ABSTRACT
Objective: COVID19 is associated with vascular inflammation. IFN-alpha (IFNa) and IFN-lambda3 (IFNl3) are potent cytokines produced in viral infections. Their effects involve interferon-stimulated genes (ISGs) and may influence expression of angiotensin-converting enzyme 2 (ACE2), the receptor for S-protein (S1P) of SARS-CoV-2. We hypothesized that S1P-induced immune/inflammatory responses in endothelial cells (EC) are mediated via IFN-activated pathways Design and methods: Human ECs were stimulated with S1P (1 mg/mL), IFNa (100ng/mL) or IFNl3 (100IU/mL). Because ACE2, ADAM17 and TMPRSS2 are important for SARS-CoV-2 infection, we used inhibitors of ADAM17 (marimastat, 3.8 nM), ACE2 (MLN4760, 440pM), and TMPRSS2 (camostat, 50 mM). Gene and protein expression was investigated by real-time PCR and immunoblotting, respectively. Vascular function was assessed in mesenteric arteries from wild-type (WT) normotensive and hypertensive (LinA3) mice and in ISG15-deficient (ISG15KO) mice. Results: S1P increased expression of IFNa (3-fold), IFNl3 (4-fold) and ISGs (2-fold) in EC (p < 0.05). EC responses to IFNa (ISG15: 16-fold) were greater than to IFNl3 (ISG15: 1.7-fold) (p < 0.05). S1P increased gene expression of IL-6 (1.3-fold), TNFa (6.2-fold) and IL-1b (3.3-fold), effects that were amplified by IFNs. Only the ADAM17 inhibitor marimastat inhibited S1P effects. IFNa and IFNl3 increase protein expression of ADAM17 (27%) and TMPRSS2 (38%). No changes were observed on ACE2 expression. This was associated with increased phosphorylation of Stat1 (134%), Stat2 (102%), ERK1/2 (42%). EC production of IL-6 was increased by IFNa (1,230pg/mL) and IFNl3 (1,124pg/mL) vs control (591pg/mL). Nitric oxide generation and eNOS phosphorylation (Ser1177) were reduced by IFNa (40%) and IFNl3 (40%). Vascular functional responses demonstrated that endothelium-dependent vasorelaxation (% Emax) in vessels from WT-mice stimulated with IFNa (67%) and IFNl3 (71%) were reduced vs control (82%) (p < 0.05). Responses were not altered in vessels from ISG15KO mice. Increased contraction was observed only in vessels from hypertensive mice treated with IFNa (9.1 ± 0.5mN vs control: 7.3 ± 0.3mN) (p < 0.05). Conclusions: In ECs, S1P, IFNa and IFNl3 increased ISG15 and IL-6 by mechanisms dependent on ADAM17. IFNs amplifies endothelial cell inflammatory responses and induced vascular dysfunction through ISG15-dependent mechanisms, with augmented effects in hypertension. Our findings demonstrate that S1P induces immune/inflammatory responses that may be important in endotheliitis associated with COVID-19. This may be especially important in the presence of cardiovascular risk factors, including hypertension.
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Due to the high incidence of kidney disease, there is an urgent need to develop wearable artificial kidneys. This need is further exacerbated by the coronavirus disease 2019 pandemic. However, the dialysate regeneration system of the wearable artificial kidney has a low adsorption capacity for urea, which severely limits its application. Therefore, nanomaterials that can effectively remove uremic toxins, especially urea, to regenerate dialysate are required and should be further investigated and developed. Herein, flower-like molybdenum disulphide (MoS2) nanosheets decorated with highly dispersed cerium oxide (CeO2) were prepared (MoS2/CeO2), and their adsorption performances for urea, creatinine, and uric acid were studied in detail. Due to the open interlayer structures and the combination of MoS2 and CeO2, which can provide abundant adsorption active sites, the MoS2/CeO2 nanomaterials present excellent uremic toxin adsorption activities. Further, uremic toxin adsorption capacities were also assessed using a self-made fixed bed device under dynamic conditions, with the aim of developing MoS2/CeO2 for the practical adsorption of uremic toxins. In addition, the biocompatibility of MoS2/CeO2 was systematically analyzed using hemocompatibility and cytotoxicity assays. Our data suggest that MoS2/CeO2 can be safely used for applications requiring close contact with blood. Our findings confirm that novel 2-dimensional nanomaterial adsorbents have significant potential for dialysis fluid regeneration. © 2022
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Finding effective and safe medicines to fight SARS-CoV-2 infection is an urgent task. RPH-137 is an original trap fusion protein against SARS-CoV-2 virus. It comprises the angiotensin-converting enzyme type 2 extracellular domain and the human IgG1 Fc fragment. The aim of the study was to carry out a preclinical evaluation of the efficacy of RPH-137 and molnupiravir against SARS-CoV-2 infection. Materials and methods: the authors analysed RPH-137 expressed in a stable CHO cell line and molnupiravir used as an active pharmaceutical ingredient. Drug-mediated inhibition of virus-induced cytotoxicity was assessed in Vero cell culture. In vivo efficacy assessments were performed in Syrian hamsters. The animals were infected intranasally with SARS-CoV-2 (PIK35 clinical isolate) in the dose of 5 log TCID50. The authors evaluated body weight measurements, lung-body weight ratios, and lung histopathology findings and determined viral RNA levels in oropharyngeal swabs by RT-PCR using the amplification cycle threshold (Ct). The statistical analyses involved one- and two-way ANOVA, Student's t-test, and Mann–Whitney test. Results: RPH-137 and molnupiravir inhibited the cytopathic effect of SARS-CoV-2 in Vero cells;the EC50 values of RPH-137 amounted to 4.69 μg/mL (21.3 nM) and 16.24 μg/mL (73.8 nM) for 50 TCID50 and 200 TCID50, respectively, whereas the EC50 values of molnupiravir were 0.63 μg/mL (1900 nM) for both doses. Intramuscular RPH-137 (30 and 80 mg/kg) had no effect on the infection process in Syrian hamsters. The comparison with the challenge control group showed that intraperitoneal RPH-137 (100 mg/kg) had statistically significant effects on a number of parameters, including a 27% reduction in inflammation and a 30% reduction in the total lesion area of the lungs by Day 7. Intragastric molnupiravir (300 mg/kg twice daily) significantly inhibited SARS-CoV-2 infection. Conclusions: both RPH-137 and molnupiravir inhibited the cytopathic effect of SARS-CoV-2 in Vero cells. In Syrian hamsters, molnupiravir demonstrated a more pronounced inhibition of SARS-CoV-2 than RPH-137. However, RPH-137 had statistically significant effects on a range of parameters. This offers additional perspectives for further research.
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Melioidosis is a particularly dangerous infection with endemic distribution caused by the Gram-negative microorganism from the pathogenicity group II Burkholderia pseudomallei. In endemic countries, melioidosis holds one of the leading places in mortality rate after HIV, tuberculosis and, in recent years, COVID-19. The natural ecological pathogen niches are located in tropical and subtropical climate zones, primarily in Southeast Asia and Australia, where its existence as a species is maintained in moist soil and water in a certain temperature environmental range. However, at present, more and more often cases of melioidosis are registered outside endemic territories, which emphasizes the relevance of improving the means and methods of laboratory diagnostics of this disease both for countries located in the zone of natural foci as well as for local healthcare of the countries after importation of this poorly known infection into their territory. In such countries, including the Russian Federation, the population has no natural immunity to the pathogen, and therefore this infection acquires even greater clinical and epidemiological significance. In the Volgograd Plague Control Research Institute, an erythrocyte antigenic melioidosis diagnostic agent for IHA was designed allowing to detect the presence of serum melioidosis antibodies. The diagnostic agent was obtained on the basis of a biological carrier - ram erythrocytes sensitized with isolated protein antigenic complexes of B. pseudomallei. The high analytical characteristics of the diagnostic agent were confirmed on sera models of immunized and recovering experimental animals. Using the obtained set of reagents, the level of serum antibodies against the causative agent of melioidosis was studied in residents from the 3 provinces of Vietnam (Ha Giang, Lang Son and Quang Ninh), as well as in control group composed of residents of the Volgograd region. In samples obtained from a non-endemic region, not more than 25% of cases contained IHA titers at lower than 1:10 dilution, which is apparently due to cross-reactivity of serum immunoglobulins. Positive serum samples from clinically healthy residents of Ha Giang, Lang Son and Quang Ninh provinces were at a titer of 1:10 detected in 71.5%, in dilutions of 1:20-1:80 - in 28.5% of cases. Thus, we believe that serum antibody titer of 1:80 established in the IHA results, has a diagnostic significance, reflecting the intensity of the anti-melioidosis populational immunity. Copyright © 2022 Saint Petersburg Pasteur Institute. All rights reserved.
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The aim of this paper was to prepare specific monoclonal antibody (mAb) against African swine fever virus (ASFV) p54 protein. The p54 protein was expressed in Escherichia coli expression system and used as the antigen in mAb production. The spleen cells from the immunized BALB/c mice were fused with myeloma cells SP2/0. To screen the positive hybridoma cells, the purified p54 protein was used as envelope antigen for indirect ELISA. After four times' subcloning, the supernatant of hybridoma cells were used to identify mAb subtype, ascites were prepared via in vivo induction method in mice and then the mAb was purified. The titer of the mAb was detected by indirect ELISA, and the specificity of the mAb was identified by cross reactivity assay, IFA and Western blot. According to the predicted secondary structure of p54 protein, using the stepwise truncation method identified the epitope region of mAbs, and labeled the region in tertiary structure of p54 protein. Results were as follows: six hybridoma cells secreting p54 monoclonal antibody were successfully screened and named 28G12-1, 31G7-1, 31G7-2, 35F10-1, 35F10-2, 38D3-1, respectively. The heavy chains of 28G12-1, 31G7-1, and 31G7-2 were IgG2a type, the heavy chains of 35F10-1, 35F10-2, 38D3-1 were IgG1 type, light chains were all kappa chains. The lowest titer of mAb was 1:25 600, and having no cross reaction with PRRSV, PRV, PEDV, PPV, SADS-CoV, PCV2, the specificity was strong. All six monoclonal antibodies could recognize the 127-146 aa on carboxyl end. In this study, ASFV p54 protein and p54 monoclonal antibody were successfully obtained, and the epitopes of six mAbs were identified, these experimental data laid a foundation for the functional research of p54 protein and the study of ASFV epitope vaccine. Copyright © 2023 Editorial Board, Institute of Animal Science of the Chinese Academy of Agricultural Sciences. All rights reserved.
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Background/Purpose: Kikuchi-Fujimoto disease (KFD) is a rare, self-limited histiocytic necrotizing lymphadenitis. Although it is of uncertain aetiology, it is associated with viral infections and autoimmune diseases. Hence, it is crucial to identify KFD from other conditions with lymphadenopathy. Here we present a case of KFD after COVID-19 infection. Method(s): Medical records were traced and reviewed Results: A previously healthy 13-year- old girl was admitted in April 2022 with four weeks of fever, dry cough, loss of weight, followed by 1 week history of painful cervical lymphadenopathy and nonspecific maculopapular rash. She received her second dose of Covid 19 vaccine in January 2022. Unfortunately, she was diagnosed with CAT II, COVID 19 infection in March 2022. There was no history of allergy, recent traveling and cat scratch injury. Clinically there was no strawberry tongue, erythema of the lips, conjunctivitis or distal extremities changes to suggest Kawasaki disease. She was initially diagnosed with infection related lymphadenitis, treated with oral azithromycin for three days and intravenous ceftriazone for one week with no improvement. Her laboratory results showed hypochromic microcystic anaemia with leucopenia, raised inflammatory markers and lactate-dehydrogenese levels. Extensive workup for infection was unremarkable. Immunology test showed ANA, ANCA, ENA were negative with normal complements. Ultrasound abdomen was normal. Excisional lymph node biopsy revealed confluent areas of necrosis surrounded by histiocytes (CD68+) with absent of neutrophils. No granuloma or atypical lymphoid cells seen. Based on histopathology report, diagnosis of KFD was established. As she was not able tolerate orally, IV hydrocortisone was started and subsequently switched to oral prednisolone. She responded well to corticosteroids with fever subsided within a day and cervical lymphadenopathy reducing in size and resolved in one month. Prednisolone was able to taper off by two months. She showed complete recovery with no recurrence during follow-up. Conclusion(s): In persistent febrile painful lymphadenopathy, excision lymph node biopsy is essential to establish definite diagnosis. This case highlights the possible association between COVID-19 and KFD.
ABSTRACT
Melioidosis is a particularly dangerous infection with endemic distribution caused by the Gram-negative microorganism from the pathogenicity group II Burkholderia pseudomallei. In endemic countries, melioidosis holds one of the leading places in mortality rate after HIV, tuberculosis and, in recent years, COVID-19. The natural ecological pathogen niches are located in tropical and subtropical climate zones, primarily in Southeast Asia and Australia, where its existence as a species is maintained in moist soil and water in a certain temperature environmental range. However, at present, more and more often cases of melioidosis are registered outside endemic territories, which emphasizes the relevance of improving the means and methods of laboratory diagnostics of this disease both for countries located in the zone of natural foci as well as for local healthcare of the countries after importation of this poorly known infection into their territory. In such countries, including the Russian Federation, the population has no natural immunity to the pathogen, and therefore this infection acquires even greater clinical and epidemiological significance. In the Volgograd Plague Control Research Institute, an erythrocyte antigenic melioidosis diagnostic agent for IHA was designed allowing to detect the presence of serum melioidosis antibodies. The diagnostic agent was obtained on the basis of a biological carrier - ram erythrocytes sensitized with isolated protein antigenic complexes of B. pseudomallei. The high analytical characteristics of the diagnostic agent were confirmed on sera models of immunized and recovering experimental animals. Using the obtained set of reagents, the level of serum antibodies against the causative agent of melioidosis was studied in residents from the 3 provinces of Vietnam (Ha Giang, Lang Son and Quang Ninh), as well as in control group composed of residents of the Volgograd region. In samples obtained from a non-endemic region, not more than 25% of cases contained IHA titers at lower than 1:10 dilution, which is apparently due to cross-reactivity of serum immunoglobulins. Positive serum samples from clinically healthy residents of Ha Giang, Lang Son and Quang Ninh provinces were at a titer of 1:10 detected in 71.5%, in dilutions of 1:20-1:80 - in 28.5% of cases. Thus, we believe that serum antibody titer of 1:80 established in the IHA results, has a diagnostic significance, reflecting the intensity of the anti-melioidosis populational immunity. Copyright © 2022 Saint Petersburg Pasteur Institute. All rights reserved.
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Heightened sympathetic drive (dysautonomia) is a hallmark of several cardiovascular diseases including SARS-CoV-2. It is also a powerful prognostic predictor for arrhythmia and sudden cardiac death, especially in patients with channelopathies (long QT syndrome-LQTS, and catecholaminergic polymorphic ventricular tachycardia-CPVT). However, little is known about the molecular targets underlying this dysautonomia. We have identified a novel pathway using a combination of single cell and bulk RNAseq, neurochemistry, FRET imaging and single cell electrophysiology. This pathway involves impairment of cyclic nucleotide coupled phosphodiesterases (PDE) linked to enhanced intracellular calcium transients and exocytosis from rat sympathetic neurons. In particular, the adaptor protein Nos1-ap, Pde2A, and Ace2 are associated with sympathetic hyperexcitability. These proteins are also conserved in human stellates from patients with LQTS and CPVT, although their role in neuronal-myocyte cellular function is unknown. We have developed a unique human iPSC sympathetic-cardiac co-culture model for target discovery in LQTS and CPVT. The lecture will highlight the use of gene manipulation of these proteins to determine their role in driving abnormal transmission and arrhythmia.
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Aim/Introduction: A hallmark of coronavirus disease 2019 (COVID-19) is lower respiratory tract infection and pneumonia. Infection of nonhuman primates mirrors mild-to-moderate human disease determined with concordant immunologic, virologic, and lung histopathologic findings in combination with imaging abnormalities. Particularly in the lower respiratory tract, medical imaging of pulmonary disease contributes uniquely to the understanding and measuring of the consequences of an infection. Advanced medical imaging, such as PET-CT, of the lungs of SARS-CoV-2-exposed nonhuman primates shows great promise in detecting and longitudinally evaluating disease in a non-invasive manner. The aim of this study was to investigate the application of [18F]DPA714 for visualizing the inflammation processes in the lungs of SARS-CoV-2-infected rhesus monkeys. Material(s) and Method(s): Four experimentally SARS-CoV-2 infected rhesus monkeys were followed for seven weeks post infection (pi) with a weekly PETCT using a MultiScan Large Field of View Extreme Resolution Research Imager (LFER) 150 (Mediso Medical Imaging Systems Ltd., Budapest, Hungary) with [18F]DPA714 as radiotracer. Following a retrospectively gated CT, an intravenous bolus of approximately 180 MBq [18F]DPA714 (molar activity: 77.4 GBq/mumol (range 39.8- 150.6 GBq/mumol, radioactivity concentration: of 277.0+/-172.8 MBq/ mL, a radiochemical purity > 98.0%) was administered. Two PET images, ten minutes each, of a single field-of-view covering the chest area, were obtained ten and thirty minutes after injection. To confirm the infection of SARS-CoV-2 , both nasal and tracheal swabs and blood samples were obtained. Result(s): All animals tested positive for SARS-CoV-2 in both swabs on multiple timepoints pi. The initial development of pulmonary lesions was already detected at the first scan 2-days pi. PET revealed an increased tracer uptake in the pulmonary lesions and mediastinal lymph nodes of all animals starting from the first scan pi onwards. However, also an increased uptake was detected in the lung tissue surrounding the lesions, which persisted until day 30 and then subsided by day 37-44 pi. In parallel, a similar pattern of increased expression of activation markers was observed on dendritic cells in blood. Conclusion(s): This study illustrates that [18F]DPA714 is a valuable radiotracer to visualize SARS-CoV-2-associated pulmonary inflammation even during mild-to-moderate disease. In addition, the inflammatory process detected in anatomically unaffected lung tissue coincided with dendritic cell activation detected in blood samples. Our data indicate that [18F]DPA714 can be applied to visualize the pulmonary tract following a SARS-CoV-2 infection in rhesus monkeys and may be translated to the clinic as diagnostic tracer for this indication too.
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Aim/Introduction: COVID-19 patients may present with lymphopenia. The degree of lymphopenia, in particular reduced CD8+ T-cell numbers, is strongly correlated with clinical deterioration and ICU admission [1,2]. It has been postulated that lymphopenia in COVID-19 is caused by;1) sequestration of CD8+ T-cells in peripheral tissues (e.g. lung) [3], 2) accelerated maturation and exhaustion [4,5] or 3) decreased lymphopoiesis [6]. The lack of data on in vivodistribution of CD8+ T-cells hampers a more thorough understanding of this critical prognostic factor. We aim to assess differences in the in vivo distribution of CD8+ T-cells in patients with proven SARS-CoV-2 presenting with lymphopenia or with normal lymphocyte counts, using [89Zr]Df-crefmirlimab PET/CT imaging. Material(s) and Method(s): This is a prospective, observational non-randomized pilot study in hospitalized patients with microbiologically proven SARS-CoV-2 infection. The prior use of immune suppressive medication was an exclusion criterion. Whole body [89Zr]Df-crefmirlimab PET/CT scan were acquired at 24 hrs after intravenous injection of 1.5mg protein dose labelled with 37 MBq (1 mCi)89Zr. Peripheral blood samples were collected for multi-colour flowcytometry to phenotype homing receptors and immune senescence markers, and transcriptomics. Result(s): Three patients were enrolled (all male, average 83 yrs (78-89 yrs)), admitted at 5.3 days (2-10 days) after onset of symptoms and scanned at 2.7 days (2-4 days) after admission. Two patients completed vaccination (2x plus booster), one patient was not vaccinated. Two patients had with lymphocyte count <1.0 x10e9/L and one patient had normal lymphocyte counts on admission. One patient required oxygen suppletion 3L/min. PET/CT scans showed remarkable differences in uptake in the upper respiratory tract versus lower respiratory tract, involvement of distant organs and distribution of CD8+ T-cells across secondary lymphoid organs, spleen and hematopoietic system. Quantification of the scans, flow cytometry and transcriptomic analyses are ongoing. Conclusion(s): In vivo imaging of CD8+ T-cells in hospitalized COVID-19 patients reveals distinct patterns of CD8+ T-cell distribution in early stages of localized infection versus systemic involvement at later stages. Translational data on T-cell phenotyping is currently processed and will be presented.
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The proceedings contain 278 papers. The topics discussed include: genotoxic preventive potential of ethanol leaves extract of Annona muricata on N-Nitroso-N-Ethylurea pro-leukemia carcinogenin mice by bone marrow micronucleus assay;performance evaluation of TOSOH G11 high performance liquid chromatography analyzer (beta thalassemia analysis mode);the accuracy of manual platelet counting;new insights in the biochemical analysis of stored irradiated blood;can somatic mutations be used in the diagnosis of myelodysplastic syndrome?;assessment of reference intervals for platelet aggregation tests on samples with low platelet counts;hematological profile of patients with COVID-19 at Oran University hospital in Algeria by hematology analyzer BC-6800;prevalence of myeloproliferative neoplasm mutations among Pakistani population;utility of reticulated platelets (RETPLTS) and their qualitative parameters on ADVIA 2120I siemens in the differential diagnosis of thrombocytopenia;evaluation of reticulated platelets (RETPLTS) and their qualitative parameters on ADVIA 2120I siemens: a new diagnostics tool;and evaluation of the ability of the differential count on two hematology analyzers to detect leukemias, verified by bone marrow and peripheral blood smear evaluation of the ability of the differential count on two hematology analyzers to detect leukemias, verified by bone marrow and peripheral blood smears.
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Coronavirus can also harm the vascular system and cause bleeding;this damage to the vascular system might be followed by dysfunctions in other body systems, therefore not a few COVID-19 survivors have experienced a stroke as a complication of COVID-19. Bleeding that occurs in the nose is a common condition that occurs as a result of a person's injury, allergies, or platelet levels that are declining. There are several simple ways to stop this bleeding, but for Indonesians betel leaf is a widely chosen traditional treatment. The subjects of experimental animal studies were divided into 4 groups. The results of the Anova test analysis showed that there was a significant difference in average platelet levels (p=0.05) in the 4 groups. The results showed that the lowest average thrombocytes and hematocrit levels were found in group 2, namely the group that was given LPS without being given betel leaf essential oil. The highest average thrombocytes and hematocrit levels were found in group 3, namely the group that was first given betel leaf essential oil, after which it was only applied LPS. Red Betel Leaf Essential Oil has the potential as an anti-bleeding in research subjects induced with infectious materials characterized by increasing average platelet levels and Hematocrit as components that play a role in the blood clotting process. Copyright © 2023, Anka Publishers. All rights reserved.
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Purpose: Hearing loss is a major global health issue affecting around 1.5 billion people worldwide, with an increasing prevalence. Acquired hearing loss is attributed to different environmental factors including ageing, noise exposure, and the intake of ototoxic medicines. Some commonly used medications can considerably affect the auditory system, resulting in cochlear and central damage that can lead to permanent hearing loss. More than 600 classes of medications are ototoxic. The most used in clinical practice are chemotherapeutics (cisplatin) and aminoglycoside antibiotics (such as gentamicin). Some investigated drugs for Covid-19 treatment (hydroxychloroquine, HCQ) and certain drug delivery agents like cyclodextrins (CD) have also been reported to induce auditory side effects. The aim of these in vivo studies was to provide functional and histological data on auditory assessments related to cisplatin, gentamicin, HCQ, and CD, when administered similarly to clinical protocol. Method(s): The studies were conducted in Wistar rats and Albino guinea pigs: * Cisplatin was administered by intraperitoneal route at 10 mg/kg in rats * Gentamicin was administered by intramuscular route at 160 mg/kg for 5 days in rats * HCQ was administered at 62 mg/kg per os daily for five days in rats * A cyclodextrin-based formulation was administered by transtym-panic route at 4 mg/mL 1 h and 30 h after noise exposure in guinea pigs Hearing was assessed using the techniques of Distortion Product Otoacoustic Emissions (DPOAE) and Auditory Brainstem Responses (ABR) at several timepoints. DPOAE are acoustic signals created and amplified by the cochlear epithelium and measured in the ear canal. DPOAE reflect the activity of outer hair cells (OHC). ABR is an electrophysiological measure of the sensorineural activity of the auditory pathway from the cochlea to the central auditory structures in response to a sound stimulus, recorded as electric potentials from scalp electrodes. A cochleogram, an FDA-recommended histological analysis for hair cell counting, was performed at the end of certain studies. Result(s): Results based on ABR thresholds, DPOAE amplitudes, and the cochleogram, showed different patterns of auditory side-effects. Cisplatin induced immediate and permanent hearing loss;gentamicin displayed delayed side-effects on auditory measures;HCQ did not affect Outer Hair Cells but might have had an effect on neurons. CD had an immediate and prolonged effect on hearing. Conclusion(s): This short presentation will help you learn the current available methods to measure hearing in preclinical in-vivo trials using two complementary functional read-outs and a histological analysis, and to determine the different sites of damage. Copyright © 2022 Elsevier B.V.
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Understanding the impact of nanomaterials on the immune system is of paramount importance. We recently proposed the "nano-immunity-by-design" approach according to which the characterization of 2D nanomaterials is based not only on their physicochemical parameters but also on immune profiling. Immune profiling can be performed using high-dimensional approaches such as single-cell mass cytometry on graphene and other 2D materials including transition metal carbides/carbonitrides (MXenes). Our studies revealed that amino functionalization of graphene oxide (GO) increased the immune compatibility of this material (Orecchioni et al. Nat Commun. 2017). Moreover, by functionalizing GO with AgInS2 nanocrystals, detection by single-cell mass cytometry was enabled in a large variety of primary immune cells (Orecchioni et al. Small. 2020). Recently, we reported on the immune modulation by Mxenes, including Ti3C2Tx, in combination with their antiviral properties against SARS-CoV-2 by using single-cell mass cytometry and other high-dimensional approaches (Unal et al. Nano Today. 2021). The present talk will also cover recent (unpublished) findings from the EU-funded projects, G-IMMUNOMICS and CARBO-IMmap, on a variety of 2D materials, including Mxenes, MoS2, WS2, and other materials, on human immune cells as well as in murine and porcine models. Our results illustrate that the chemical and immunological design of 2D materials offers new strategies for their safe exploitation in medicine. Copyright © 2022 Elsevier B.V.
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BACKGROUND AND AIM: Increased Vascular Endothelial Growth Factor (VEGFA) Gene Expression (GE) has been documented in SARS-CoV2 infection. We wished to understand the relationship of VEGFA and VEGF B GE in both Murine SARS-CoV and Human SARS-CoV-2 in-vitro models of infection. METHOD(S): Secondary analysis of datasets from mice given nasal installation of SARS-CoV (MA15), MS1 (GSE33266MCV-1) and MS2 (GSE68820) from pulmonary tissues was undertaken. This allowed viral dose and temporal response analysis, respectively. Also studied were In-vitro Human hACE2 cells infected with SARS-CoV2 (dataset INV, GSE169158). Gene expression (GE) VEGF sub-types were analysed using Qlucore Omics Explorer (QOE) and gene enrichment functional profiling through the g:Profiler online platform. RESULT(S): For Murine studies, MA15 instillation compared to controls in MS1, lead to down-regulation of both VEGFB (MA15 10
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Background. Benefit of COVID-19 vaccines is limited by need for freezing, high cost, requirement for multiple boosters, and waning immunity. An unmet need for safe and effective vaccines that can be quickly deployed worldwide during a pandemic exists. A barrier to developing scalable low-cost vaccines is the restricted access to vaccine- or adjuvant-formulations due to protection by intellectual property rights. This limits research to understand their mechanism of action and potential applicability towards vulnerable populations or emerging pathogens. Methods. We studied cellular and molecular mechanisms of action of adjuvant formulations developed for global open access in 4 distinct but complementary in vitro platforms that are human, age-specific, and enable the same individual to serve as control and test condition;generating data on adjuvant-induced responses in vitro that predict activity in vivo. Results. Whole blood assay that models magnitude of innate immune activation and identifies cell types activated showed that liposomal co-formulation of MPL +QS-21 activated monocytes and natural killer cells and induced cytokine production;tissue construct assay that models monocyte extravasation and autonomous differentiation into dendritic cells (DC) showed that only MPL+QS-21 co-formulation promoted CD14+ cells towards DC phenotype;monocyte-derived DC (MoDC) assay that interrogates immune activation type showed that MPL+QS-21 co-formulation in lipid nanoparticles promoted MoDC maturation by increasing CD40, CD86, CCR7 and HLA-DR expression and Th1-polarizing cytokine secretion;and dendritic cell-T cell interface assay that assesses potential of formulations to re-activate SARS-CoV-2 Spike antigen-specific T cells showed that only MPL+QS-21 coformulation enhanced activation of antigen-specific CD4+ and CD8+ T cells. Conclusion. Thus, human in vitro modeling provides new insight into the mechanism of action and synergistic effects of MPL+QS-21, and positions us to study them in vulnerable populations to assess potential age-specific application on vaccine development. Precision vaccinology coupled with global access may enable marked public health progress by accelerating, de-risking, and advancing affordable adjuvanted vaccines to the most vulnerable.
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Background. Immune dysregulation associated with COVID-19 includes immune cell activation, inflammatory cytokine release, and neutrophil extracellular trap release (NETosis), which are mediated by spleen tyrosine kinase (SYK) (Fig 1). Fostamatinib, an oral spleen tyrosine kinase (SYK) inhibitor, was approved for immune thrombocytopenia (ITP) in 2018, and the Phase 3 trials showed a lower than expected rate of thrombosis.1 Clinical studies showed a reduction in IL-6 in patients with rheumatoid arthritis.2 The active metabolite of fostamatinib (R406) protected against LPS-induced acute lung injury and thrombosis in mice3,4 and reduced MUC1 in a mouse model of ALI.5 Fostamatinib demonstrated abrogation of the hyperimmune response caused by anti-spike IgG,6 including reduction in hyperactivation of platelets7 and NETosis in neutrophils8 in in vitro studies using plasma from patients with severe COVID-19. A phase 2 study (NCT04579393) evaluated fostamatinib vs. placebo (all received standard of care [SOC]) in 59 hospitalized patients with COVID-19 and demonstrated reduction in mortality, ordinal scale scores, and number of days in the intensive care unit (ICU) as well as meeting the primary endpoint of safety.9 A phase 3 clinical study (NCT04629703) of fostamatinib for the treatment of COVID-19 is underway. (Figure Presented) Methods. A Phase 3, randomized, double-blind, placebo-controlled, adaptive design, multi-center study (NCT04629703) is underway to evaluate fostamatinib in 308 adult patients hospitalized with COVID-19 and on oxygen without intubation (Fig 2). Patients will receive fostamatinib 150 mg BID or placebo for 14 days;both arms receive SOC. At baseline, the clinical status score (8-point ordinal scale) had to be 5 or 6. Patients >= 65 years had to have >= 1 risk factor for severe disease and adults < 65 had to have >= 3. The primary outcome is days on oxygen (Day 1 to 29). Other endpoints include change in clinical status score, days in the ICU, time to hospital discharge, all-cause mortality, oxygen-free status and safety. Fostamatinib is investigational for COVID-19. Results. Blinded data from this trial in progress are as of 2 December 2021. See Fig 3. Conclusion. Final results of this Phase 3 trial are anticipated in 2022.
ABSTRACT
Background. Macrophages, including lung alveolar macrophages (AM), and monocytes are the first lines of defense against SARS-CoV-2. Several reports have suggested that SARS-CoV-2 can hijack AM and monocytes for replication and viral spread, which may, in turn, drive the cytokine storm associated with severe COVID-19. Herein, we describe one of many advantageous features that EDP-235, a novel and potent SARS-CoV-2 3C-like protease (3CLpro) inhibitor under development as a once-daily oral antiviral therapy for COVID-19, displays - excellent penetration into macrophages and monocytes. Methods. Intracellular uptake of EDP-235 was tested side-by-side with nirmatrelvir in rat lung AM, human monocytes and human macrophages. To determine the in vivo drug distribution into lung AM, rats were dosed orally with 25 mg/kg of EDP-235 or nirmatrelvir and plasma andAMdrug levels were analyzed by LC/MS/MS. Results. The ratios of intracellular to extracellular concentrations of EDP-235 in rat lung AM, human monocytes and human macrophages were 22.8, 22.7 and 30.5, respectively. In contrast, nirmatrelvir had ratios of 1.2 to 1.5 in these cells. Consistent with the in vitro observations, EDP-235 showed favorable rat AM penetration with an AUC0-24 ratio of 28.4 (AM over plasma), and nirmatrelvir had much less rat AM penetration with an AUC0-24 ratio of 0.5 (AM over plasma). EDP-235 had respective AUC0-24 values of 9.6 and 271.9 h*mug/mL in rat plasma and AM, while the AUC0-24 values of nirmatrelvir in rat plasma and AM were 2.7 and 1.2 h*mug/mL, respectively. Conclusion. EDP-235, a novel and potent SARS-CoV-2 3CL protease inhibitor, demonstrated excellent penetration into monocytes and macrophages, including lung AM. EDP-235 has the potential to eliminate the viral replication of SARS-CoV-2 in these critical immune cells, thus mitigating macrophage-mediated cytokine storm in high-risk COVID-19 patients. Clinical trials with EDP-235 for COVID-19 treatment and prevention are ongoing.
ABSTRACT
Background. Vaccines aim to induce immune responses that prevent disease. They may also clear chronic infections or reduce tumor progression. Vaccine adjuvants augment immune responses, in general, by providing stimulatory signals. Our focus has been on a different type of adjuvant that enhances vaccine-induced T cell responses by modulating the herpes virus entry mediator (HVEM) pathway. The B and T cell attenuator (BTLA) is expressed on naive T cells and, upon binding to HVEM on antigen presenting cells, dampens signaling through the T cell receptor. HVEM binds with a different domain to the co-stimulator LIGHT. Within a trimolar BTLA-LIGHT/HVEM complex, inhibition prevails. Herpes simplex virus (HSV-1) glycoprotein D (gD) attaches to the BTLA binding site of HVEM and as it has higher binding affinity outcompetes BTLA binding and allows for co-stimulation through LIGHT. This results in enhanced signaling through the T cell receptor and thereby augments and broadens CD8+ T cell responses as we showed with chimpanzee adenovirus (AdC) vector vaccines for several viral antigens. Methods. Immunogenicity in rodents was evaluated following one or two immunizations with AdC vectors expressing antigens of HIV (gag), HPV-16 (E7/6/5), HBV (core & pol), influenza virus (nucleoprotein) and SARS-CoV2 (nucleoprotein), with or without gD. Vaccine-induced CD8+ T cell responses, including their magnitude, functions, duration, and breadth were characterized. Vaccine efficacy was also evaluated. Results. Vaccination with gD-antigen fusion proteins increased CD8+ T cell frequencies to all of the antigens tested (Fig) and improved efficacy. Addition of gD increased stimulation of CD8+ T cells to subdominant epitopes and thereby enhanced breadth of responses. Conclusion. Checkpoint modification of the HVEM pathway with a gD-antigen fusion protein produces potent, prolonged, and broad responses of CD8+ T cells to immunodominant and subdominant epitopes. The latter is especially important for chronic viral infections, where, due to exhaustion of T cells to dominant epitopes therapeutic efficacy of vaccines may rely on expansion of T cells to subdominant epitopes. Clinical studies to evaluate therapeutic vaccination for chronic HBV are planned. (Figure Presented).
ABSTRACT
BACKGROUND: Although mRNA vaccines have been deployed with great success against COVID-19, unlocking this technology against glioblastoma will necessitate new lipid-nanoparticle formulations that overcome cancer tolerance and immunosuppression. OBJECTIVE/METHODS: We sought to develop a novel mRNA vaccine system to make tolerogenic tumor antigens appear more dangerous through use of unmodified nucleosides (pathogen associated molecular patterns, PAMPs) and highly cationic lipid shells that elicit a systemic damage response against cancer antigens. RESULT(S): We developed a novel vaccine formulation that increases payload packaging of tumor amplified mRNA into multilamellar (onion-shaped) particles for systemic (intravenous) administration. We demonstrate significant immunogenicity and efficacy of multilamellar RNA-NPs in syngeneic murine models for high-grade glioma (KR158b-pp65), and diffuse midline glioma (H3K27M DMG). Remarkably, RNA-NPs significantly improve median survival outcomes of DMG bearing mice beginning therapy at endpoint (Day 35 after midline intracranial implantation). Unlike prototypical mRNA vaccines that activate endosomal toll-like receptors (i.e. TLR7), multilamellar RNA-NPs elicit immunologic response predominantly through intracellular pathogen recognition receptors (RIG-I);long-term survival benefits from RNA-NPs were completely abrogated in RIG-I knockout mice. In canines (pet dogs) with spontaneous gliomas, RNA-NPs elicit massive recruitment/activation of peripheral blood mononuclear cells (PBMCs) which correlate with their trafficking into lymphoreticular organs (in follow-up murine studies). In canines receiving neoadjuvant RNA-NPs, prior to glioma biopsy, we see significant reprogramming of the glioma microenvironment with increased gene signatures for antigen processing/ presentation, interferon signaling and cytotoxicity. Upon translation into human clinical trials for glioblastoma patients (NCT04573140), RNA-NPs elicit rapid (within hours) release of cytokines (e.g. IL-1, IL-6, IL-12 TNF- alpha, interferons) and chemokines (e.g. MIP1alpha, MCP-1, IP-10), which correlate with mobilization of PBMCs and activation of dendritic cells/CD8 lymphocytes. CONCLUSION(S): First-in-human application of systemic multilamellar RNA-NP vaccines results in significant biologic effects and rapid immunologic reprogramming.