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Ionizable amino lipids are a major constituent of the lipid nanoparticles for delivering nucleic acid therapeutics (e.g., DLin-MC3-DMA in ONPATTRO , ALC-0315 in Comirnaty , SM-102 in Spikevax ). Scarcity of lipids that are suitable for cell therapy, vaccination, and gene therapies continue to be a problem in advancing many potential diagnostic/therapeutic/vaccine candidates to the clinic. Herein, we describe the development of novel ionizable lipids to be used as functional excipients for designing vehicles for nucleic acid therapeutics/vaccines in vivo or ex vivo use in cell therapy applications. We first studied the transfection efficiency (TE) of LNP-based mRNA formulations of these ionizable lipid candidates in primary human T cells and established a workflow for engineering of primary immune T cells. We then adapted this workflow towards bioengineering of CAR constructs to T cells towards non-viral CAR T therapy. Lipids were also tested in rodents for vaccine applications using self-amplifying RNA (saRNA) encoding various antigens. We have then evaluated various ionizable lipid candidates and their biodistribution along with the mRNA/DNA translation exploration using various LNP compositions. Further, using ionizable lipids from the library, we have shown gene editing of various targets in rodents. We believe that these studies will pave the path to the advancement in nucleic acid based therapeutics and vaccines, or cell gene therapy agents for early diagnosis and detection of cancer, and for targeted genomic medicines towards cancer treatment and diagnosis.
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Background & Aim: Immunological characteristics of COVID-19 show pathological hyperinflammation associated with lymphopenia and dysfunctional T cell responses. These features provide a rationale for restoring functional T cell immunity in COVID-19 patients by adoptive transfer of SARS-CoV-2 specific T cells. Methods, Results & Conclusion(s): To generate SARS-CoV-2 specific T cells, we isolated peripheral blood mononuclear cells from 7 COVID-19 recovered and 13 unexposed donors. Consequently, we stimulated cells with SARS-CoV-2 peptide mixtures covering spike, membrane and nucleocapsid proteins. Then, we culture expanded cells with IL-2 for 21 days. We assessed immunophenotypes, cytokine profiles, antigen specificity of the final cell products. Our results show that SARSCoV- 2 specific T cells could be expanded in both COVID-19 recovered and unexposed groups. Immunophenotypes were similar in both groups showing CD4+ T cell dominance, but CD8+ and CD3+CD56+ T cells were also present. Antigen specificity was determined by ELISPOT, intracellular cytokine assay, and cytotoxicity assays. One out of 14 individuals who were previously unexposed to SARS-CoV-2 failed to show antigen specificity. Moreover, ex-vivo expanded SARS-CoV-2 specific T cells mainly consisted of central and effector memory subsets with reduced alloreactivity against HLA-unmatched cells suggesting the possibility for the development of third-party partial HLA-matching products. In conclusion, our findings show that SARSCoV- 2 specific T cell can be readily expanded from both COVID-19 and unexposed individuals and can therefore be manufactured as a biopharmaceutical product to treat severe COVID-19 patients.Copyright © 2023 International Society for Cell & Gene Therapy
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Introduction/Aim: The spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enables it to recognise and bind host receptors. These dynamics have been modelled in various cell types and immortalised lines, but rarely in primary airway epithelial cells (AEC), and especially not in children. Therefore, this study on AEC recapitulated earlier work testing the hypothesis that exposure to the spike protein would induce airway immune responses in airway cells of young children. Method(s): Primary AEC monolayer cultures from healthy children (n = 5, <10 years old, males = 5) were exposed to the spike protein S1 subunit (0.01, 1, and 10 mug/mL) over 48 h. Induced inflammatory cytokines, interleukin (IL) 6 and IL8, and viral-associated chemokines, CCL5 and CXCL10 were measured via ELISA. Basal receptor gene expression (ACE2 and TMPRSS2) was measured in monolayer (n = 5) and terminally differentiated (air-liquid interface [ALI];n = 5) cell models as well as in ex-vivo cells obtained directly from nasal brushings (n = 71). Generalised linear modelling, accounting for individual variability, identified any statistical difference (p < 0.05). Result(s): Exposure to the spike protein resulted in no increase in IL6 and IL8 production, however a significant (p < 0.05) decrease was observed at the highest dose tested (10 mug/mL). CXCL10 was only significantly induced at the highest dose (10 mug/mL) whereas CCL5 was not induced. When compared to ex-vivo samples, baseline expression of ACE2 and TMPRSS2 was significantly lower in monolayer cultures (~57- and ~4- fold respectively, p < 0.05), whereas ALI cultures had similar expression levels. Conclusion(s): The use of recombinant spike protein and monolayer cultures appears to not accurately model SARS-CoV-2 spike protein-host interactions. The lack of inflammatory responses may be attributed to the lower receptor gene expression in monolayer cultures. Future studies should utilise live virus and ALI cultures as a more biologically relevant model to study virus-host interactions.
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The proceedings contain 63 papers. The topics discussed include: doxorubicin cardiotoxicity in human organotypic cardiac slices is modulated by P38 MAPK inhibition in a sex- and isoform-specific manner;validation of a modified response evaluation criteria in solid tumors after stereotactic ablative radiosurgery for lung cancer;safer use of aspirin in older adults, need for a consensus;efficacy of facemasks in prevention of COVID-19: a systematic review;practice patterns of rapid influenza diagnostic test;equity and inclusion in patient centered outcomes research: lessons from the adaptable study at Montefiore site;a solution to decrease potentially inappropriate medications (PIM) use during hospitalization;predictors of misperceptions, risk perceptions, and personal risk perceptions about COVID-19 by country, education and income;cognitive function and the consumption of probiotic foods in older adults: an NHANES study;and registered dietitian nutritionist care impacts nutrition-related outcomes for patients with cancer in the outpatient setting.
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Introduction: More than 8 million lives are claimed annually by various respiratory diseases including lung cancer. While therapeutics is the first line of defence, treatment failure always remains challenging and research studies face a lag of transition from preclinical to clinical phase. This is partly due to the inadequate representation of the preclinical models in clinical trials. In this proof-of-concept study, we sought to use an ex-vivo model to identify lung pathologies and therapeutically screen them in a rodent model. Method(s): Briefly, the heart-lung tissues were extracted and decellularized using a detergent-based decellularization technique. Subsequently, lungs were seeded and cultured (6-10 days) with human cell lines: BEAS-2B, A549, and Calu3, demonstrating healthy lung, cancerous state, and congenital pathologies (cystic fibrosis), respectively. By altering the cultural conditions and exploiting the unique characteristics of these cell lines, we were able to model a variety of novel pathological models in ex vivo, such as advanced-stage solid tumours and the primary phase of infection via SARS-COV2. We also validated the above-mentioned observations by histology and immunofluorescence staining. Another novel part of our study includes a qualitative screening of efficacy and impact of important Therapeutics (anti-neoplastic)- Cisplatin and Wogonin, in our cancer models. Result(s): Using A549 and BEAS-2B cells, we were able to model different stages of Non-small cell lung cancer, qualitatively validated the resemblance to clinical samples and monitor the impact of different therapeutics on these models. The qualitative assessment also demonstrated different levels of cell death depending on the efficacy of the drugs. Contribution to research : Collectively this study demonstrates the remarkable versatility and strength of the ex vivo model in representing important lung pathologies and screening therapeutics in the preclinical phase.
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Objectives: In this study, we developed angiotensin-converting enzyme 2 (ACE2)-specific, peptide-derived 68Ga- and 18F-labeled radiotracers, motivated by the hypotheses that ACE2 is an important determinant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) susceptibility and that modulation of ACE2 in coronavirus disease 2019 (COVID-19) drives severe organ injury. Our current efforts are focusing on broader dissemination of ACE2-targeted PET radiotracers based on chelation of [18F]AlF enabling advanced murine and potentially human studies. Method(s): A series of NOTA-conjugated peptides derived from the known ACE2 inhibitor DX600 were synthesized, with variable linker identity. Since DX600 bears 2 cystine residues, both linear and cyclic peptides were studied. An ACE2 inhibition assay was used to identify lead compounds, which were labeled with 68Ga and 18F-AlF to generate the corresponding peptide radiotracers (68Ga-NOTA-PEP). The most potent 68Ga and 18F-AlF DX600 derivatives were subsequently studied in a humanized ACE2 (hACE2) transgenic model. Result(s): Cyclic DX-600-derived peptides had markedly lower half-maximal inhibitory concentrations than their linear counterparts. The 3 cyclic peptides with triglycine, aminocaproate, and polyethylene glycol linkers had calculated half-maximal inhibitory concentrations similar to or lower than the parent DX600 molecule. Peptides were readily labeled with 68Ga and 18F-AlF, and the biodistribution of both tracers was determined in an hACE2 transgenic murine cohort. Pharmacologic concentrations of coadministered NOTA-PEP (blocking) showed a significant reduction of 68Ga-NOTA-PEP4 signals in the heart, liver, lungs, and small intestine. Ex vivo hACE2 activity in these organs was confirmed as a correlate to in vivo results. The biodistribution of both tracers was similar, with apparent blocking observed in the lungs using the 18F-AlF peptide that needs to be verified via additional experiments. Conclusion(s): NOTA-conjugated cyclic peptides derived from the known ACE2 inhibitor DX600 retain their activity when N-conjugated for 68Ga or 18F-AlF chelation. In vivo studies in a transgenic hACE2 murine model using the lead tracer, 68Ga-NOTA-PEP4, showed specific binding in the heart, liver, lungs and intestine-organs known to be affected in SARS-CoV-2 infection. Blocking studies using the 18F-AlF labeled correlate showed modulation of PET signals in the normal lungs. These results suggest that 68Ga-NOTA-PEP4 or the 18F-AlF correlate could be used to detect organ-specific suppression of ACE2 in SARS-CoV-2-infected murine models and COVID-19 patients.Copyright © 2023 Southern Society for Clinical Investigation.
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Allergic and hypersensitivity reactions induced by COVID-19 vaccines are increasingly reported and some patients may develop prolonged urticarial reactions following COVID-19 vaccination. Herein, we investigated the risk factors and immune mechanisms for patients with COVID-19 vaccines-induced immediate allergy and chronic urticaria (CU). We prospectively recruited and analyzed 129 patients with COVID-19 vaccine-induced immediate allergic and urticarial reactions as well as 115 COVID-19 vaccines-tolerant individuals from multiple medical centers during 2021-2022. The clinical manifestations included acute urticaria, anaphylaxis, and delayed to chronic urticaria developed after COVID-19 vaccinations. The serum levels of histamine, IL-2, IL-4, IL-6, IL-8, IL-17A, TARC, and PARC were significantly elevated in allergic patients comparing to tolerant subjects (P-values=4.5x10-5-0.039). Ex vivo basophil revealed that basophils from allergic patients could be significantly activated by COVID-19 vaccine excipients (polyethylene glycol 2000 and polysorbate 80) or spike protein (P-values from 3.5x10-4 to 0.043). Further BAT study stimulated by patients' autoserum showed positive in 81.3% of patients with CU induced by COVID-19 vaccination (P=4.2x10-13), and the reactions could be attenuated by anti-IgE antibody. Autoantibodies screening also identified the significantly increased of IgE-anti-IL-24, IgG-anti-FceRI, IgG-anti-TPO, and IgG-anti-thyroid-related proteins in COVID-19 vaccines-induced CU patients comparing to SARS-COV-2 vaccines-tolerant controls (P-values= 4.6x10-10-0.048). Patients with COVID-19 vaccines-induced recalcitrant CU patients could be successfully treated with anti-IgE therapy. In conclusion, our results revealed that multiple vaccine components, inflammatory cytokines, and autoreactive IgG/IgE antibodies contribute to COVID-19 vaccine-induced immediate allergic and autoimmune urticarial reactions (Minor revision in Journal of Autoimmunity [IF=14.551]).Copyright © 2023
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Background: Although increasing evidence confirms neuropsychiatric manifestations associated mainly with severe COVID-19 infection, long-term neuropsychiatric dysfunction (recently characterized as part of "long COVID-19" syndrome) has been frequently observed after mild infection. Method(s): We performed a broad translational investigation, employing brain imaging and cognitive tests in 81 living COVID-19 patients (mildly infected individuals) as well as flow cytometry, respirometry, microscopy, proteomics, and metabolomics in postmortem brain samples, and in preclinical in vitro and ex vivo models. Result(s): We observed orbitofrontal cortical atrophy, neurocognitive impairment, excessive fatigue and anxiety symptoms in living individuals. Postmortem brain tissue from 26 individuals who died of COVID-19 revealed histopathological signs of brain damage. Five individuals out of the 26 exhibited foci of SARS- CoV-2 infection and replication, particularly in astrocytes. Supporting the hypothesis of astrocyte infection, neural stem cell-derived human astrocytes in vitro are susceptible to SARS-CoV-2 infection through a non-canonical mechanism that involves spike-NRP1 interaction. SARS-CoV-2-infected astrocytes manifested changes in energy metabolism and in key proteins and metabolites used to fuel neurons, as well as in the biogenesis of neurotransmitters. Moreover, human astrocyte infection elicits a secretory phenotype that significantly reduces neuronal viability. Conclusion(s): Our data support the model in which COVID-19 alter cortical thickness, promoting psychiatric symptoms. In addition, SARS-CoV-2 is able to reach the brain, infects astrocytes, and consequently, leads to neuronal death or dysfunction. These deregulated processes could contribute to the structural and functional alterations seen in the brains of COVID-19 patients. Funding Source: Sao Paulo Research Foundation (FAPESP) Keywords: COVID-19, Anxiety, Astrocytes, Multi-omics, Brain Magnetic Resonance Imaging (MRI)Copyright © 2023
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The development of single use flexible bronchoscopes (SUFB) has proceeded with pace over the last 2 years. Concerns regarding infection related to standard bronchoscopes with subsequent COVID-19 pandemic accelerated global uptake with multiple companies releasing SUFB. There has been no ex-vivo comparison of SUFBs to date. We obtained samples of all commercially available SUFBs (TSC© , Boston Scientific©, Ambu©, Vathin© and Pentax © prototype SUFB). We compared technical metrics using a custom-built bench toolkit engineered to allow standardisation. Angulation was analysed by a force meter to ascertain the effort needed to fully flex the scopes while empty and while accessed by both a forceps and cytology brush. The Ambu aScope 4 has the best performance in measured thumb force (Mean 4.15Nm/100 ). The Pentax EB15- S01 has the smallest outer diameter and the largest working channel but has the greatest loss in angulation when its working channel is occupied (-33 / -67 , -100 ). The Pentax EB15-S01 deviated the most from its reported specifications. The Vathin H-SteriScope provided the most angulation overall, including with its working channel in use (180 / 186 , 366 ). This research helps to inform the practical usability of each bronchoscope when deciding which SUFB is best for the physicians intended end use. Further research should look at perceived qualitative assessment of SUFB by clinicians.
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Rational: Evidence of neutrophil dysfunction in COVID-19 is based on transcriptomics. Cell functions are interwoven pathways, so understanding the effect of COVID-19 across neutrophil function may identify therapeutic targets. We examined neutrophil phenotype and function in 41 hospitalised, non-ICU COVID-19 patients versus 23 age-matched controls (AMC) and 26 community acquired pneumonia (CAP) patients. Method(s): Isolated neutrophils underwent ex vivo analyses for migration, phagocytosis and NETosis, and the effect of PI3K inhibition. Circulating DNAse 1 activity and levels of cfDNA were measured. Result(s): Compared to AMC and CAP, COVID-19 neutrophils demonstrated elevated transmigration (p=0.0397, A) and NETosis (p=0.0366, B), but impaired phagocytosis (p=0.0236, C) associated with impaired ROS generation (p<0.0001). COVID-19 and CAP patients showed increased systemic markers of NETosis including increased cfDNA (p=0.0153) and impaired DNAse activity (p<0.0.001, D). Ex vivo inhibition of PI3K gamma and delta reduced NET release by COVID-19 neutrophils (p=0.0156). Conclusion(s): COVID-19 is associated with neutrophil dysfunction across all main effector functions, with elevated migration, impaired antimicrobial responses and elevated NETosis. These changes represent a clear mechanism for tissue damage and highlight that targeting neutrophil function via PI3k may help modulate COVID-19 severity. (Figure Presented).
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Background: Vaccines against SARS-CoV-2 virus significantly reduce morbidity and mortality of the pandemic. But with millions of people vaccinated in a short period of time, even very rare side effects like the clotting disorder vaccine-induced thrombotic thrombocytopenia (VITT) became apparent. We recently identified an increase in procoagulant platelets in these patients, which is even higher than in a previously reported cohort of COVID-19 patients. Method(s): 8 patients (4 female and 4 male) who were hospitalized with suspected thrombotic complications 5 to 16 days after ChAdOx1 nCoV-19 vaccination were included in this study. The median age was 38 years. All patients had thrombocytopenia at admission. Three had a fatal outcome and five were successfully treated. The blood samples were analyzed by using enzyme immune assays, flow cytometry, ex vivo thrombus formation assay and heparin-induced platelet aggregation assay. Result(s): All sera from VITT patients contained antibodies against PF4 [OD 3.0+/-0.68] with the ability to activate platelets (8/8). Sera induced significant increase in procoagulant markers (CD62P and phosphatidylserine externalization) [CD62P/PS positive PLTs: 40.82+/-7.02%] compared to COVID-19 patients [FI CD62P/PS positive PLTs:15.71+/-7.70];p=0.8977. The formation of procoagulant platelets could be significantly reduced by use of the monoclonal IV.3 antibody as well as IVIG [FI CD62P/PS positive PLTs:1.01+/-0.36];p=0.0001. In thrombus formation model, IgGs from VITT patients induced increase platelet surface area (8.64+/-0.53, SAC+/-SEM) compared to control (0.72+/-0.0.07, SAC+/-SEM);p=0.001), which was inhibited by IVIG (4.07+/-0.51, p= 0.001). Conclusion(s): Our ex vivo microfluidic thrombus formation model supports the significance of procoagulant platelet in the pathogenesis of VITT. It may offer significant clinical implications and therapeutic options like evaluation of IVIG as a recommended therapy or other drugs for treatment of clinical picture of VITT.
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The role of the renin-angiotensin system (RAS) in coronavirus disease 2019 (COVID-19) has received much attention, because the angiotensin-converting enzyme 2 (ACE2) has been identified as the main receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has been speculated that, in COVID-19, RAS dysregulation in favor of angiotensin II (Ang-II)-mediated signaling might result in severe tissue inflammation and lung injury. Likewise, the role of a pre-existing therapy with angiotensin-converting enzyme 1 inhibitors (ACEi) or Ang- II type 1 receptor blockers (ARBs) in COVID-19 is largely unclear. We evaluated the effects of the ACEi enalapril (ENA) and the ARB losartan (LOS) on SARS-CoV-2 infection in human ex vivo-cultured, precision-cut lung slices (PCLS) obtained from normal human lung tissue. The PCLS were pretreated for 5 days with vehicle, LOS or ENA (300 muM), followed by mock infection or infection with SARS-CoV-2 and (continued) incubation with vehicle, LOS or ENA for 1 or 2 days. Thereafter, PCLS were harvested for analysis of viral replication, inflammatory responses, endoplasmic reticulum (ER) stress and apoptosis pathways. Both LOS and ENA significantly reduced viral replication in PCLS, with ENA being more potent. LOS was more efficient than ENA in reducing the expression of IL1B, CCL2, CXCL2 and TNFA, but not of IL6, whereas ENA preferentially caused a reduction of IL6 and CCL2 in SARS-CoV-2-infected PCLS. Furthermore, ENA, but not LOS, significantly decreased the expression of viral entry factors, ACE2 and transmembrane serine protease 2 (TMPRSS2), in infected PCLS, both of which were found to be robustly induced upon SARS-CoV-2 infection. Importantly, LOS or ENA did not exert apoptosis or other cytotoxic effects. Renin-angiotensin system-antagonizing drugs do not seem to exert detrimental effects during SARS-CoV-2 infection.On the contrary, in an ex vivo model of human PCLS, such treatment was found to dampen SARS-CoV-2 infection and consecutive inflammation.
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The palatine tonsils (hereinafter referred to as "tonsils") serve as a reservoir for viral infections and play roles in the immune system's first line of defense. The aims of this study were to establish tonsil epithelial cell-derived organoids and examine their feasibility as an ex vivo model for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The tonsil organoids successfully recapitulated the key characteristics of the tonsil epithelium, including cellular composition, histologic properties, and biomarker distribution. Notably, the basal layer cells of the organoids express molecules essential for SARS-CoV-2 entry, such as angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2) and furin, being susceptible to the viral infection. Changes in the gene expression profile in tonsil organoids revealed that 395 genes associated with oncostatin M signaling and lipid metabolism were highly upregulated within 72 h after SARS-CoV-2 infection. Notably, remdesivir suppressed the viral RNA copy number in organoid culture supernatants and intracellular viral protein levels in a dosedependent manner. Here, we suggest that tonsil epithelial organoids could provide a preclinical and translational research platform for investigating SARS-CoV-2 infectivity and transmissibility or for evaluating antiviral candidates.
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Background: Blood product in therapeutic transfusion are now commonly acknowledged to present biologically active constituents during processes of preparation. In the midst of worldwide COVI-19 pandemic, preliminary evidence, suggest that convalescent plasma may lessen the severity of COVID-19, particularly concerning patients with profound B-cell lymphopenia and prolonged COVID-19 symptoms. Aim(s): This study examined the influence of photochemical pathogen reduction treatment (PRT) using amotosalen-HCl and UVA light vs untreated control convalescent plasma (n = 72 -paired samples) -cFFP. Method(s): This study investigated the soluble inflammatory factors: SCD40L, IFN-alpha, IFN-beta, IFN-gamma, IL-1 beta, IL-6, IL-8, IL-10, IL-18, TNF-alpha and ex-vivo inflammatory bioactivity on endothelial cells. Result(s): We observed that IL-8 concentrations were significantly decreased in cFFP w PRT, whereas IL-18 concentration was increased. We observed after activation with cFFP w PRT and w/o PRT no significant modulation of IL-6 released by endothelial cells. CD54 and CD31 expression in the presence of cFFP (w or w/o PRT) is close to negative controls, even if CD54 and CD31 were significant decreased in presence of cFFP w vs w/o PRT. Conclusion(s): It appears valuable to carry on investigations, of IL-18 and IL-8, on both the physiopathology of PRT convalescent plasma treated and post marketing clinical trials. Further research, including a careful clinical evaluation of CCP-treated patients, will be required to further define the clinical relevance of these findings.
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Background: COVID-19 convalescent plasma (CCP) contains neutralizing anti-SARS- CoV- 2 antibodies that may be useful as COVID-19 passive immunotherapy in patients at risk of developing severe disease. Aim(s): Such plasma from convalescent patients may also have additional immune-modulatory properties when transfused to COVID-19 patients. Method(s): CCP (n = 766) were compared to control non-convalescent plasma (n = 166) for soluble inflammatory markers, ex-vivo inflammatory bioactivity on endothelial cells, neutralizing auto-Ab to type I IFNs, and reported adverse events in the recipients. Result(s): CCP exhibited significantly higher IL-6 and TNF-alpha (0.531+/-0.04 vs 0.271+/-0.04;p = 0.0061 and 0.900+/-0.07 vs 0.283+/-0.07 pg/ml;p < 0.0001), respectively) and lower IL-10 (0.731+/-0.07 vs 1.22+/-0.19 pg/ ml, p = 0.0034) levels than control plasma. Other inflammatory markers as well as ex-vivo bioactivity did not differ significantly between CCP and control plasma. Neutralizing auto-Abs against type I IFNs were detected in 14/766 (1.8 %) CCP. They were not associated with reported adverse events when transfused (n = 14). Inflammatory markers and bioactivity in CCP with or without auto-Ab, or in CCP associated or not with adverse events in transfused patients, did not differ significantly. Overall, CCP exhibited moderately increased inflammatory markers compared to control plasma with no discernable differences in ex-vivo bioactivity. Auto-Ab to type I IFNs, detected in a small fraction of CCP, were not associated with reported adverse events or differences in inflammatory markers. Conclusion(s): Further defining the clinical relevance of these findings will require further studies including careful clinical evaluation of patients treated with CCP.
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Background: Vaccines against SARS-CoV- 2 virus reduce morbidity and mortality of the pandemic. But with millions of people vaccinated in a short period of time, even very rare side effects like the clotting disorder vaccine-induced thrombotic thrombocytopenia (VITT) became apparent. We recently identified an increase in procoagulant platelets in these patients. Aim(s): Investigation of the impact of procoagulant platelets in thrombus formation. Method(s): 8 patients (4 female, 4 male) who were hospitalized with suspected thrombotic complications 5 to 16 days after ChAdOx1 nCoV-19 vaccination were included in this study. The blood samples were analyzed by using enzyme immune assays, flow cytometry, ex vivo thrombus formation assay and heparin-induced platelet aggregation assay. Result(s): The median age was 38 years. All patients had thrombocytopenia at admission. Three had a fatal outcome and five were successfully treated. All sera from VITT patients contained high titer antibodies against platelet factor 4 (PF4) [OD: 3.0 +/- 0.68] with the ability to activate platelets in the HIPA assay (8/8). Sera from VITT patients induced significant increase in procoagulant markers (P-selectin [CD62P] and phosphatidylserine externalization) [% CD62P/ PS positive PLTs: 40.82 +/- 7.02] compared to COVID-19 patients [% CD62P/PS positive PLTs: 15.71 +/- 7.70]. The generation of procoagulant platelets was PF4 dependent. The formation of procoagulant platelets could be significantly reduced by use of the monoclonal IV.3 [% CD62P/PS positive PLTs: 1.05 +/- 0.21];p = 0.0001 antibody as well as IVIG [% CD62P/PS positive PLTs: 1.01 +/- 0.36];p = 0.0001. In thrombus formation model, IgGs from VITT patients induced increase platelet surface area (Mean % SAC +/- SEM: 10.38 +/- 1.30) compared to control IgG, which was inhibited by IVIG (4.08 +/- 0.96), p = 0.001. Conclusion(s): Our ex vivo microfluidic thrombus formation model supports the significance of procoagulant platelet in the pathogenesis of VITT. It may offer significant clinical implications and therapeutic options like evaluation of IVIG as a recommended therapy or other drugs for treatment of clinical picture of VITT. (Table Presented).
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Background: With increasing number of vaccinations against SARS-CoV-2, rare but life threatening thrombotic events at unusual sites have been reported, and collectively this phenomenon is termed as vaccine-induced immune thrombotic thrombocytopenia (VITT). Pathophysiology of VITT is similar to that of heparin-induced thrombocytopenia (HIT), and associated with platelet-activating antibodies against platelet factor 4 (PF4). Aim(s): Current guidelines for anticoagulation in VITT patients are issued accordingly, with a focus on non-heparin anticoagulants. In this study, we investigated the interactions of heparin, danaparoid, fondaparinux and argatroban with VITT-Ab/ PF4 complexes. Method(s): We utilized an in-house enzyme immunoassays (EIA) to estimate antibody binding, inhibition and dissociation of preformed PF4-VITT complexes. Using biolayer interferometry (BLI), we analyzed binding kinetics and dissociation of complexes in real time. In a flow-based ex vivo model, we assessed the impact of anticoagulants on VITT-mediated thrombus formation. Result(s): We found that heparin and danaparoid not only inhibited VITT IgG binding to PF4 but were also able to effectively dissociate preformed PF4/IgG complexes in EIA. In BLI, binding of PF4 specific antibodies was observed for all VITT samples tested, and we found remarkable changes in their dissociation after addition of various anticoagulants. Furthermore, IgGs from VITT patients induce increased thrombus formation in comparison to the healthy controls (mean % SAC +/- SEM: 11.59 +/- 0.57 vs. 1.99 +/- 0.34 respectively, p < 0.001), which can further be effectively inhibited with danaparoid and heparin (mean % SAC +/- SEM 2.82 +/- 0.50 and 1.85 +/- 0.56. p < 0.001). Fondaparinux and argatroban inhibited thrombus formation;however, they did not affect antibody binding. Conclusion(s): Taken together, our data shed a light on suitability of anticoagulants in VITT, and indicate that negatively charged anticoagulants can disrupt VITT-Ab/ PF4 interactions, which might serve as an approach to reduce antibody-mediated complications in VITT. Our results should be confirmed, however, in a clinical setting before a recommendation regarding the selection of anticoagulation in VITT patients could be made.
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Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with an increased risk of venous and arterial thrombosis but the underlying mechanism if still unclear. Aim(s): The study would identify a mechanism implicated in platelet activation and thrombus growth during SARS-CoV-2 infection. Method(s): We performed a cross-sectional analysis of platelet function in 30 SARS-CoV-2 and 20 healthy subjects (HS) by measuring Nox2-derived oxidative stress and thromboxane (Tx) B2 and investigated if administration of monoclonal antibodies against the Spike(S) protein of SARS-CoV-2 affects platelet activation. Furthermore, we investigated in vitro if the Spike(S) protein of SARS-CoV-2 or plasma from SARS-CoV-2 enhanced platelet activation. Result(s): Ex vivo studies showed enhanced platelet Nox2-derived oxidative stress and TxB2 biosynthesis and under laminar flow platelet-dependent thrombus growth in SARS-CoV-2 compared to controls;both effects were lowered by Nox2 and Toll-like receptor 4(TLR4) inhibitors. Two hours after administration of monoclonal antibodies a significant inhibition of platelet activation was observed in SARS-CoV-2 patients compared to untreated ones. In vitro study showed that S protein functionally interacts with platelet TLR4, and a docking simulation analysis suggested that TLR4 binds to S protein via three receptor-binding domains;furthermore, in platelets from SARS-CoV-2 S protein co-immunoprecipitated with TLR4. Plasma from SARS-CoV-2 patients incubated with normal platelets enhanced platelet activation and Nox2-related oxidative stress, an effect blunted by TNF-alpha inhibitor;this effect was recapitulated by an in vitro study documenting that TNF-alpha alone promoted platelet activation and amplified the platelet response to S protein via p47phox up-regulation. Conclusion(s): The study identifies two TLR4-dependent and independent pathways promoting platelet-dependent thrombus growth and suggests inhibition of TLR4 or p47phox as a tool to counteract thrombosis in SARS-CoV-2.
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Introduction: Liver cirrhosis entails elevated risk of COVID-19-associated mortality. This study determined T cell-mediated and antibody reactivity against the spike 1 (S1) protein of SARS-CoV-2 among 48 cirrhotic patients and 39 healthy controls after two and three doses of mRNA COVID-19 vaccination. Aims & Methods: SARS-CoV-2-specific T cell reactivity was measured by induced level of T cell-derived interferon-gamma (IFN-gamma) in blood cells stimulated ex vivo with multimeric peptides spanning the N-terminal portion of S1. S1-induced IFN-gamma was quantified before and after the 1st and 2nd vaccination (BNT162b2, Pfizer-BioNTech or mRNA-1273, Moderna), while serum IgG against the receptor-binding domain (RBD) within S1 (anti-RBD-S1 IgG) were quantified after the 1st, 2nd and third dose of the vaccine. Result(s): T cell reactivity against S1 was reduced in cirrhotic patients after the 1st (P<0.001 vs controls) and 2nd (P<0.001) vaccination. Sixty-eight % of patients lacked detectable S1-specific T cell reactivity after the 1st vaccination vs. 19% in controls (OR 0.11, HR 0.03-0.48, P=0.003) and 36% remained devoid of reactivity after the 2nd vaccination vs. 6% in controls (OR 0.12, HR 0.03-0.59, P=0.009). T cell reactivity in cirrhosis remained significantly impaired after correction for potential confounders in multivariable analysis. Advanced cirrhosis (Child-Pugh class B) was associated with absent or lower T cell responses (P<0.05 vs. Child-Pugh class A). The deficiency of T cell reactivity was paralleled by lower levels of anti-RBD-S1 IgG after the 1st (P<0.001 vs. controls) and 2nd (P<0.05) vaccination. Anti-RBD IgG levels were increased significantly after the 3rd compared to the 2nd dose (median 944 vs. 563 BAU/ml, p=0.002). Hybrid immunity, i.e., the combined effect of vaccination and naturally acquired COVID-19, was associated with significantly higher antibody levels (>5680 (821->5680) vs 944 (5-5675)) BAU/ml, p=0.0001) as compared to antibody levels achieved through 3 doses of vaccination alone. The time elapsed between vaccination to blood draw after the 3rd dose of the vaccine was significantly longer than after the 2nd dose (90 vs 118 days, p < 0.001). Conclusion(s): Cirrhotic patients show deficient T cell reactivity against SARS-CoV-2 antigens along with diminished levels of anti-RBD-S1 IgG after dual COVID-19 vaccination. Nevertheless, a third dose of mRNA COVID-19 vaccine generally results in high antibody levels, and hybrid immunity following naturally acquired infection elicits further augmented levels in patients with cirrhosis. As immune waning is of concern with regards to COVID-19, continued vigilance as well as iterated booster vaccine doses for these vulnerable patients is likely prudent.