, yet normo-hypotension, increased levels of ACE2 and metabolic alkalosis with likely increased intracellular pH. We reported that during the first wave of COVID-19 in early 2020 none of our cohort of 128 GS/BS patients from the major hotspots in Northern Italy had been infected or suffered any major COVID-19 symptoms and in a second survey on the same cohort in 2021, we reported only eight positives, four asymptomatic and four with very light symptoms This study aims to investigate potential mechanisms as ACE2 glycosylation and Cat-L activity related to patients’ metabolic alkalosis and viral entry/infection.METHODMononuclear cells ACE2 glycosylation (Western blot) and blood Cat-L activity (ELISA) from 20 GS/BS patients have been compared with those from 15 healthy subjects.RESULTSNon-glycosylated ACE2 was higher in GS/BS (0.82 ± 0.19 d.u. versus 0.67 ± 0.13, P = 0.01); glycosylated ACE2 was not different (0.85 ± 0.28 in GS/BS versus 0.73 ± 0.23, P = 0.19). Cat-L activity was lower in GS/BS (3.90 ± 1.13 r.f.u. versus 5.31 ± 0.8, P <0 0.001) and inversely correlated with blood bicarbonate (HCO3−), while a negative correlation between glycosylated ACE2 and HCO3− approaches statistical significance (P = 0.08).CONCLUSIONGS/BS's metabolic alkalosis, likely by increasing intracellular pH, influences the glycosylation of ACE2 and the activity of Cat-L, providing a mechanistic explanation for the near-complete absence of COVID-19 or its symptoms reported in our cohort. These findings provide a rationale for pursuing the identification and/or synthesis of new drugs that specifically target ACE2 glycosylation and/or proteases involved in SARS-CoV-2 infection.

Dietary Empagliflozin Regulates Angiotensin Converting Enzyme 2 (ACE2) and Transmembrane Serine Protease 2 (TMPRSS2) in Kidney Cortex of Mice

Persons with type 2 diabetes mellitus (T2D) are at greater risk for poor prognosis, including renal pathology, when infected with Covid‐19 virus. A newer class of medications for T2D is the gliflozin class, including Empagliflozin (EMPA), which inhibits the renal proximal tubule sodium glucose cotransporter, type 2 (SGLT2) resulting in glucosuria and reduction in hyperglycemia. Whether these medications alter susceptibility and responses to Covid‐19, and similar infections, is not known. The angiotensin 2 converting enzyme (ACE2) is the receptor for Covid‐19, while transmembrane serine protease 2 (TMPRSS2) is a proteinase expressed on the surface of epithelial cells and facilitates virus uptake. In general, ACE2 is thought to be renoprotective, as it converts Ang I (Ang 1‐10) to Ang 1‐9 and Ang II (Ang 1‐8) to Ang 1‐7. Ang 1‐8, in particular, is a potent vasoconstrictor and natriferic agent. Our aim was to determine how chronic EMPA affected ACE2 and TMPRSS2 protein levels and ACE2 activity in the kidney cortex of mice. Adult male (M) and female (F) mice of three strains (n = 8/group), i.e., C57Bl6 (C57), Swiss Webster (SW), and Tally Ho/Jng (TH) were fed high‐fat control diet (C, 60% milk‐fat) or EMPA‐containing high‐fat diet (E, 0.01%) for 12 weeks. TH mice are insulin resistant and obese, with a tendency to develop T2D over time. SW are lean and the closest genetically related background strain to TH. EMPA increased glucosuria but not body weight (p = 0.38 for treatment, 3‐way ANOVA); however kidney weight (normalized to body weight) was 2‐22% increased (depending on group) by EMPA (p = 0.002 for treatment). Kidney cortex protein levels of ACE2 and TMPRSS2 were determined by conventional western blotting (to observe band size and antibody specificity) and by dot blotting (to compare all samples on the same blot). Dot blotting revealed significantly higher ACE2 protein in males (p < 0.0001) relative to females and in the TH and SW strains (p < 0.0001), relative to C57. There was a significant sex‐by‐strain interaction in that the TH females were not significantly lower than TH males. Similarly, cortical TMPRSS2 was lower in females (p < 0.0001) and in C57 mice (p < 0.0001), but also reduced by EMPA (p = 0.030). ACE2 activity was determined in 10 μg kidney cortex by calculating the slope (over 2 hours) of the difference in fluorescence generated in the presence of the ACE2 substrate (Mca‐YVADAPK(Dnp)‐OH) with the addition of the ACE2‐specific antagonist (MLN) minus fluorescence with addition of both captopril (ACE antagonist) plus MLN. Slopes were: (RFU/min, mean ± sem): MC57C‐ 19.9 ± 5.5; MC57E‐ 31.8 ± 5.2; MSWC‐ 20.2 ± 1.9; MSWE‐ 25.7 ± 3.6; MTHC‐ 18.8 ± 3.6; MTHE‐ 34.7 ± 11.3; FC57C‐ 4.7 ± 1.2; FC57E‐ 9.7 ± 3.4; FSWC‐ 4.9 ± 1.2; FSWE‐ 4.4 ± 1.8; FTHC‐ 9.0 ± 1.8; and FTHE‐ 16.2 ± 2.3. Three‐way ANOVA revealed significant effects of both sex (p < 0.0001) and treatment (p = 0.0052) in that Empagliflozin and male sex increased the slope (activity). Overall, male sex was the greatest determinant of differences in ACE2 protein and activity and TMPRSS2 protein. EMPA increased ACE2 activity, but reduced TMPRSS2 protein levels. In general, sex differences were attenuated in the TH strain. There is the potential for these relative increases to play a role in increased sensitivity of male, obese, and/or T2D subjects to pathology associated with Covid‐19; however, the beneficial effect of enhanced ACE2 to reduce Ang II may also provide countering protection.

Lack of Xdh Leads to Alterations in Renin‐Angiotensin‐Aldosterone System and Kidney Injury

Uric acid (UA) homeostasis has been elucidated as a key player in chronic kidney disease and cardiovascular diseases, yet definitive mechanisms are unknown. Previous studies in rodents and humans have shown that increased levels of serum UA predict an activated intrarenal Renin‐Angiotensin‐Aldosterone System (RAAS). In hyperuricemic rats, nephropathy was reported with glomerular hypertrophy and interstitial inflammation accompanied by increased renal renin expression. To explore whether hypouricemia leads to RAAS changes and kidney damage, we utilized a novel rat model with genetic ablation of the Xdh gene in the Dahl salt‐sensitive rat background (SSXdh‐/‐). The Xdh gene encodes for the Xanthine Oxidoreductase enzyme group, which produces UA from xanthine and hypoxanthine. We have shown that the SSXdh‐/‐model is hypouricemic and has severe kidney damage. The SSXdh‐/‐ rats demonstrated significant kidney function decline (SSXdh+/+&amp; SSXdh‐/‐respectively, diuresis: 2.7 ± 0.9 &amp; 14.4 ± 5.1 ml; creatinine clearance: 0.51 ± 0.19 &amp; 0.12 ± 0.04 ml/min; and plasma Na+: 136 ± 1.8 &amp; 150 ± 3.7 mmol/l). Quantification of RAAS components was done using liquid chromatography‐tandem mass spectrometry. The comparison of plasma from SSXdh+/+vs SSXdh‐/‐rats revealed significantly lower levels of angiotensin I (650 ± 109, 178 ± 25 pmol/l), Ang II (583 ± 70, 171 ± 16 pmol/l), and renin (indirect measurements using Ang I + Ang II: 1233 ± 172, 349 ± 40 pmol/l). Aldosterone level was increased in SSXdh‐/‐rats (870 ± 115 vs. 4106 ± 1038 pmol/l). The aldosterone/ Ang II ratio was significantly increased in the homozygous rats suggesting enhanced adrenal function (2 ± 0.1, 25 ± 6.3). To further evaluate the contribution of RAAS and its downstream signaling mechanisms, we performed RNA‐Seq analysis on kidney cortex tissue, which showed a number of RAAS‐related genes being differentially expressed between the SSXdh+/+and SSXdh‐/‐ rats. In hypouricemic rats, there was an increase in expression levels of Ace, Ccl2, Fos, Map3k1, Ptger2,and Stat3,and a decrease of Agtr1a, Hsd11b1,and Renb. Therefore, our results show that lack of Xdh/UA leads to kidney injury and functional decline with substantial remodeling in RAAS.

ACE2 and TMPRSS2 Immunolocalization and COVID-19-Related Thyroid Disorder.

Simple SummaryWe analyzed the underlying mechanism of thyroidal manifestations in patients with COVID-19 using ACE2 and TMPRSS2 immunostaining. Prior to our work, few studies had investigated the mRNA expression of ACE2 in the thyroid gland. However, there has been no tissue-level study based on histological staining of ACE2 and TMPRSS2. Here, we found for the first time that ACE2 and TMPRSS2 proteins were not expressed in thyroid follicular cells and that ACE2 was exclusively expressed in thyroidal pericytes by immunolocalization of ACE2 and TMPRSS2 in the human thyroid tissue. The results of the present study demonstrate that SARS-CoV-2 does not directly invade thyroid follicular cells, but microcirculatory damage caused by pericyte infection may affect surrounding follicles in the thyroid gland.Thyroid dysfunction has been reported to be an extrapulmonary symptom of COVID-19. It is important to identify the tissue subset that expresses angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2), which are essential for host infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in order to understand the viral pathogenesis of COVID-19-related thyroid dysfunction. We investigated the expression and distribution of ACE2- and TMPRSS2-expressing cells in the thyroid gland. RT-PCR and Western blotting were performed on human thyroid follicular cells (Nthy-ori3-1) and rat thyroid tissues to detect the expression levels of ACE and TMPRSS2 mRNA and proteins. We also analyzed the expression patterns of ACE2 and TMPRSS2 in 9 Sprague-Dawley rats and 15 human thyroid tissues, including 5 normal, 5 with Hashimoto’s thyroiditis, and 5 with Graves’ disease, by immunohistochemistry (IHC) and immunofluorescence. Both ACE2 and TMPRSS2 mRNAs and proteins were detected in the thyroid tissue. However, ACE2 and TMPRSS2 proteins were not expressed in thyroid follicular cells. In IHC, ACE2 and TMPRSS2 were not stained in the follicular cells. No cells co-expressed ACE2 and TMPRSS2. ACE2 was expressed in pericytes between follicles, and TMPRSS2 was mainly stained in the colloid inside the follicle. There was no difference in expression between the normal thyroid, Hashimoto’s thyroiditis, and Graves’ disease. SARS-CoV-2 does not directly invade the thyroid follicular cells. Whether SARS-CoV-2 infection of pericytes can affect COVID-19-related thyroid dysfunction warrants further study.

The Effect of Hydroalcoholic Seed Extract of Securigera Securidaca on the Hepatic Renin-Angiotensin System in the Streptozotocin-Induced Diabetic Animal Model

Background: The aim of the study was to investigate the effect of hydroalcoholic seed extract of Securigera securidaca (S. securidaca) (HESS) on liver local reninangiotensin system (RAS) in streptozotocin-induced diabetic rats. Methods: Three groups of diabetic male Wistar rats were treated with different doses of HESS (100, 200, 400 mg/Kg-BW), and the results were compared with diabetic and healthy control groups. To test the effects of HESS on liver local RAS as well as its alternative pathway, the tissue levels of renin, angiotensin-converting enzyme (ACE), ACE2, angiotensin-II (Ang-II), and Ang-(1-7) were measured. The oxidative state of liver tissue was evaluated by biomarkers of malondialdehyde (MDA), total oxidant status (TOS), and total antioxidant status (TAS). Due to the association between local RAS activity and tissue inflammation, the production of interleukins (IL) IL-1, IL-6, tumor necrosis factor alfa (TNF-a), and IL-10 in the liver was assayed in the experimental group. Results: Dose-dependent effects of HESS showed that the highest dose of the extract had a reducing effect on the hepatic levels of local RAS components including angiotensinogen, ACE, and Ang-II. Surprisingly, despite the decrease in tissue level of ACE2, an increase in Ang-(1-7) tissue concentration was observed. Decreased local RAS activity through treatment with the highest dose of HESS was associated with decreased tissue levels of proinflammatory cytokines (IL-1, IL-6, TNF-a), and increased anti-inflammatory cytokine (IL-10). Most of the effects of the extract are attributed to its antioxidant properties. Conclusions: S. securidaca seed can be suggested as a suitable drug supplement to prevent hepatic complications of diabetes.

Correlation between Urinary Angiotensin Converting Enzyme-2 &amp; Micro Albuminuria in Non-Diabetic-Hypertensive and Type 2 Diabetic-Hypertensive patients

Background: The Renin Angiotensin System (RAS) is one of the main contributors to insulin resistance which leads to T2DM and its complications. The ACE2/Angiotensin-(1-7)/Mas axis has lately been anticipated to function as a negative regulator of the RAS, consequently, rendering a shielding task against progress of type 2 diabetes, as well as, lowering blood pressure. Aim: To correlate urinary angiotensin 2 levels and microalbuminuria in Type 2 Diabetic and non-Diabetic-hypertensive patients. Methods: A particular population of selected subjects was categorized into 2 groups, with 49 subjects in each group. Diabetic and hypertensive patients were selected from the SIMS diabetic Clinic and medical wards, Lahore. Anthrometric parameters, as well as, BSR were recorded in the clinics. Blood and urine samples were collected and stored for evaluating the biochemical parameters in the Physiology Laboratory, UHS. Results: Microalbuminuria, as well as urinary ACE2 levels in group A (non-diabetic-hypertensive subjects), were greater than in group B (diabetic-hypertensive subjects). There was a significant positive correlation between uACE2 levels and microalbuminuria in group A patients. Conclusion: Although the current study reinforced the fact that both T2DM and hypertension are risk factors for CKD, contrary to our prediction based on evidence gained from various research studies, we were unable to prove that urinary ACE 2 levels are higher in patients with hypertension compounded with diabetes mellitus. Keywords: The Renin Angiotensin System (RAS), angiotensin converting enzyme 2, microalbuminuria levels.

P037 A patient with systemic inflammation, relapsing polychondritis and pancytopaenia: VEXAS

Abstract Background/Aims We report a patient presenting with an inflammatory illness consistent with relapsing polychondritis (RP) and a pancytopaenia. Testing of his bone marrow revealed confirmation of the somatic mutation for the newly recognised condition VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. VEXAS is an acquired, adult-onset autoinflammatory syndrome which is progressive and frequently fatal. First reported in October 2020 by Beck et al, it results from somatic mutations at methionine-41 (p.Met41) of UBA1 gene, the major E1 enzyme that initiates ubiquitylation. Clinical features of VEXAS include periodic fevers, neutrophilic dermatoses, pulmonary infiltrates, chondritis and venous thromboembolic events. Bone marrow biopsy (BMB) shows vacuoles in myeloid precursor cells, and these cause hematologic abnormalities like myelodysplastic syndrome, multiple myeloma, or monoclonal gammopathy of unknown significance. Methods Clinical details were collected retrospectively. UBA1 sequencing: UBA1 exon 3 (NM_153280) was amplified from using High-Fidelity Phusion Polymerase (Life Tech) and primers UBA1-3F and UBA1-3R. PCR products were sequenced using the BigDye v3.1 terminator sequencing kit on an ABI3130xl (Life Tech). Electropherograms were analysed using 4Peaks software. Results A previously fit 63-year-old gentleman presented with a three-month history of recurrent fevers, night sweats and intermittent cough. At presentation he also had left testicular pain. Bloods showed neutropenia (1.9x10^9/L), normocytic anaemia (128g/L) and raised CRP (98mg/L). However, his symptoms progressed with headaches, myalgia, conjunctivitis, nasal chondritis and sinusitis. His CRP rose to 149mg/L and ferritin was 886ng/mL. He had a negative PET scan, autoimmune screen, myeloma screen and normal levels of complement, CK and ACE. Furthermore, multiple blood cultures and infectious disease screening tests were negative. He subsequently developed a left auricular chondritis. Within a week of commencing prednisolone 40mg and methotrexate 15mg his symptoms improved by 80% and his CRP was 10mg/L. At 15mg of prednisolone his CRP rose (134mg/L) and his fevers, night sweats and arthralgia returned. His prednisolone was then increased to 20mg. Over the next nine months his disease progressed despite prednisolone and methotrexate. He developed worsening cytopenias (Hb86/L, plts105x109/L, MCV117fL), bilateral sclerouveitis, inflammatory joint pains, vasculitic rash, chest pain and dyspnoea on exertion. CTPA and HRCT showed no significant abnormalities. VEXAS syndrome was diagnosed after BMB testing identified the UBA1 mutation c.122T&amp;gt;c, p.Met41Thr. His treatment now encompasses prednisolone and Tocilizumab. His cytopenias are transfusion dependent, he has been under review to consider BMT although is currently stable clinically. Conclusion VEXAS syndrome should be considered in men over 50 with treatment-refractory giant cell arteritis, RP, polyarteritis nodosa and Sweet’s syndrome. VEXAS has a significant mortality and developing effective treatments is a priority. Given there is an available confirmatory test early recognition and subsequent testing for the UBA1 mutation will help physicians to identify appropriate treatment options and avoid unnecessary investigations. Disclosure S. Sajawal: None. M. Green: None. S. Savic: Consultancies; Novartis, SOBI, TAKEDA. Member of speakers’ bureau; Novartis, SOBI. Grants/research support; SOBI.

Regulation of Proinflammatory Molecules and Tissue Factor by SARS-CoV-2 Spike Protein in Human Placental Cells: Implications for SARS-CoV-2 Pathogenesis in Pregnant Women.

SARS-CoV-2 infects cells via binding to ACE2 and TMPRSS2, which allows the virus to fuse with host cells. The viral RNA is detected in the placenta of SARS-CoV-2-infected pregnant women and infection is associated with adverse pregnancy complications. Therefore, we hypothesize that SARS-CoV-2 infection of placental cells induces pro-inflammatory cytokine release to contribute to placental dysfunction and impaired pregnancy outcomes. First, expression of ACE2 and TMPRSS2 was measured by qPCR in human primary cultured term cytotrophoblasts (CTBs), syncytiotrophoblast (STBs), term and first trimester decidual cells (TDCs and FTDCs, respectively), endometrial stromal cells (HESCs) as well as trophoblast cell lines HTR8, JEG3, placental microvascular endothelial cells (PMVECs) and endometrial endothelial cells (HEECs). Later, cultured HTR8, JEG3, PMVECs and HEECs were treated with 10, 100, 1000 ng/ml of recombinant (rh-) SARS-CoV-2 S-protein ± 10 ng/ml rh-IFNγ. Pro-inflammatory cytokines IL-1β, 6 and 8, chemokines CCL2, CCL5, CXCL9 and CXCL10 as well as tissue factor (F3), the primary initiator of the extrinsic coagulation cascade, were measured by qPCR as well as secreted IL-6 and IL-8 levels were measured by ELISA. Immunohistochemical staining for SARS-CoV-2 spike protein was performed in placental specimens from SARS-CoV-2–positive and normal pregnancies. ACE2 levels were significantly higher in CTBs and STBs vs. TDCs, FTDCs and HESCs, while TMPRSS2 levels were not detected in TDCs, FTDCs and HESCs. HTR8 and JEG3 express ACE2 and TMPRSS2, while PMVECs and HEECs express only ACE2, but not TMPRSS2. rh-S-protein increased proinflammatory cytokines and chemokines levels in both trophoblast and endothelial cells, whereas rh-S-protein only elevated F3 levels in endothelial cells. rh-IFNγ ± rh-S-protein augments expression of cytokines and chemokines in trophoblast and endothelial cells. Elevated F3 expression by rh-IFNγ ± S-protein was observed only in PMVECs. In placental specimens from SARS-CoV-2-infected mothers, endothelial cells displayed higher immunoreactivity against spike protein. These findings indicated that SARS-CoV-2 infection in placental cells: 1) induces pro-inflammatory cytokine and chemokine release, which may contribute to the cytokine storm observed in severely infected pregnant women and related placental dysfunction; and 2) elevates F3 expression that may trigger systemic or placental thrombosis.

Whole Exome Sequencing Identifies a Rare Mutation in NACAD as a Possible Cause of COVID orchitis in Brothers

IntroductionLittle is known of SARS-CoV2 (COVID-19) pathogenicity, but organ inflammation syndromes have been well observed and documented in the lungs, heart, liver, kidneys, and testes. While the blood-testis barrier is known to protect testes from antigens, current evidence suggests COVID-19 can affect the testis. Reports of bilateral orchitis are growing. Current hypotheses consider causes of (1) cellular induced cytokine storm or (2) IgG deposits in the vascular endothelium to be at blame but causal genes or underlying genetic susceptibility have not yet been identified.ObjectiveThis project sought to uncover genetic explanations as to why certain men face increased susceptibility to developing COVID orchitis.MethodsWe identified and examined six COVID-19 patients who all were confirmed with polymerase chain reaction (PCR), including three COVID-19 (+) men without orchitis (controls) and three COVID (+) men with orchitis (bilateral testicular pain for at least 5 days around the time of testing PCR positive). Of note, among the three men with COVID-19 who had orchitis, two of them were siblings. DNA extraction and whole exome sequencing were performed on blood using the QIAmp blood maxi kit on five of the six patients. Variants were prioritized by being shared between the three patients affected with orchitis, absent in controls, and introducing nonsense, frameshift, splicing or non‐synonymous amino acid changes and less than 10% in population prevalence. Based on WES findings, DuoSet® Human ACE2 reagent kit 2 (catalog number: DY933-05) was purchased from R&D Systems, USA, and used to measure the level of soluble ACE2 in the plasma samples.ResultsThe average age of the men in the study was 25 years old. The average duration of COVID symptoms (fever, sore throat, cough, body aches) were 7 days. Among the men who developed bilateral testis pain, the symptoms lasted for an average of 22 days. A list of 16 variants was generated that found to be shared between the two siblings with COVID orchitis along with the unrelated subject with COVID orchitis, and not present in the two controls. Among the 16 variants, a nonsynonymous non-frameshit deletion in NACAD variant on chromosome 7 with a frequency of 3.9% prevalence in ExAC was prioritized based on known involvement in the ACE2 pathway, read depth, and genotype quality. Phenotypically, we found that circulating levels of soluble ACE2 was 3.72 ng/ml among men who had COVID orchitis and was lower than men who developed COVID without orchitis.ConclusionsWe observed a stop mutation in NACAD in 2 brothers and 1 unrelated man who developed COVID orchitis. Interestingly, we found lower circulating ACE2 serum levels in both brothers with orchitis and the one nonrelated orchitis subject but normal serum levels in all controls. NACAD when involved with cellular ability to shuttle out ACE2 becomes critical for COVID symptomatology. With decreased transcellular and extracellular transport of ACE2 being possible in subjects with the gene mutation, it can be postulated more ACE2 will be found intracellularly leading to increased cellular entry of SARS CoV-2 and possibility of orchitis sequelae.DisclosureNo

Structural and functional impact by SARS-CoV-2 Omicron spike mutations.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), bearing an unusually high number of mutations, has become a dominant strain in many countries within several weeks. We report here structural, functional, and antigenic properties of its full-length spike (S) protein with a native sequence in comparison with those of previously prevalent variants. Omicron S requires a substantially higher level of host receptor ACE2 for efficient membrane fusion than other variants, possibly explaining its unexpected cellular tropism. Mutations not only remodel the antigenic structure of the N-terminal domain of the S protein but also alter the surface of the receptor-binding domain in a way not seen in other variants, consistent with its remarkable resistance to neutralizing antibodies. These results suggest that Omicron S has acquired an extraordinary ability to evade host immunity by excessive mutations, which also compromise its fusogenic capability.

Strong ACE-2 expression in the choroidal vessels: do high choroid plexuses serve as a gateway for SARS-CoV-2 infection on the human brain?

Previous studies have confirmed the key mechanism by which SARS-CoV-2 enters human cells. It is well established that ACE2 is the receptor that can mark the beginning of the infection. In light of this, the organs that express higher levels of ACE2 are generally considered at higher risk, while those with lower levels should be somehow more protected. This - if related to the scarcity of ace2-expressing cells in the brain - seems to contrast with the presence of a variety of neurological symptoms that follow infection with ace2. The aim of this work was to analyze ACE2 expression in the human brain, focusing on the choroid plexuses. Twenty brain samples were obtained at autopsy from ten human fetuses and from ten adult subjects. All samples were selected to contain the choroid plexus. Specimens were fixed in 10% formalin, routinely processed and paraffin embedded. 5-micron sections were stained with Hematoxylin and Eosin (H&E) and immunostained with a commercial anti-human ACE2 rabbit monoclonal antibody at 1:100 dilution. We analyzed 20 samples by immunohistochemistry, and we noted that, as far as fetal samples are concerned, a strong reactivity for ACE2 was detected in the myxoid stroma of the choroid plexuses and in the endothelial cells in fetuses. The complete absence of the ACE2 marker was detected in epithelial cells, neurons and glial cells of the cerebral cortex, both in fetuses and in adults. Whereas a strong but selective reactivity for ACE2 was also detected in adult choroid plexuses, mainly localized in the endothelial cells of the choroid capillaries. Our study shows a strong expression of ACE in the fetal and adult brain choroid plexuses. This new histopathological finding may clarify the susceptibility of the human brain to SARS-COV-2 infection. Our data indicate the choroid plexus as the entry gate of virus for in the human brain; therefore, the entrance of SARS-CoV-2 into the cerebrospinal fluid through the choroid plexuses might represent the mechanism utilized by this coronavirus to cause direct injury to brain cells.

ACE2 and ANGII levels in patients with COVID-19 based on thoracic tomography findings and PCR test results

Reverse transcriptase polymerase chain reaction tests and thoracic tomography have been widely employed in the diagnosis of the disease, but doubts about their sensitivity still persist. Also there are controversial results about ACE2 and AngII levels according to the severity of disease. In this study, we aimed to analyze the ACE2 and AngII levels in patients with suspected COVID-19 based on polymerase chain reaction test results and thoracic tomography findings and to examine their relationship with disease severity. Patients with suspected COVID-19 in the emergency department were divided into 4 groups according to thoracic tomography findings and PCR test results. The in-hospital mortality of patients was recorded. ACE2 and AngII levels in patients were analyzed according to groups and severity of the disease. ACE2 levels for the patients with suspected COVID-19 were significantly lower than in the control group, but AngII levels were higher (not statistically significant). The mean age and male sex ratio of patients who developed acute respiratory distress syndrome (ARDS) and died were significantly higher than those who survived. Whereas there was no difference in ACE2 levels in patients with severe diseases such as ARDS and mortality, their AngII levels were significantly lower. It can be suggested that decreased ACE2 levels combined with increased AngII levels are determinative at disease onset and in the development of lung damage. However, decreased AngII levels are more determinative in patients with severe diseases such as ARDS and mortality.

ACE2 Serum Levels as Predictor of Infectability and Outcome in COVID-19.

BackgroundCOVID‐19 can generate a broad spectrum of severity and symptoms. Many studies analysed the determinants of severity but not among some types of symptoms. More importantly, very few studies analysed patients highly exposed to the virus that nonetheless remain uninfected.MethodsWe analysed serum levels of ACE2, Angiotensin II and anti-Spike antibodies in 2 different cohorts at high risk of viral exposure, highly exposed but uninfected subjects, either high risk health care workers or persons cohabiting with infected close relatives and seropositive patients with symptoms. We tested the ability of the sera of these subjects to neutralize lentivirus pseudotyped with the Spike-protein.ResultsWe found that the serum levels of ACE2 are significantly higher in highly exposed but uninfected subjects. Moreover, sera from this seronegative persons can neutralize SARS-CoV-2 infection in cellular assays more strongly that sera from non-exposed negative controls eventhough they do not have anti-CoV-2 IgG antibodies suggesting that high levels of ACE2 in serum may somewhat protect against an active infection without generating a conventional antibody response. Finally, we show that among patients with symptoms, ACE2 levels were significantly higher in infected patients who developed cutaneous as compared with respiratory symptoms and ACE2 was also higher in those with milder symptoms.ConclusionsThese findings suggest that soluble ACE2 could be used as a potential biomarker to predict SARS-CoV-2 infection risk and to discriminate COVID-19 disease subtypes.

The Traditional Chinese Medicine Formula Jing Guan Fang for Preventing SARS-CoV-2 Infection: From Clinical Observation to Basic Research.

COVID-19 is a global epidemic. Developing adjuvant therapies which could prevent the virus from binding to cells may impair viral infection. This study produces a traditional Chinese medicine formula, Jing Guan Fang (JGF), based on ancient medical texts, and examines the efficacy and the mechanism by which JGF prevents viral infections. JGF reduces COVID-19 like symptoms. Functional studies show that JGF inhibits the formation of syncytium and reduces the formation of viral plaque. JGF is not toxic in vitro and in vivo. Mechanistically, JGF induces lysosomal-dependent ACE2 degradation and suppresses mRNA and the protein levels of TMPRSS2 in human lung WI-38 and MRC-5 cells. Mice that inhale JGF exhibit reduced ACE2 and TMPRSS2 protein levels in lung tissues. Together, these findings suggest that JGF may improve the COVID-19 like symptoms and inhibit viral infection. Moreover, JGF may be applicable as an adjuvant preventive strategy against SARS-CoV-2 infection in addition to the use of vaccines.

A Novel Modulator of the Renin-Angiotensin System, Benzoylaconitine, Attenuates Hypertension by Targeting ACE/ACE2 in Enhancing Vasodilation and Alleviating Vascular Inflammation.

The monoester alkaloids in Aconitum carmichaelii, including benzoylaconitine (BAC), benzoylmesaconine, and benzoylhypaconitine, were found to have anti-hypertensive effects in spontaneously hypertension rats (SHRs), of which BAC is the strongest. However, its antihypertensive target and underlying molecular mechanisms remain unclear. In this study, first, we screened the antihypertensive targets of BAC by using the CVDPlatform (www.cbligand.org/CVD) and found that ACE/ACE2 are the most possible targets. Then, we verified the effect of BAC on ACE/ACE2 by virtual docking, SPR, enzyme activity assay, and HUVECs cell experiment. We found that BAC could bind with ACE/ACE2, inhibit ACE activity and protein expression, and activate ACE2 enzyme activity. Using vascular function test in vitro, we found that BAC could target ACE/ACE2 to enhance endothelium-dependent vasorelaxation. In BAC-treated SHRs, the levels of ACE and AngII in serum were reduced while Ang (1–7) was increased significantly, and the expression of ACE was reduced, which suggested that BAC can inhibit ACE and activate ACE2 to inhibit AngI to AngII and promote AngII to Ang (1–7) to inhibit vasoconstriction and finally attenuate hypertension. Furthermore, the signaling pathways with regard to vasorelaxation and vascular inflammation were investigated. The results showed that BAC could significantly activate Akt/eNOS, increase NO production, and promote endothelial-related vasodilation; BAC could also reduce inflammatory factors TNF-α and IL6, inhibition of COX-2 expression, and IKB-α phosphorylation to reduce vascular inflammation in SHRs. In brief, BAC targets ACE/ACE2 to enhance endothelium-dependent vasorelaxation and reduce vascular inflammation to attenuate hypertension as a potential modulator of the renin–angiotensin system.

Hippocampus RNA Sequencing of Pentylenetetrazole-Kindled Rats and Upon Treatment of Novel Chemical Q808.

The expression of genes altered in epilepsy remains incomplete, particularly in the hippocampus, which exhibits exquisite vulnerability to epilepsy. Q808 is an innovation chemical compound that has potent anti-convulsant effect. Exploring its mechanism can not only explore the pathogenesis of epilepsy but also provide a theoretical basis for its clinical application. The present study aimed to use RNA sequencing (RNA-seq) to reveal the gene transcriptomic profile of chronic pentylenetetrazole (PTZ)-kindled seizure rats and the difference of the PTZ model rat before and after treatment with Q808. Quantitative real-time PCR (qRT-PCR) was performed to validate the RNA-seq results. The protein level was estimated with Western blot. Hippocampal transcriptomic analysis showed that 289 differentially expressed genes (DEGs) were confirmed in the PTZ-kindled seizure group compared with the vehicle control. Gene cluster analysis identified most of the DEGs linked to neuronal apoptosis, neurogenesis, neuronal projections, and neurotransmitter regulation. After analysis across the three groups, 23 hub genes and 21 pathways were identified, and qRT-PCR analysis confirmed that most of the mRNA levels of hub genes were consistent with the RNA-seq results. Q808 treatment increased the level of ACE, a GABA-related protein. Our analysis showed the comprehensive compendium of genes and pathways differentially expressed for PTZ-kindled seizure rats and upon Q808 treatment in PTZ-kindled seizure, which may provide a theoretical basis to explore the mechanism and unique efficacy of Q808 and the pathophysiology of epilepsy in the future.

Early reduction of SARS-CoV-2-replication in bronchial epithelium by kinin B2 receptor antagonism

SARS-CoV-2 has evolved to enter the host via the ACE2 receptor which is part of the kinin-kallikrein pathway. This complex pathway is only poorly understood in context of immune regulation but critical to control infection. This study examines SARS-CoV-2-infection and epithelial mechanisms of the kinin-kallikrein-system at the kinin B2 receptor level in SARS-CoV-2-infection that is of direct translational relevance. From acute SARS-CoV-2-positive study participants and -negative controls, transcriptomes of nasal curettages were analyzed. Primary airway epithelial cells (NHBEs) were infected with SARS-CoV-2 and treated with the approved B2R-antagonist icatibant. SARS-CoV-2 RNA RT-qPCR, cytotoxicity assays, plaque assays, and transcriptome analyses were performed. The treatment effect was further studied in a murine airway inflammation model in vivo. Here, we report a broad and strong upregulation of kallikreins and the kinin B2 receptor (B2R) in the nasal mucosa of acutely symptomatic SARS-CoV-2-positive study participants. A B2R-antagonist impeded SARS-CoV-2 replication and spread in NHBEs, as determined in plaque assays on Vero-E6 cells. B2R-antagonism reduced the expression of SARS-CoV-2 entry receptor ACE2, G protein–coupled receptor signaling, and ion transport in vitro and in a murine airway inflammation in vivo model. In summary, this study provides evidence that treatment with B2R-antagonists protects airway epithelial cells from SARS-CoV-2 by inhibiting its replication and spread, through the reduction of ACE2 levels and the interference with several cellular signaling processes. Future clinical studies need to shed light on the airway protection potential of approved B2R-antagonists, like icatibant, in the treatment of early-stage COVID-19.Graphical Key messagesInduction of kinin B2 receptor in the nose of SARS-CoV-2-positive patients.Treatment with B2R-antagonist protects airway epithelial cells from SARS-CoV-2.B2R-antagonist reduces ACE2 levels in vivo and ex vivo.Protection by B2R-antagonist is mediated by inhibiting viral replication and spread.

Salivary ACE2 and TMPRSS2 link to periodontal status and metabolic parameters

AbstractBackgroundPeriodontitis links extensively to various non‐communicable diseases such as diabetes and cardiovascular disease and most recently COVID‐19 complications. Two important proteins in humans, ACE2 and TMPRSS2, have been associated with various health problems and coronavirus infection. This study was to investigate the soluble ACE2 and TMPRSS2 in saliva and serum as well as their association with periodontal status and systemic conditions.MethodsThe demographic background, lifestyles, full‐mouth periodontal status and medical parameters in 139 subjects were examined and recorded. Saliva and serum samples were collected for determining the levels of ACE2, TMPRSS2 and MMP8.FindingsThe salivary levels of ACE2 increased with the severity and complexity of periodontitis and correlated positively with alveolar bone loss and salivary MMP8 as an inflammatory biomarker. While, the salivary levels of TMPRSS2 were negatively correlated with body mass index (BMI), cholesterol/high‐density lipoprotein (HDL) ratio, low‐density lipoprotein (LDL), salivary MMP8 and number of remaining teeth. No inter‐correlation existed among ACE2 and TMPRSS2 levels in both saliva and serum.ConclusionsThe present study demonstrated the disparate correlation of the soluble ACE2 and TMPRSS2 with periodontal and systemic parameters. Salivary ACE2 could differentiate periodontal health and severe periodontitis, while salivary TMPRSS2 prominently linked to the metabolic parameters of obesity and cardiovascular disease. Our findings imply the potential application of the biofluidic soluble form of SARS‐CoV‐2 entry proteins in assessing oral or systemic conditions and delivering appropriate healthcare.

Abstract 14: Cause of PTH independent hypercalcemia other than malignancy and granulomatous disorders

Case Report: We report cases of five patients. All females in the age group of 75 to 80 years. They presented to us in altered sensorium. Underlying comorbidities were hypertension and diabetes. After ruling out most probable diagnosis which was stroke, next differential was dyselectrolytemia. We found hypercalcemia in all the patients which was PTH independent with S. Calcium value >12 mg/dl. Prolonged intake of calcium and Vit D supplements was ruled out Keeping age in consideration, the most common differential of PTH independent hypercalcemia malignancy followed by granulomatous disease were ruled out in all the patients by doing PET CT/MR. Activated Vit D and ACE levels were also negative. No evidence of hyperthyroidism or adrenal insufficiency. We managed on the lines of acute hypercalcemia. 4 patients responded well and 1 continued to be in altered sensorium due to superadded sepsis. So excluding other causes, we had made an impression of uncommon cause of hypercalcemia which is immobilization. Although these females had not undergone any trauma. They were mostly bed bound. The pathophysiology of Immobilization related hypercalcemia (IH) is unclear. It is said that muscle activity transmits signal for bone formation through osteocytes and with immobility, mechanical stimulation is reduced, causing unopposed resorption.Conclusion: IH is an unknown complication in immobile patients. If untreated, patients can land up with complications of hypercalcemia.