RESUMO
The non-classical Major Histocompatibility Complex class II (MHCII) protein, H2-M, edits peptides bound to conventional MHCII in favor of stable peptide/MHCII (p/MHCII) complexes. Here, we show that H2-M deficiency affects B-1 cell survival, reduces cell renewal capacity, and alters immunoglobulin repertoire, allowing for the selection of cells specific for highly abundant epitopes, but not low-frequency epitopes. H2-M-deficient B-1 cells have shorter CDR3 length, higher content of positively charged amino acids, shorter junctional regions, less mutation frequency, and a skewed clonal distribution. Mechanistically, H2-M loss reduces plasma membrane p/MHCII association with B cell receptors (BCR) on B-1 cells and diminishes integrated BCR signal strength, a key determinant of B-1 cell selection, maturation, and maintenance. Thus, H2-M:MHCII interaction serves as a cell-intrinsic regulator of BCR signaling and influences the selection of the B-1 cell clonal repertoire.
Assuntos
Linfócitos B/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Ativação Linfocitária/imunologia , CamundongosRESUMO
Tissue-resident macrophages (TRMΦ) are important immune sentinels responsible for maintaining tissue and immune homeostasis within their specific niche. Recently, the origins of TRMΦ have undergone intense scrutiny, in which now most TRMΦ are thought to originate early during embryonic development independent of hematopoietic stem cells (HSCs). We previously characterized two distinct subsets of mouse peritoneal cavity macrophages (MΦ) (large and small peritoneal MΦ) whose origins and relationship to both fetal and adult long-term (LT) HSCs have not been fully investigated. In this study, we employ highly purified LT-HSC transplantation and in vivo lineage tracing to show a dual ontogeny for large and small peritoneal MΦ, in which the initial wave of peritoneal MΦ is seeded from yolk sac-derived precursors, which later require LT-HSCs for regeneration. In contrast, transplanted fetal and adult LT-HSCs are not able to regenerate brain-resident microglia. Thus, we demonstrate that LT-HSCs retain the potential to develop into TRMΦ, but their requirement is tissue specific in the peritoneum and brain.
Assuntos
Encéfalo/citologia , Células-Tronco Hematopoéticas/fisiologia , Macrófagos/fisiologia , Peritônio/citologia , Animais , Linhagem da Célula , Desenvolvimento Embrionário , Feminino , Camundongos , Especificidade de Órgãos/fisiologia , Gravidez , RegeneraçãoRESUMO
Disruption of vulnerable atherosclerotic plaques often leads to myocardial infarction and stroke, the leading causes of morbidity and mortality in the United States. A diagnostic method that detects high-risk atherosclerotic plaques at early stages could prevent these sequelae. The abundance of immune cells in the arterial wall, especially inflammatory Ly-6Chi monocytes and foamy macrophages, is indicative of plaque inflammation, and may be associated with plaque vulnerability. Hence, we sought to develop a new method that specifically targets these immune cells to offer clinically-relevant diagnostic information about cardiovascular disease. We combine ultra-selective nanoparticle targeting of Ly-6Chi monocytes and foamy macrophages with clinically-viable photoacoustic imaging (PAI) in order to precisely and specifically image inflamed plaques ex vivo in a mouse model that mimics human vulnerable plaques histopathologically. Within the plaques, high-dimensional single-cell flow cytometry (13-parameter) showed that our nanoparticles were almost-exclusively taken up by the Ly-6Chi monocytes and foamy macrophages that heavily infiltrate plaques. PAI identified inflamed atherosclerotic plaques that display ~6-fold greater signal compared to controls (P<0.001) six hours after intravenous injection of ultra-selective carbon nanotubes, with in vivo corroboration via optical imaging. Our highly selective strategy may provide a targeted, non-invasive imaging strategy to accurately identify and diagnose inflamed atherosclerotic lesions.
RESUMO
BACKGROUND: Up-regulated interleukin 6 (IL-6) signaling, immune system activation, and pronociceptive autoantibodies are characteristic of complex regional pain syndrome (CRPS). IL-6 is known to promote B cell differentiation, thus we hypothesized that IL-6 signaling plays a crucial role in the development of adaptive immune responses and nociceptive sensitization in a murine tibia fracture model of CRPS. METHODS: Mice deficient in IL-6 expression (IL-6-/-) or B cell deficient (muMT) underwent tibia fracture and 3 weeks of cast immobilization or sham injury. The deposition of IgM in fractured limbs was followed using Western blotting, and passive serum transfer to muMT fracture mice was used to detect nociception-supporting autoantibodies. Lymph nodes were assessed for hypertrophy, IL-6 expression was measured using qPCR and ELISA, and germinal center formation was evaluated using FACS and immunohistochemistry. The therapeutic effects of exogenous neutralizing anti-IL-6 antibodies were also evaluated in the CRPS fracture model. RESULTS: Functional IL-6 signaling was required for the post fracture development of nociceptive sensitization, vascular changes, and IgM immune complex deposition in the skin of injured limbs. Passive transfer of sera from wild-type, but not IL-6-/- fracture mice into muMT fracture mice caused enhanced allodynia and postural unweighting. IL-6-/- fracture mice displayed reduced popliteal lymphadenopathy after fracture. Germinal center responses were detected in the popliteal lymph nodes of wild-type, but not in IL-6-/- fracture mice. We observed that IL-6 expression was dramatically enhanced in popliteal lymph node tissue after fracture. Conversely, administration of anti-IL-6 antibodies reduced nociceptive and vascular changes after fracture and inhibited lymphadenopathy. CONCLUSIONS: Collectively, these data support the hypothesis that IL-6 signaling in the fracture limb of mice is required for germinal center formation, IgM autoantibody production and nociceptive sensitization. Anti-IL-6 therapies might, therefore, reduce pain after limb fracture or in the setting of CRPS.
Assuntos
Síndromes da Dor Regional Complexa , Nociceptividade , Animais , Modelos Animais de Doenças , Centro Germinativo , Imunoglobulina M , Masculino , Camundongos , TíbiaRESUMO
CTLA-4 is an important regulator of T-cell function. Here, we report that expression of this immune-regulator in mouse B-1a cells has a critical function in maintaining self-tolerance by regulating these early-developing B cells that express a repertoire enriched for auto-reactivity. Selective deletion of CTLA-4 from B cells results in mice that spontaneously develop autoantibodies, T follicular helper (Tfh) cells and germinal centers (GCs) in the spleen, and autoimmune pathology later in life. This impaired immune homeostasis results from B-1a cell dysfunction upon loss of CTLA-4. Therefore, CTLA-4-deficient B-1a cells up-regulate epigenetic and transcriptional activation programs and show increased self-replenishment. These activated cells further internalize surface IgM, differentiate into antigen-presenting cells and, when reconstituted in normal IgH-allotype congenic recipient mice, induce GCs and Tfh cells expressing a highly selected repertoire. These findings show that CTLA-4 regulation of B-1a cells is a crucial immune-regulatory mechanism.
Assuntos
Subpopulações de Linfócitos B/imunologia , Antígeno CTLA-4/imunologia , Homeostase/imunologia , Sistema Imunitário/imunologia , Tolerância Imunológica/imunologia , Animais , Subpopulações de Linfócitos B/metabolismo , Antígeno CTLA-4/genética , Antígeno CTLA-4/metabolismo , Diferenciação Celular/imunologia , Centro Germinativo/citologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Sistema Imunitário/citologia , Sistema Imunitário/metabolismo , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
Emerging evidence suggests that Complex Regional Pain Syndrome (CRPS) is in part a post-traumatic autoimmune disease mediated by an adaptive immune response after limb injuries. We previously observed in a murine tibial fracture model of CRPS that pain-related behaviors were dependent upon adaptive immune mechanisms including the neuropeptide-dependent production of IgM for 5 months after injury. However, the time course of induction of this immune response and the demonstration of germinal center formation in lymphoid organs has not been evaluated. Using the murine fracture model, we employed behavioral tests of nociceptive sensitization and limb dysfunction, serum passive transfer techniques, western blot analysis of IgM accumulation, fluorescence-activated cell sorting (FACS) of lymphoid tissues and immunohistochemistry to follow the temporal activation of the adaptive immune response over the first 3 weeks after fracture. We observed that: 1) IgM protein levels in the skin of the fractured mice were elevated at 3 weeks post fracture, but not at earlier time points, 2) serum from fracture mice at 3 weeks, but not 1 and 2 weeks post fracture, had pro-nociceptive effects when passively transferred to fractured muMT mice lacking B cells, 3) fracture induced popliteal lymphadenopathy occurred ipsilateral to fracture beginning at 1 week and peaking at 3 weeks post fracture, 4) a germinal center reaction was detected by FACS analysis in the popliteal lymph nodes from injured limbs by 3 weeks post fracture but not in other lymphoid tissues, 5) germinal center formation was characterized by the induction of T follicular helper cells (Tfh) and germinal center B cells in the popliteal lymph nodes of the injured but not contralateral limbs, and 6) fracture mice treated with the Tfh signaling inhibitor FK506 had impaired germinal center reactions, reduced IgM levels, reduced nociceptive sensitization, and no pronociceptive serum effects after administration to fractured muMT mice. Collectively these data demonstrate that tibia fracture induces an adaptive autoimmune response characterized by popliteal lymph node germinal center formation and Tfh cell dependent B cell activation, resulting in nociceptive sensitization within 3 weeks.
Assuntos
Centro Germinativo , Fraturas da Tíbia , Imunidade Adaptativa , Animais , Modelos Animais de Doenças , Membro Posterior , Imunoglobulinas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nociceptividade , Linfócitos T Auxiliares-Indutores , TíbiaRESUMO
The current paradigm that a single long-term hematopoietic stem cell can regenerate all components of the mammalian immune system has been challenged by recent findings in mice. These findings show that adult tissue-resident macrophages and innate-like lymphocytes develop early in fetal hematopoiesis from progenitors that emerge prior to, and apparently independently of, conventional long-term hematopoietic stem cells. Here, we discuss these recent findings, which show that an early and distinct wave of hematopoiesis occurs for all major hematopoietic lineages. These data provide evidence that fetal hematopoietic progenitors not derived from the bona fide long-term hematopoietic stem cells give rise to tissue-resident immune cells that persist throughout adulthood. We also discuss recent insights into B lymphocyte development and attempt to synthesize seemingly contradictory recent findings on the origins of innate-like B-1a lymphocytes during fetal hematopoiesis.
Assuntos
Subpopulações de Linfócitos B/citologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Macrófagos/citologia , Animais , Linhagem da Célula , Embrião de Mamíferos/embriologia , CamundongosRESUMO
When examining datasets of any dimensionality, researchers frequently aim to identify individual subsets (clusters) of objects within the dataset. The ubiquity of multidimensional data has motivated the replacement of user-guided clustering with fully automated clustering. The fully automated methods are designed to make clustering more accurate, standardized and faster. However, the adoption of these methods is still limited by the lack of intuitive visualization and cluster matching methods that would allow users to readily interpret fully automatically generated clusters. To address these issues, we developed a fully automated subset identification and characterization (SIC) pipeline providing robust cluster matching and data visualization tools for high-dimensional flow/mass cytometry (and other) data. This pipeline automatically (and intuitively) generates two-dimensional representations of high-dimensional datasets that are safe from the curse of dimensionality. This new approach allows more robust and reproducible data analysis,+ facilitating the development of new gold standard practices across laboratories and institutions.
Assuntos
Análise por Conglomerados , Visualização de Dados , Citometria de Fluxo/métodos , Reconhecimento Automatizado de Padrão/métodos , Animais , Biomarcadores Tumorais/sangue , Células da Medula Óssea , Humanos , Leucemia Mieloide Aguda/sangue , Linfócitos/citologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/citologia , Cavidade Peritoneal/citologiaRESUMO
Part of the flow/mass cytometry data analysis process is aligning (matching) cell subsets between relevant samples. Current methods address this cluster-matching problem in ways that are either computationally expensive, affected by the curse of dimensionality, or fail when population patterns significantly vary between samples. Here, we introduce a quadratic form (QF)-based cluster matching algorithm (QFMatch) that is computationally efficient and accommodates cases where population locations differ significantly (or even disappear or appear) from sample to sample. We demonstrate the effectiveness of QFMatch by evaluating sample datasets from immunology studies. The algorithm is based on a novel multivariate extension of the quadratic form distance for the comparison of flow cytometry data sets. We show that this QF distance has attractive computational and statistical properties that make it well suited for analysis tasks that involve the comparison of flow/mass cytometry samples.
Assuntos
Análise por Conglomerados , Biologia Computacional/estatística & dados numéricos , Interpretação Estatística de Dados , Citometria de Fluxo/estatística & dados numéricos , Algoritmos , Humanos , ImunofenotipagemRESUMO
BACKGROUND: IgD is an enigmatic antibody isotype best known when coexpressed with IgM on naive B cells. However, increased soluble IgD (sIgD) levels and increased IgD+IgM- B-cell populations have been described in the human upper respiratory mucosa. OBJECTIVE: We assessed whether levels of sIgD and IgD+ B cell counts are altered in nasal tissue from patients with chronic rhinosinusitis (CRS). We further characterized IgD+ B-cell populations and explored clinical and local inflammatory factors associated with tissue sIgD levels. METHODS: sIgD levels were measured by means of ELISA in nasal tissues, nasal lavage fluid, sera, and supernatants of dissociated nasal tissues. IgD+ cells were identified by using immunofluorescence and flow cytometry. Inflammatory mediator levels in tissues were assessed by using real-time PCR and multiplex immunoassays. Bacterial cultures from the middle meatus were performed. Underlying medical history and medicine use were obtained from medical records. RESULTS: sIgD levels and numbers of IgD+ cells were significantly increased in uncinate tissue (UT) of patients with chronic rhinosinusitis without nasal polyps (CRSsNP) compared with that of control subjects (4-fold, P < .05). IgD+ cells were densely scattered in the periglandular regions of UT from patients with CRSsNP. We also found that IgD+CD19+CD38bright plasmablast numbers were significantly increased in tissues from patients with CRSsNP compared with control tissues (P < .05). Among numerous factors tested, IL-2 levels were increased in UT from patients with CRSsNP and were positively correlated with tissue IgD levels. Additionally, supernatants of IL-2-stimulated dissociated tissue from patients with CRSsNP had significantly increased sIgD levels compared with those in IL-2-stimulated dissociated control tissue ex vivo (P < .05). Tissue from patients with CRS with preoperative antibiotic use or those with pathogenic bacteria showed higher IgD levels compared with tissue from patients without these variables (P < .05). CONCLUSION: sIgD levels and IgD+CD19+CD38bright plasmablast counts were increased in nasal tissue of patients with CRSsNP. IgD levels were associated with increased IL-2 levels and the presence of pathogenic bacteria. These findings suggest that IgD might contribute to enhancement mucosal immunity or inflammation or respond to bacterial infections in patients with CRS, especially CRSsNP.
Assuntos
Linfócitos B/imunologia , Imunoglobulina D/metabolismo , Mucosa Nasal/imunologia , Pólipos Nasais/imunologia , Sistema Respiratório/patologia , Rinite/imunologia , Sinusite/imunologia , ADP-Ribosil Ciclase 1/metabolismo , Adulto , Idoso , Antígenos CD19/metabolismo , Células Cultivadas , Doença Crônica , Feminino , Humanos , Interleucina-2/metabolismo , Masculino , Pessoa de Meia-Idade , Regulação para Cima , Adulto JovemRESUMO
Changes in the frequencies of cell subsets that (co)express characteristic biomarkers, or levels of the biomarkers on the subsets, are widely used as indices of drug response, disease prognosis, stem cell reconstitution, etc. However, although the currently available computational "gating" tools accurately reveal subset frequencies and marker expression levels, they fail to enable statistically reliable judgements as to whether these frequencies and expression levels differ significantly between/among subject groups. Here we introduce flow cytometry data analysis pipeline which includes the Earth Mover's Distance (EMD) metric as solution to this problem. Well known as an informative quantitative measure of differences between distributions, we present three exemplary studies showing that EMD 1) reveals clinically-relevant shifts in two markers on blood basophils responding to an offending allergen; 2) shows that ablative tumor radiation induces significant changes in the murine colon cancer tumor microenvironment; and, 3) ranks immunological differences in mouse peritoneal cavity cells harvested from three genetically distinct mouse strains.
Assuntos
Biomarcadores/metabolismo , Algoritmos , Citometria de Fluxo , ProbabilidadeRESUMO
B cells are key components of cellular and humoral immunity and, like all lymphocytes, are thought to originate and renew from hematopoietic stem cells (HSCs). However, our recent single-HSC transfer studies demonstrate that adult bone marrow HSCs do not regenerate B-1a, a subset of tissue B cells required for protection against pneumonia, influenza, and other infections. Since B-1a are regenerated by transfers of fetal liver, the question arises as to whether B-1a derive from fetal, but not adult, HSCs. Here we show that, similar to adult HSCs, fetal HSCs selectively fail to regenerate B-1a. We also show that, in humanized mice, human fetal liver regenerates tissue B cells that are phenotypically similar to murine B-1a, raising the question of whether human HSC transplantation, the mainstay of such models, is sufficient to regenerate human B-1a. Thus, our studies overtly challenge the current paradigm that HSCs give rise to all components of the immune system.
Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Células-Tronco Fetais/transplante , Transplante de Tecido Fetal/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Animais , Animais Recém-Nascidos , Linfócitos B/citologia , Linfócitos B/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/imunologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Fetais/imunologia , Células-Tronco Fetais/metabolismo , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Fígado/citologia , Fígado/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
The diagnosis of cystic fibrosis (CF) patients with allergic bronchopulmonary aspergillosis (ABPA) is clinically challenging, due to the absence of an objective biological test. Since blood basophils play a major role in allergic responses, we hypothesised that changes in their surface activation pattern discriminate between CF patients with and without ABPA.We conducted a prospective longitudinal study (Stanford cohort) comparing basophil activation test CD203c levels by flow cytometry before and after activation with Aspergillus fumigatus allergen extract or recombinant Asp f1 in 20 CF patients with ABPA (CF-ABPA) and in two comparison groups: CF patients with A. fumigatus colonisation (AC) but without ABPA (CF-AC; n=13) and CF patients without either AC or ABPA (CF; n=12). Patients were tested every 6â months and when ill with pulmonary exacerbation. We also conducted cross-sectional validation in a separate patient set (Dublin cohort).Basophil CD203c surface expression reliably discriminated CF-ABPA from CF-AC and CF over time. Ex vivo stimulation with A. fumigatus extract or recombinant Asp f1 produced similar results within the Stanford (p<0.0001) and the Dublin cohorts. CF-ABPA patients were likelier to have elevated specific IgE to A. fumigatus and were less frequently co-infected with Staphylococcus aureus.Basophil CD203c upregulation is a suitable diagnostic and stable monitoring biomarker of ABPA in CF.
Assuntos
Aspergilose Broncopulmonar Alérgica/metabolismo , Basófilos/metabolismo , Portador Sadio/metabolismo , Fibrose Cística/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Aspergilose Pulmonar/metabolismo , Pirofosfatases/metabolismo , Adolescente , Adulto , Alérgenos/farmacologia , Aspergilose Broncopulmonar Alérgica/complicações , Aspergilose Broncopulmonar Alérgica/diagnóstico , Aspergillus fumigatus/imunologia , Basófilos/efeitos dos fármacos , Biomarcadores/metabolismo , Portador Sadio/diagnóstico , Estudos de Casos e Controles , Criança , Estudos de Coortes , Fibrose Cística/complicações , Fibrose Cística/diagnóstico , Feminino , Citometria de Fluxo , Humanos , Masculino , Estudos Prospectivos , Aspergilose Pulmonar/complicações , Aspergilose Pulmonar/diagnóstico , Adulto JovemRESUMO
Amyloid fibrils composed of peptides as short as six amino acids are therapeutic in experimental autoimmune encephalomyelitis (EAE), reducing paralysis and inflammation, while inducing several pathways of immune suppression. Intraperitoneal injection of fibrils selectively activates B-1a lymphocytes and two populations of resident macrophages (MΦs), increasing IL-10 production, and triggering their exodus from the peritoneum. The importance of IL-10-producing B-1a cells in this effective therapy was established in loss-of-function experiments where neither B-cell-deficient (µMT) nor IL10(-/-) mice with EAE responded to the fibrils. In gain-of-function experiments, B-1a cells, adoptively transferred to µMT mice with EAE, restored their therapeutic efficacy when Amylin 28-33 was administered. Stimulation of adoptively transferred bioluminescent MΦs and B-1a cells by amyloid fibrils resulted in rapid (within 60 min of injection) trafficking of both cell types to draining lymph nodes. Analysis of gene expression indicated that the fibrils activated the CD40/B-cell receptor pathway in B-1a cells and induced a set of immune-suppressive cell-surface proteins, including BTLA, IRF4, and Siglec G. Collectively, these data indicate that the fibrils activate B-1a cells and F4/80(+) MΦs, resulting in their migration to the lymph nodes, where IL-10 and cell-surface receptors associated with immune-suppression limit antigen presentation and T-cell activation. These mechanisms culminate in reduction of paralytic signs of EAE.
Assuntos
Amiloide/farmacologia , Linfócitos B/imunologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Ativação Linfocitária/efeitos dos fármacos , Transferência Adotiva , Amiloide/metabolismo , Amiloide/uso terapêutico , Animais , Encefalomielite Autoimune Experimental/imunologia , Endocitose , Feminino , Interleucina-10/fisiologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Processes that define immunoglobulin repertoires are commonly presumed to be the same for all murine B cells. However, studies here that couple high-dimensional FACS sorting with large-scale quantitative IgH deep-sequencing demonstrate that B-1a IgH repertoire differs dramatically from the follicular and marginal zone B cells repertoires and is defined by distinct mechanisms. We track B-1a cells from their early appearance in neonatal spleen to their long-term residence in adult peritoneum and spleen. We show that de novo B-1a IgH rearrangement mainly occurs during the first few weeks of life, after which their repertoire continues to evolve profoundly, including convergent selection of certain V(D)J rearrangements encoding specific CDR3 peptides in all adults and progressive introduction of hypermutation and class-switching as animals age. This V(D)J selection and AID-mediated diversification operate comparably in germ-free and conventional mice, indicating these unique B-1a repertoire-defining mechanisms are driven by antigens that are not derived from microbiota.
Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Variação Genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Animais , Rearranjo Gênico do Linfócito B , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Peritônio/imunologia , Baço/imunologiaRESUMO
Preclinical Research Although acetaminophen (APAP) is an effective analgesic and anti-pyretic, APAP overdose is the most frequent cause of serious, often lethal, drug-induced hepatotoxicity. Administration of N-acetyl cysteine (NAC) within 8 hours of APAP overdose effectively mitigates APAP-induced hepatotoxicity. Thus, preventing APAP toxicity before it occurs by formulating APAP with NAC is logical and, as we show here in a mouse model, is effective in preventing APAP toxicity. Thus, toxic oral APAP doses sufficient to cause severe widespread liver damage do not cause significant damage when administered concurrently with equal amounts of NAC, that is, in the NAC-APAP treated animals, hepatic transaminases increase only marginally and liver architecture remains fully intact. Thus, we conclude that concomitant oral dosing with APAP and NAC can provide a convenient and effective way of preventing toxicity associated with large dosage of APAP. From a public health perspective, these findings support the concept that a co-formulation of APAP plus NAC is a viable over-the-counter (OTC) alternative to the current practice of providing APAP OTC and treating APAP toxicity if/when it occurs. In essence, our findings indicate that replacing the current OTC APAP with a safe and functional APAP/NAC formulation could prevent the accidental and intentional APAP toxicity that occurs today.