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Current antigen delivery platforms, such as alum and nanoparticles, are not readily tunable, thus may not generate optimal adaptive immune responses. We created an antigen delivery platform by loading lyophilized Microporous Annealed Particle (MAP) with aqueous solution containing target antigens. Upon administration of antigen loaded MAP (VaxMAP), the biomaterial reconstitution forms an instant antigen-loaded porous scaffold area with a sustained release profile to maximize humoral immunity. VaxMAP induced CD4+ T follicular helper (Tfh) cells and germinal center (GC) B cell responses in the lymph nodes similar to Alum. VaxMAP loaded with SARS-CoV-2 spike protein improved the magnitude, neutralization, and duration of anti-receptor binding domain antibodies compared to Alum vaccinated mice. A single injection of Influenza specific HA1-loaded-VaxMAP enhanced neutralizing antibodies and elicited greater protection against influenza virus challenge than HA1-loaded-Alum. Thus, VaxMAP is a platform that can be used to promote adaptive immune cell responses to generate more robust neutralizing antibodies, and better protection upon pathogen challenge.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Imunidade Humoral , Glicoproteína da Espícula de Coronavírus , Animais , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Camundongos , COVID-19/prevenção & controle , COVID-19/imunologia , Porosidade , Feminino , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Materiais Biocompatíveis/química , Camundongos Endogâmicos BALB C , Linfócitos B/imunologia , SARS-CoV-2/imunologia , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/administração & dosagem , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controleRESUMO
Current antigen delivery platforms, such as alum and nanoparticles, are not readily tunable, thus may not generate optimal adaptive immune responses. We created an antigen delivery platform by loading lyophilized Microporous Annealed Particle (MAP) with aqueous solution containing target antigens. Upon administration of antigen loaded MAP (VaxMAP), the biomaterial reconstitution forms an instant antigen-loaded porous scaffold area with a sustained release profile to maximize humoral immunity. VaxMAP induced CD4+ T follicular helper (Tfh) cells and germinal center (GC) B cell responses in the lymph nodes similar to Alum. VaxMAP loaded with SARS-CoV-2 spike protein improved the magnitude and duration of anti-receptor binding domain antibodies compared to Alum and mRNA-vaccinated mice. A single injection of Influenza specific HA1-loaded-VaxMAP enhanced neutralizing antibodies and elicited greater protection against influenza virus challenge than HA1-loaded-Alum. Thus, VaxMAP is a platform that can be used to promote adaptive immune cell responses to generate more robust neutralizing antibodies, and better protection upon pathogen challenge.
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Acral dermatoses, including hyperkeratotic palmoplantar eczema (HPE), palmoplantar psoriasis (PP), and mycosis fungoides palmaris et plantaris (MFPP), can be challenging to diagnose clinically and histopathologically. In this setting, cytokine biomarkers may be able to help provide diagnostic clarity. Therefore, we evaluated IL-17A, IFN-γ, and IL-13 expression in PP, HPE, and MFPP and compared their expression profiles with nonacral sites. We used biopsy specimens from the Yale Dermatopathology database, selecting cases of HPE (n = 12), PP (n = 8), MFPP (n = 8), normal acral skin (n = 9), nonacral eczema (n = 10), and nonacral psoriasis (n = 10) with classic clinical and histopathologic features. IL17A mRNA expression by RNA in situ hybridization differentiated PP (median score 63.1 [interquartile range 9.4-104.1]) from HPE (0.8 [0-6.0]; P = 0.003), MFPP (0.6 [0-2.6]; P = 0.003), and normal acral skin (0 [0-0]; P < 0.001). Unexpectedly, both PP and HPE showed co-expression of IFNG and IL13 mRNA. In contrast, nonacral psoriasis and eczema showed divergent patterns of IFNG and IL13 mRNA expression. Taken together, we show that IL17A mRNA expression may be a useful biomarker of PP, and we further show that acral dermatoses exhibit distinct immunology compared to nonacral sites, with implications for clinical management.
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T follicular helper (TFH) cells are the conventional drivers of protective, germinal center (GC)based antiviral antibody responses. However, loss of TFH cells and GCs has been observed in patients with severe COVID-19. As T cellB cell interactions and immunoglobulin class switching still occur in these patients, noncanonical pathways of antibody production may be operative during SARS-CoV-2 infection. We found that both TFH-dependent and -independent antibodies were induced against SARS-CoV-2 infection, SARS-CoV-2 vaccination, and influenza A virus infection. Although TFH-independent antibodies to SARS-CoV-2 had evidence of reduced somatic hypermutation, they were still high affinity, durable, and reactive against diverse spike-derived epitopes and were capable of neutralizing both homologous SARS-CoV-2 and the B.1.351 (beta) variant of concern. We found by epitope mapping and B cell receptor sequencing that TFH cells focused the B cell response, and therefore, in the absence of TFH cells, a more diverse clonal repertoire was maintained. These data support an alternative pathway for the induction of B cell responses during viral infection that enables effective, neutralizing antibody production to complement traditional GC-derived antibodies that might compensate for GCs damaged by viral inflammation.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Células T Auxiliares Foliculares/imunologia , Sequência de Aminoácidos , Animais , Formação de Anticorpos/imunologia , Linfócitos B/imunologia , Vacinas contra COVID-19/imunologia , Centro Germinativo/imunologia , Humanos , Ativação Linfocitária/imunologia , Camundongos , Linfócitos T Auxiliares-IndutoresRESUMO
Objective. To evaluate the interrater reliability of a universal evaluator rubric used to assess student pharmacist communication skills during patient education sessions.Methods. Six US schools and colleges of pharmacy each submitted 10 student videos of a simulated community pharmacy patient education session and recruited two raters in each of the five rater groups (faculty, standardized patients, postgraduate year one residents, student pharmacists, and pharmacy preceptors). Raters used a rubric containing 20 items and a global assessment to evaluate student communication of 12 videos. Agreement was computed for individual items and overall rubric score within each rater group, and for each item across all rater groups. Average overall rubric agreement scores were compared between rater groups. Agreement coefficient scores were categorized as no to minimal, weak, moderate, strong, or almost perfect agreement.Results. Fifty-five raters representing five rater groups and six pharmacy schools evaluated student communication. Item agreement analysis for all raters revealed five items with no to minimal or weak agreement, 10 items with moderate agreement, one item with strong agreement, and five items with almost perfect agreement. Overall average agreement across all rater groups was 0.73 (95% CI, 0.66-0.81). The preceptor rater group exhibited the lowest agreement score of 0.68 (95% CI, 0.58-0.78), which significantly deviated from the overall average.Conclusion. While strong or almost perfect agreement scores were not observed for all rubric items, overall average interrater reliability results support the use of this rubric in a variety of raters to assess student pharmacist communication skills during patient education sessions.
Assuntos
Educação em Farmácia , Estudantes de Farmácia , Comunicação , Avaliação Educacional , Humanos , Farmacêuticos , Reprodutibilidade dos TestesRESUMO
When students fail to meet minimum competence standards on summative pharmacy skills-based assessments, remediation can be used to ensure student readiness for progression. Skills-based remediation is challenging as a high volume of resources is required to develop an action plan that addresses the heterogeneity in student needs and to create and execute another assessment equivalent to the initial assessment. Although many Doctor of Pharmacy (PharmD) programs face these same challenges, there is no consensus on how to best address them. Recently, faculty from six PharmD programs convened to share ideas and approaches to overcoming these challenges. This commentary aims to define remediation as it pertains to summative skills-based assessments, share our consensus views regarding remediation best practices, and highlight areas where there is more work to be done. Our intent is to advance the ongoing conversation and empower institutions to develop their own effective and impactful skills-based remediation policies, procedures, and activities.
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Educação em Farmácia , Farmácia , Estudantes de Farmácia , Currículo , Docentes , Humanos , LaboratóriosRESUMO
There are currently limited Food and Drug Administration (FDA)-approved drugs and vaccines for the treatment or prevention of Coronavirus Disease 2019 (COVID-19). Enhanced understanding of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and pathogenesis is critical for the development of therapeutics. To provide insight into viral replication, cell tropism, and host-viral interactions of SARS-CoV-2, we performed single-cell (sc) RNA sequencing (RNA-seq) of experimentally infected human bronchial epithelial cells (HBECs) in air-liquid interface (ALI) cultures over a time course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as a major target at the onset of infection, which we confirmed by electron and immunofluorescence microscopy. Over the course of infection, the cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell-intrinsic expression of type I and type III interferons (IFNs) and interleukin (IL)-6 but not IL-1. This results in expression of interferon-stimulated genes (ISGs) in both infected and bystander cells. This provides a detailed characterization of genes, cell types, and cell state changes associated with SARS-CoV-2 infection in the human airway.
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Brônquios/patologia , COVID-19/diagnóstico , Expressão Gênica , SARS-CoV-2/isolamento & purificação , Análise de Célula Única/métodos , Adulto , Brônquios/virologia , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Células Cultivadas , Epitélio/patologia , Epitélio/virologia , Humanos , Imunidade Inata , Estudos Longitudinais , SARS-CoV-2/genética , Transcriptoma , Tropismo ViralRESUMO
Identifying drugs that regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and its symptoms has been a pressing area of investigation during the coronavirus disease 2019 (COVID-19) pandemic. Nonsteroidal anti-inflammatory drugs (NSAIDs), which are frequently used for the relief of pain and inflammation, could modulate both SARS-CoV-2 infection and the host response to the virus. NSAIDs inhibit the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), which mediate the production of prostaglandins (PGs). As PGs play diverse biological roles in homeostasis and inflammatory responses, inhibiting PG production with NSAIDs could affect COVID-19 pathogenesis in multiple ways, including: (1) altering susceptibility to infection by modifying expression of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for SARS-CoV-2; (2) regulating replication of SARS-CoV-2 in host cells; and (3) modulating the immune response to SARS-CoV-2. Here, we investigate these potential roles. We demonstrate that SARS-CoV-2 infection upregulates COX-2 in diverse human cell culture and mouse systems. However, suppression of COX-2 by two commonly used NSAIDs, ibuprofen and meloxicam, had no effect on ACE2 expression, viral entry, or viral replication. In contrast, in a mouse model of SARS-CoV-2 infection, NSAID treatment reduced production of pro-inflammatory cytokines and impaired the humoral immune response to SARS-CoV-2 as demonstrated by reduced neutralizing antibody titers. Our findings indicate that NSAID treatment may influence COVID-19 outcomes by dampening the inflammatory response and production of protective antibodies rather than modifying susceptibility to infection or viral replication.ImportancePublic health officials have raised concerns about the use of nonsteroidal anti-inflammatory drugs (NSAIDs) for treating symptoms of coronavirus disease 2019 (COVID-19). NSAIDs inhibit the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), which are critical for the generation of prostaglandins - lipid molecules with diverse roles in homeostasis and inflammation. Inhibition of prostaglandin production by NSAIDs could therefore have multiple effects on COVID-19 pathogenesis. Here, we demonstrate that NSAID treatment reduced both the antibody and pro-inflammatory cytokine response to SARS-CoV-2 infection. The ability of NSAIDs to modulate the immune response to SARS-CoV-2 infection has important implications for COVID-19 pathogenesis in patients. Whether this occurs in humans and whether it is beneficial or detrimental to the host remains an important area of future investigation. This also raises the possibility that NSAIDs may alter the immune response to SARS-CoV-2 vaccination.
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Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.
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Infecções por Coronavirus/genética , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno , SARS-CoV-2/fisiologia , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/imunologia , COVID-19/virologia , Linhagem Celular , Chlorocebus aethiops , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Coronavirus/classificação , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Técnicas de Inativação de Genes , Redes Reguladoras de Genes , Células HEK293 , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Células Vero , Internalização do VírusRESUMO
Anaphylaxis is a life-threatening allergic reaction caused by cross-linking of high-affinity IgE antibodies on the surface of mast cells and basophils. Understanding the cellular mechanisms that lead to high-affinity IgE production is required to develop better therapeutics for preventing this severe reaction. A recently discovered population of T follicular helper Tfh13 cells regulates the production of high-affinity IgE in mouse models of allergy and can also be found in patients with allergies with IgE antibodies against food or aeroallergens. Here we describe optimized protocols for identifying Tfh13 cells in both mice and humans.
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Separação Celular/métodos , Citometria de Fluxo/métodos , Células T Auxiliares Foliculares , Subpopulações de Linfócitos T , Animais , Humanos , CamundongosRESUMO
Prime editing enables diverse genomic alterations to be written into target sites without requiring double-strand breaks or donor templates. The design of prime-editing guide RNAs (pegRNAs), which must be customized for each edit, can however be complex and time consuming. Compared with single guide RNAs (sgRNAs), pegRNAs have an additional 3' extension composed of a primer binding site and a reverse-transcription template. Here we report a web tool, which we named pegFinder ( http://pegfinder.sidichenlab.org ), for the rapid design of pegRNAs from reference and edited DNA sequences. pegFinder can incorporate sgRNA on-target and off-target scoring predictions into its ranking system, and nominates secondary nicking sgRNAs for increasing editing efficiency. CRISPR-associated protein 9 variants with expanded targeting ranges are also supported. To facilitate downstream experimentation, pegFinder produces a comprehensive table of candidate pegRNAs, along with oligonucleotide sequences for cloning.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , RNA Guia de Cinetoplastídeos/genética , Software , Algoritmos , Proteína 9 Associada à CRISPR/genética , Células HEK293 , Humanos , InternetRESUMO
Soluble forms of angiotensin-converting enzyme 2 (ACE2) have recently been shown to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We report on an improved soluble ACE2, termed a "microbody," in which the ACE2 ectodomain is fused to Fc domain 3 of the immunoglobulin (Ig) heavy chain. The protein is smaller than previously described ACE2-Ig Fc fusion proteins and contains an H345A mutation in the ACE2 catalytic active site that inactivates the enzyme without reducing its affinity for the SARS-CoV-2 spike. The disulfide-bonded ACE2 microbody protein inhibits entry of SARS-CoV-2 spike protein pseudotyped virus and replication of live SARS-CoV-2 in vitro and in a mouse model. Its potency is 10-fold higher than soluble ACE2, and it can act after virus bound to the cell. The microbody inhibits the entry of ß coronaviruses and virus with the variant D614G spike. The ACE2 microbody may be a valuable therapeutic for coronavirus disease 2019 (COVID-19) that is active against viral variants and future coronaviruses.
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Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/farmacologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Microcorpos/metabolismo , SARS-CoV-2/efeitos dos fármacos , Sequência de Aminoácidos , Animais , COVID-19/prevenção & controle , COVID-19/virologia , Modelos Animais de Doenças , Dissulfetos/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Transgênicos , Domínios Proteicos , Multimerização Proteica , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Vírion/metabolismo , Internalização do Vírus/efeitos dos fármacosRESUMO
Identifying drugs that regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and its symptoms has been a pressing area of investigation during the coronavirus disease 2019 (COVID-19) pandemic. Nonsteroidal anti-inflammatory drugs (NSAIDs), which are frequently used for the relief of pain and inflammation, could modulate both SARS-CoV-2 infection and the host response to the virus. NSAIDs inhibit the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), which mediate the production of prostaglandins (PGs). PGE2, one of the most abundant PGs, has diverse biological roles in homeostasis and inflammatory responses. Previous studies have shown that NSAID treatment or inhibition of PGE2 receptor signaling leads to upregulation of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for SARS-CoV-2, thus raising concerns that NSAIDs could increase susceptibility to infection. COX/PGE2 signaling has also been shown to regulate the replication of many viruses, but it is not yet known whether it plays a role in SARS-CoV-2 replication. The purpose of this study was to dissect the effect of NSAIDs on COVID-19 in terms of SARS-CoV-2 entry and replication. We found that SARS-CoV-2 infection induced COX-2 upregulation in diverse human cell culture and mouse systems. However, suppression of COX-2/PGE2 signaling by two commonly used NSAIDs, ibuprofen and meloxicam, had no effect on ACE2 expression, viral entry, or viral replication. Our findings suggest that COX-2 signaling driven by SARS-CoV-2 may instead play a role in regulating the lung inflammation and injury observed in COVID-19 patients.
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Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here we performed a genome-wide CRISPR screen with SARS-CoV-2 and identified known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered novel pro-viral genes and pathways including the SWI/SNF chromatin remodeling complex and key components of the TGF-ß signaling pathway. Small molecule inhibitors of these pathways prevented SARS-CoV-2-induced cell death. We also revealed that the alarmin HMGB1 is critical for SARS-CoV-2 replication. In contrast, loss of the histone H3.3 chaperone complex sensitized cells to virus-induced death. Together this study reveals potential therapeutic targets for SARS-CoV-2 and highlights host genes that may regulate COVID-19 pathogenesis.
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Allergies to food and environmental antigens have steeply grown to epidemic proportions. IgE antibodies are key mediators of allergic disease, including life-threatening anaphylaxis. There is now compelling evidence that one of the hallmarks of anaphylaxis-inducing IgE molecules is their high affinity for allergen, and the cellular pathway to high-affinity IgE is typically through sequential switching of IgG B cells. Further, in contrast to the previously held paradigm that a subset of CD4+ T cells called Th2 cells promotes IgE responses, recent studies suggest that T follicular helper cells are crucial for inducing anaphylactic IgE. Here we discuss recent studies that have enabled us to understand the nature, induction, and regulation of this enigmatic antibody isotype in allergic sensitization.
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Imunoglobulina E/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Anafilaxia/imunologia , Anafilaxia/parasitologia , Animais , Diferenciação Celular , Plasticidade Celular , Humanos , Hipersensibilidade/imunologia , Hipersensibilidade/parasitologiaRESUMO
Objective. To use an expert consensus-building process to develop a rubric used by multiple evaluator types to assess Doctor of Pharmacy students' patient communication skills.Methods. Faculty and staff members from six schools and colleges of pharmacy collaborated on a multi-step expert consensus-building process to create the final version of a communication rubric. First, faculty and patient content experts evaluated each item in the rubric for its relevance, criticality, and global comprehensiveness using a five-point Likert scale (0=not at all, 4=to a high extent). Descriptive statistics were used to analyze the resulting data. Faculty members evaluated the results and came to a consensus on the second version of the rubric. A corresponding codebook was developed and refined through a two-phase process.Results. The initial communication rubric was evaluated by 13 expert reviewers. Mean global comprehensiveness on the rubric was 3.83 for faculty experts and 3.5 for patient experts. After evaluating results from the expert consensus-building process, 14 items on the rubric did not change, five items were revised, three items were removed, and two items were added. The second version of the instrument included 20 items in six topic areas. A codebook was finalized to increase scoring consistency for the 20 communication items.Conclusion. Overall, content experts concluded that the rubric had high global comprehensiveness. Collaboration involving faculty members from multiple schools of pharmacy resulted in a 20-item communication rubric and codebook that can be used to increase consistency in scoring student pharmacists' patient communication skills.
Assuntos
Educação em Farmácia , Estudantes de Farmácia , Comunicação , Consenso , Avaliação Educacional , HumanosRESUMO
Cross-linking of high-affinity immunoglobulin E (IgE) results in the life-threatening allergic reaction anaphylaxis. Yet the cellular mechanisms that induce B cells to produce IgE in response to allergens remain poorly understood. T follicular helper (TFH) cells direct the affinity and isotype of antibodies produced by B cells. Although TFH cell-derived interleukin-4 (IL-4) is necessary for IgE production, it is not sufficient. We report a rare population of IL-13-producing TFH cells present in mice and humans with IgE to allergens, but not when allergen-specific IgE was absent or only low-affinity. These "TFH13" cells have an unusual cytokine profile (IL-13hiIL-4hiIL-5hiIL-21lo) and coexpress the transcription factors BCL6 and GATA3. TFH13 cells are required for production of high- but not low-affinity IgE and subsequent allergen-induced anaphylaxis. Blocking TFH13 cells may represent an alternative therapeutic target to ameliorate anaphylaxis.
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Anafilaxia/imunologia , Imunoglobulina E/imunologia , Interleucina-13/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Adolescente , Animais , Criança , Fator de Transcrição GATA3/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Interleucina-13/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas Proto-Oncogênicas c-bcl-6/metabolismoRESUMO
T follicular helper cells produce interleukin-10 during chronic viral infections to support the humoral response.