ABSTRACT
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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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.
ABSTRACT
Lectins are a group of highly specific carbohydrate-binding proteins with a wide spectrum of action, involved in the so-called «first line» of body defense. These unique biomolecules show high specificity for various mono- and oligosaccharides, primarily for viral and bacterial glycoconjugates. Cyanobacteria lectins are effective against enveloped viruses and are an appealing alternative to existing synthetic drugs. Virtually complete absence of resistance formation in viruses to these compounds is known. The purpose of this review is to analyze, summarize, and discuss the results of experimental studies in vivo and in vitro, illustrating the mechanisms of action and antiviral effects of lectins obtained from cyanobacteria in relation to the most dangerous and socially significant viruses: SARS-Cov-2, HIV, Ebola viruses, influenza, and hepatitis C. In addition, the article outlines some of the challenges that must be overcome in order to obtain effective antiviral drugs in the future.
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Since the recent coronavirus disease 2019 pandemic and the lifestyle changes it necessitated, the demand for mental health treatment has skyrocketed, with long wait lists for both psychological and psychiatric care. Over-the-counter supplements and home remedies are increasingly sought. In this study, we screened natural materials and blended supplements from Asia that may improve the mood and mental health of humans by testing cell viability and expression of the proopiomelanocortin gene as a marker of beta-endorphin production in rat hypothalamus neuron cells. Among 23 tested samples, 3 samples produced significantly higher cell viability in R-HTH-507 cells than the control treatment. In a real-time-polymerase chain reaction (RT-PCR) experiment, 7 samples showed significant beta-endorphin synthesis activity. This is the first report that the Asian natural materials Areca catechu, Moringa oleifera, Lignosis rhinocerus, and Aegle marmelos promote beta-endorphin synthesis;further investigation will identify the active ingredients in the blended samples. These results suggested that these Asian natural materials have great potential to expand the range of treatments for mental health. Copyright © The Author(s) 2023.
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Introduction At the onset of the Covid-19 pandemic, hospital educational activities were halted in order to focus on healthcare delivery and maintain social distancing. As a response to this disruption, BSPGHAN trainees set up the BSPGHAN Education Series, a twice- weekly virtual learning programme. The core objective of this programme was to deliver high quality paediatric gastroenterology, hepatology and nutrition (PGHAN) teaching during the pandemic. In this study, we analysed the attendances and feedback received from the education series, in order to guide future directions. Methods We reviewed the Zoom meeting attendance logs and Survey Monkey feedback forms for the BSPGHAN Education Series from April 2020 to December 2020. Results In nine months, a total of 55 talks were delivered by 43 speakers. 23 (41.8%) sessions were gastroenterologythemed, 25 (45.4%) were hepatology-themed and 7 (12.7%) were nutrition-themed. Thirteen paediatrics gastroenterology units (12 in the UK and 1 in the United States) and all 3 UK tertiary paediatric liver centres contributed to the talks. The highest contributing centres were Birmingham Children's Hospital (20 sessions), followed by King's College Hospital (9 sessions) and Leeds Children's Hospital (7 sessions). Attendance logs and feedback forms were available for 53 sessions. A total of 2369 attendances were logged, with a median of 41 attendees per session (IQR 31-54). Attendees from 22 countries have participated in these sessions. A total of 810 survey feedback forms were received, with a median of 14 forms received per session (IQR 10-18). 32% were filled in by PGHAN Grid trainees, 23% by consultants, 15% by clinical fellows. Allied Health Professionals (AHPs) comprised 6% of feedback returns. 54% of survey feedback respondents accessed the teaching sessions from home. An average of 98% (95% CI 96.3-99.2) survey respondents strongly agreed/agreed that the sessions were relevant to their learning. 97% (95% CI 96-98.7) of survey respondents strongly agreed/agreed that the sessions delivered were of high quality. Discussion The BSPGHAN series has been a positive initiative arising from the pandemic, providing access to high quality PGHAN education when local availability was paused, and giving a platform for the society internationally. Our report shows that the BSPGHAN Education Series has been wellreceived by attendees. The virtual sessions are more accessible compared to in-person teaching sessions, as evidenced by the high percentage of feedback respondents accessing the sessions from home. Looking ahead, the BSPGHAN Education Group, set up in October 2020, will play a vital role in the further development of the Education Series. Sessions are recorded and made available to BSPGHAN members on the BSPGHAN websitefurther work may include creating online learning modules centred around these recordings. AHP involvement is an area for development- for 2021, we hope to include more topics that will be relevant to their interests.
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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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Absence of a relevant animal disease model and lack of the use of supportive studies have limited the process of rapid development of SARS-CoV-2 vaccines. This study has evaluated how the choice of species selection and study design affected the outcomes of the non-clinical toxicity studies of the various SARS-CoV-2 vaccine concepts. The study has been focused on safety aspects i.e. on the Developmental and Reproductive Toxicity (DART) and Repeated Dose Toxicity (RDT) studies. Rat and rabbitwere themost commonly used species in preclinical toxicity testing. Some COVID-19 vaccine candidates show a DART study design suboptimal for antibody transfer during lactation. The toxicity studies showed no serious adverse effects.Minor effectswere expected, i.e. local reactogenicity, immune response and macroscopic findings at the injection site. In addition, comments received during the EMA assessment of the vaccines have been evaluated and consisted most frequently of commentary on study design, species selection and missing data regardless of the utilized vaccine concept. Use of supportive platform studies often substantiated the commentary on these main three categories. Animal model-based toxicity testing has shown limited value in establishing safety of the vaccines, and, more importantly, low translational value in supporting clinical development. From a 3R perspective sponsors are encouraged to focus on products from the supportive platform, both with respect to DART and RDT studies. Regulatory emphasis on data obtained from vaccines with the same platform technology data can be used to support marketing approval of new vaccines.
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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.
ABSTRACT
Case report - Introduction: Catastrophic antiphospholipid syndrome (CAPS) is a rare, life-threatening disease occurring in up to 1% of antiphospholipid syndrome (APS) cases. It was first defined in 1992 and remains a difficult to treat entity with a mortality rate of 37%. We describe a patient with systemic lupus erythematosus (SLE) and CAPS presenting with simultaneous multi-organ injuries who was successfully managed with 'triple' therapy including cyclophosphamide. Case report - Case description: A 42-year-old female presented to her local hospital with chest pain and worsening vision. She had a background of SLE, triple antibody-positive APS (previous DVT, pregnancy loss and strokes), hypertension, a metallic mitral valve, a previous myocardial infarction and pre-existing visual impairment due to a prior intra-cerebral bleed related to anticoagulation. Examination revealed a faint malar rash, cortical blindness and long tract neurological signs. Her ECG showed ischaemic changes and the admission troponin was significantly raised (3773ng/L). An echocardiogram showed new left ventricular dysfunction and a subsequent cardiac MRI was in keeping with coronary artery disease. Investigations showed an acute kidney injury, newly deranged liver function tests and a raised INR (>11, with no bleeding). Complement was normal with a low dsDNA titre. Urinalysis revealed proteinuria and a protein creatinine ratio measured 176mg/mmol. MRI diffusion weighted brain imaging showed acute bilateral occipital and left fronto-parietal infarcts. She had symptoms of a lupus flare with arthralgia and a butterfly facial rash. COVID-19 PCR tests were negative and she had not been recently vaccinated. She was diagnosed with CAPS and transferred to St Thomas' hospital intensive care. On arrival, she received 1mg intravenous vitamin K followed by triple therapy for CAPS: an unfractionated heparin infusion, oral prednisolone 40mg daily, 5 days of plasma exchange and, given her background of SLE, she was treated with intravenous cyclophosphamide (according to the EUROLUPUS regimen). Intravenous methylprednisolone was avoided due to a previous hypertensive encephalopathy reaction. She responded rapidly. Her troponin fell from a peak of 5054 to 294ng/ L, her creatinine settled at a new baseline (232umol/L) and her liver function normalised. She was switched back to warfarin due to her metallic valve and started on aspirin for cardiovascular secondary prevention. She required physical and occupational therapy due to her strokes but recovered well. Case report - Discussion: According to the 2003 criteria, CAPS can be classified as definite when there is evidence of: -3 organs involved, development of manifestations simultaneously or within a week, confirmation by imaging and/or histopathology of small vessel occlusion and positive antiphospholipid antibodies. Probable CAPS is when 3 out of the 4 criteria are present. In this case, three organs were confirmed to be involved with imaging showing cerebral and cardiac ischaemia. Her creatinine rose from a base of 190 to 289umol/L coupled with a high protein creatinine ratio confirming renal involvement. A Budd-Chiari syndrome was also suspected due to deranged liver function tests and INR, though imaging performed after therapy did not confirm this. A biopsy of any of these four organs was not feasible given the severity of her presentation and coagulopathy. There are no randomised controlled trials but data from the CAPS registry guides treatment and management follows a logical approach: anticoagulation to treat thrombosis, glucocorticoids for inflammation and plasma exchange (or IVIG) to remove the circulating autoantibodies. Triple therapy was associated with a reduced mortality compared to no treatment (28.6% versus 75%, respectively). Following analyses from the CAPS registry we also chose to treat with cyclophosphamide, which is associated with improved survival in patients with SLE. This decision was based on the clinical features of an SLE flare as opposed to serological grounds. There have b en reports of rituximab and eculizumab being used successfully in CAPS, though generally as a last resort. As complement activation is seen in animal models of antiphospholipid syndrome thrombosis and rituximab is often used in refractory SLE, they may prove to be promising agents for refractory CAPS. Case report - Key learning points: 1. Prompt recognition and early treatment is vital in managing CAPS 2. Triple therapy with anticoagulation, glucocorticoids and plasma exchange / IVIG is associated with better survival in CAPS 3. Cyclophosphamide is associated with better survival in patients with CAPS and concomitant SLE.