Co-administration of N-Acetylcysteine and Acetaminophen Efficiently Blocks Acetaminophen Toxicity.

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.

Linkage of inflammation and oxidative stress via release of glutathionylated peroxiredoxin-2, which acts as a danger signal.

The mechanism by which oxidative stress induces inflammation and vice versa is unclear but is of great importance, being apparently linked to many chronic inflammatory diseases. We show here that inflammatory stimuli induce release of oxidized peroxiredoxin-2 (PRDX2), a ubiquitous redox-active intracellular enzyme. Once released, the extracellular PRDX2 acts as a redox-dependent inflammatory mediator, triggering macrophages to produce and release TNF-α. The oxidative coupling of glutathione (GSH) to PRDX2 cysteine residues (i.e., protein glutathionylation) occurs before or during PRDX2 release, a process central to the regulation of immunity. We identified PRDX2 among the glutathionylated proteins released in vitro by LPS-stimulated macrophages using mass spectrometry proteomic methods. Consistent with being part of an inflammatory cascade, we find that PRDX2 then induces TNF-α release. Unlike classical inflammatory cytokines, PRDX2 release does not reflect LPS-mediated induction of mRNA or protein synthesis; instead, PRDX2 is constitutively present in macrophages, mainly in the reduced form, and is released in the oxidized form on LPS stimulation. Release of PRDX2 is also observed in human embryonic kidney cells treated with TNF-α. Importantly, the PRDX2 substrate thioredoxin (TRX) is also released along with PRDX2, enabling an oxidative cascade that can alter the -SH status of surface proteins and thereby facilitate activation via cytokine and Toll-like receptors. Thus, our findings suggest a model in which the release of PRDX2 and TRX from macrophages can modify the redox status of cell surface receptors and enable induction of inflammatory responses. This pathway warrants further exploration as a potential novel therapeutic target for chronic inflammatory diseases.

AutoGate: automating analysis of flow cytometry data.

Nowadays, one can hardly imagine biology and medicine without flow cytometry to measure CD4 T cell counts in HIV, follow bone marrow transplant patients, characterize leukemias, etc. Similarly, without flow cytometry, there would be a bleak future for stem cell deployment, HIV drug development and full characterization of the cells and cell interactions in the immune system. But while flow instruments have improved markedly, the development of automated tools for processing and analyzing flow data has lagged sorely behind. To address this deficit, we have developed automated flow analysis software technology, provisionally named AutoComp and AutoGate. AutoComp acquires sample and reagent labels from users or flow data files, and uses this information to complete the flow data compensation task. AutoGate replaces the manual subsetting capabilities provided by current analysis packages with newly defined statistical algorithms that automatically and accurately detect, display and delineate subsets in well-labeled and well-recognized formats (histograms, contour and dot plots). Users guide analyses by successively specifying axes (flow parameters) for data subset displays and selecting statistically defined subsets to be used for the next analysis round. Ultimately, this process generates analysis "trees" that can be applied to automatically guide analyses for similar samples. The first AutoComp/AutoGate version is currently in the hands of a small group of users at Stanford, Emory and NIH. When this "early adopter" phase is complete, the authors expect to distribute the software free of charge to .edu, .org and .gov users.

A conversation with Leonard and Leonore Herzenberg.

Leonard and Leonore Herzenberg have left an indelible mark on the fields of immunology and cell biology, both in research and clinical aspects. They are perhaps best known for developing the technologies of fluorescence flow cytometry and hybridomas. Over six decades, they made a number of important and fundamental discoveries in lymphocyte biology by applying these technologies. During this era, they immersed themselves in the sociopolitical environment, interjecting scientific rationale into public discourse about McCarthyism, nuclear fallout, war, genetics, and other politically charged topics. Their unique philosophy has shaped their lives, their science, and ultimately the scientific community. In this Conversation, we explore some of these driving forces and the impact on the laboratory.

Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs.

In the field of regenerative medicine, one of the ultimate goals is to generate functioning organs from pluripotent cells, such as ES cells or induced pluripotent stem cells (PSCs). We have recently generated functional pancreas and kidney from PSCs in pancreatogenesis- or nephrogenesis-disabled mice, providing proof of principle for organogenesis from PSCs in an embryo unable to form a specific organ. Key when applying the principles of in vivo generation to human organs is compensation for an empty developmental niche in large nonrodent mammals. Here, we show that the blastocyst complementation system can be applied in the pig using somatic cell cloning technology. Transgenic approaches permitted generation of porcine somatic cell cloned embryos with an apancreatic phenotype. Complementation of these embryos with allogenic blastomeres then created functioning pancreata in the vacant niches. These results clearly indicate that a missing organ can be generated from exogenous cells when functionally normal pluripotent cells chimerize a cloned dysorganogenetic embryo. The feasibility of blastocyst complementation using cloned porcine embryos allows experimentation toward the in vivo generation of functional organs from xenogenic PSCs in large animals.

H-Y antigen-binding B cells develop in male recipients of female hematopoietic cells and associate with chronic graft vs. host disease.

B cells are known to play an important role in pathogenesis of human chronic graft vs. host disease (cGVHD). Our group has previously shown that IgG allo-antibodies recognize Y chromosome-encoded proteins (H-Y) and a dominant H-Y epitope, DEAD box protein (DBY-2) detectable 6-12 mo after transplant in male patients who receive grafts from female donors (F→M) hematopoietic cell transplantation (HCT). Here we present FACS studies of peripheral blood mononuclear cells collected 6 mo after transplant showing that 16 of 28 (57%) F→M HCT patients have circulating donor B cells that express B-cell receptor (mainly IgM and Igλ) specific for DBY-2. The detection of these DBY-2 B cells 6 mo after HCT are associated with cGVHD development (P = 0.004). Specifically, 15 of 16 F→M with DBY-2 B cells developed cGVHD. In contrast, cGVHD developed in only 5 of the 12 who did not have DBY-2 B cells detected. This demonstrates circulating human B cells binding an alloantigen (DBY-2) and that these DBY-2-specific B cells appear before development of cGVHD in roughly half of the F→M patients. Our study suggests that detection of anti-DBY-2 B cells may predict cGVHD and that this prediction may have clinical utility. Validation of this hypothesis will require larger prospective studies.

Reversal of paralysis and reduced inflammation from peripheral administration of ß-amyloid in TH1 and TH17 versions of experimental autoimmune encephalomyelitis.

ß-Amyloid 42 (Aß42) and ß-amyloid 40 (Aß40), major components of senile plaque deposits in Alzheimer's disease, are considered neurotoxic and proinflammatory. In multiple sclerosis, Aß42 is up-regulated in brain lesions and damaged axons. We found, unexpectedly, that treatment with either Aß42 or Aß40 peptides reduced motor paralysis and brain inflammation in four different models of experimental autoimmune encephalomyelitis (EAE) with attenuation of motor paralysis, reduction of inflammatory lesions in the central nervous system (CNS), and suppression of lymphocyte activation. Aß42 and Aß40 treatments were effective in reducing ongoing paralysis induced with adoptive transfer of either autoreactive T helper 1 (T(H)1) or T(H)17 cells. High-dimensional 14-parameter flow cytometry of peripheral immune cell populations after in vivo Aß42 and Aß40 treatment revealed substantial modulations in the percentage of lymphoid and myeloid subsets during EAE. Major proinflammatory cytokines and chemokines were reduced in the blood after Aß peptide treatment. Protection conferred by Aß treatment did not require its delivery to the brain: Adoptive transfer with lymphocytes from donors treated with Aß42 attenuated EAE in wild-type recipient mice, and Aß deposition in the brain was not detected in treated EAE mice by immunohistochemical analysis. In contrast to the improvement in EAE with Aß treatment, EAE was worse in mice with genetic deletion of the amyloid precursor protein. Therefore, in the absence of Aß, there is exacerbated clinical EAE disease progression. Because Aß42 and Aß40 ameliorate experimental autoimmune inflammation targeting the CNS, we might now consider its potential anti-inflammatory role in other neuropathological conditions.

Distinct B-cell lineage commitment distinguishes adult bone marrow hematopoietic stem cells.

The question of whether a single hematopoietic stem cell (HSC) gives rise to all of the B-cell subsets [B-1a, B-1b, B-2, and marginal zone (MZ) B cells] in the mouse has been discussed for many years without resolution. Studies here finally demonstrate that individual HSCs sorted from adult bone marrow and transferred to lethally irradiated recipients clearly give rise to B-2, MZ B, and B-1b, but does not detectably reconstitute B-1a cells. These findings place B-2, MZ, and B-1b in a single adult developmental lineage and place B-1a in a separate lineage derived from HSCs that are rare or missing in adults. We discuss these findings with respect to known developmental heterogeneity in other HSC-derived lymphoid, myeloid, and erythroid lineages, and how HSC developmental heterogeneity conforms to the layered model of the evolution of the immune system that we proposed some years ago. In addition, of importance to contemporary medicine, we consider the implications that HSC developmental heterogeneity may have for selecting HSC sources for human transplantation.

Antigen-specific antibody responses in B-1a and their relationship to natural immunity.

B-1a cells are primarily thought of as natural antibody-producing cells. However, we now show that appropriate antigenic stimulation induces IgM and IgG B-1a antibody responses and long-lived T-independent antigen-specific B-1a memory that differs markedly from canonical B-2 humoral immunity. Thus, we show here that in the absence of inflammation, priming with glycolipid (FtL) from Francisella tularensis live vaccine strain induces splenic FtL-specific B-1a to mount dominant IgM and activation-induced cytidine deaminase-dependent IgG anti-FtL responses that occur within 3-5 d of FtL priming and fade within 1 wk to natural antibody levels that persist indefinitely in the absence of secondary FtL immunization. Equally surprising, FtL priming elicits long-term FtL-specific B-1a memory cells (IgM>>IgG) that migrate rapidly to the peritoneal cavity and persist there indefinitely, ready to respond to appropriately administrated secondary antigenic stimulation. Unlike B-2 responses, primary FtL-specific B-1a responses and establishment of persistent FtL-specific B-1a memory occur readily in the absence of adjuvants, IL-7, T cells, or germinal center support. However, in another marked departure from the mechanisms controlling B-2 memory responses, rechallenge with FtL in an inflammatory context is required to induce B-1a secondary antibody responses. These findings introduce previously unexplored vaccination strategies for pathogens that target the B-1a repertoire.

Antigen-specific memory in B-1a and its relationship to natural immunity.

In the companion article by Yang and colleagues [Yang Y, et al. (2012) Proc Natl Acad Sci USA, 109, 10.1073/pnas.1121631109], we have shown that priming with glycolipid (FtL) from Francisella tularensis live-vaccine strain (i) induces FtL-specific B-1a to produce robust primary responses (IgM >>IgG); (ii) establishes persistent long-term production of serum IgM and IgG anti-FtL at natural antibody levels; and (iii) elicits FtL-specific B-1a memory cells that arise in spleen but rapidly migrate to the peritoneal cavity, where they persist indefinitely but divide only rarely. Here, we show that FtL rechallenge alone induces these PerC B-1a memory cells to divide extensively and to express a unique activation signature. However, FtL rechallenge in the context of a Toll-like receptor 4 agonist-stimulated inflammatory response readily induces these memory cells to migrate to spleen and initiate production of dominant IgM anti-FtL secondary responses. Thus, studies here reveal unique mechanisms that govern B-1a memory development and expression, and introduce B-1a memory as an active participant in immune defenses. In addition, at a practical level, these studies suggest previously unexplored vaccination strategies for pathogen-associated antigens that target the B-1a repertoire.

Glut1-mediated glucose transport regulates HIV infection.

Cell cycle entry is commonly considered to positively regulate HIV-1 infection of CD4 T cells, raising the question as to how quiescent lymphocytes, representing a large portion of the viral reservoir, are infected in vivo. Factors such as the homeostatic cytokine IL-7 have been shown to render quiescent T cells permissive to HIV-1 infection, presumably by transiently stimulating their entry into the cell cycle. However, we show here that at physiological oxygen (O(2)) levels (2-5% O(2) tension in lymphoid organs), IL-7 stimulation generates an environment permissive to HIV-1 infection, despite a significantly attenuated level of cell cycle entry. We identify the IL-7-induced increase in Glut1 expression, resulting in augmented glucose uptake, as a key factor in rendering these T lymphocytes susceptible to HIV-1 infection. HIV-1 infection of human T cells is abrogated either by impairment of Glut1 signal transduction or by siRNA-mediated Glut1 down-regulation. Consistent with this, we show that the susceptibility of human thymocyte subsets to HIV-1 infection correlates with Glut1 expression; single-round infection is markedly higher in the Glut1-expressing double-positive thymocyte population than in any of the Glut1-negative subsets. Thus, our studies reveal the Glut1-mediated metabolic pathway as a critical regulator of HIV-1 infection in human CD4 T cells and thymocytes.

Distinct plasma profile of polar neutral amino acids, leucine, and glutamate in children with Autism Spectrum Disorders.

The goal of this investigation was to examine plasma amino acid (AA) levels in children with Autism Spectrum Disorders (ASD, N = 27) and neuro-typically developing controls (N = 20). We observed reduced plasma levels of most polar neutral AA and leucine in children with ASD. This AA profile conferred significant post hoc power for discriminating children with ASD from healthy children. Furthermore, statistical correlations suggested the lack of a typical decrease of glutamate and aspartate with age, and a non-typical increase of isoleucine and lysine with age in the ASD group. Findings from this limited prospective study warrant further examination of plasma AA levels in larger cross-sectional and longitudinal cohorts to adequately assess for relationships with developmental and clinical features of ASD.

Nerve growth factor: A key local regulator in the pathogenesis of inflammatory arthritis.

OBJECTIVE: The effect of nerve growth factor (NGF) and its receptor (NGFR) in inflammatory diseases is a novel research field. The purpose of this study was to investigate the role of NGF/NGFR in human T cell subpopulations and fibroblast-like synovial cells (FLS) and examine its pathophysiologic significance in psoriatic arthritis (PsA) and rheumatoid arthritis (RA). METHODS: Expression of NGF/NGFR was examined in synovial fluid (SF), FLS, peripheral blood (PB)-derived T cells, and SF-derived T cells from patients with PsA, RA, and osteoarthritis (OA). NGF levels were determined by enzyme-linked immunosorbent assay. NGF-induced T cell/FLS proliferation was examined by MTT assay. Low-affinity (p75)/high-affinity (TrkA) NGFR expression was determined by high-dimensional fluorescence-activated cell sorting. A monochlorobimane assay was used to determine the effect of NGF on T cell survival. RESULTS: Levels of NGF were higher in SF samples from PsA and RA patients as compared to SF samples from OA patients. NGF-induced FLS proliferation was more marked in PsA and RA patients. TrkA was up-regulated on activated SF T cells from PsA (mean ± SD 22 ± 6.2%) and RA (8 ± 1.3%) patients, whereas in SF samples from OA patients, TrkA+CD3+ T cells were not detectable. NGF induced the proliferation of PB T cells, induced the phosphorylation of Akt in activated T cells, and consistent with known pAkt activity, inhibited tumor necrosis factor α-induced cell death in these T cells. CONCLUSION: Based on our findings, we propose a model in which NGF secreted by FLS into PsA and RA synovium promotes the survival of activated autoreactive T cells as well as FLS proliferation. Thus, NGF has the potential to sustain the chronic inflammatory cascades of arthritis of autoimmune origin.

Distinct progenitors for B-1 and B-2 cells are present in adult mouse spleen.

Recent studies by Dorshkind, Yoder, and colleagues show that embryonic (E9) B-cell progenitors located in the yolk sac and intraembryonic hemogenic endothelium before the initiation of circulation give rise to B-1 and marginal zone B cells but do not give rise to B-2 cells. In studies here, we confirm and extend these findings by showing that distinct progenitors for B-1 and B-2 cells are present in the adult spleen. Furthermore, we show that the splenic B-cell progenitor population (lin(-)CD19(+)/B220(lo/-)/CD43(-)) that gives rise to B-1 cells is likely to be heterogeneous because, in some recipients, it also gives rise to B cells expressing the marginal zone phenotype (B220(hi)IgM(hi)IgD(lo)CD21(hi)) and to some (CD19(-)CD5(hi)) T cells. In addition to the well-known function differences between B-1 and B-2, our studies demonstrate that substantial developmental differences separate these B-cell lineages. Thus, consistent with the known dependence of B-2 development on IL-7, all B-2 progenitors express IL-7R. However, >30% of the B-1 progenitors do not express this marker, enabling the known IL-7 independent development of B-1 cells in IL-7(-/-) mice. In addition, marker expression on cells in the early stages of the B-2 development pathway (CD19(-)/c-Kit(lo/-)/Sca-1(lo/-)) in adult bone marrow distinguish it from the early stages of B-1 development (CD19(hi)/c-Kit(+)/Sca-1(+)), which occur constitutively in neonates. In adults, in vivo inflammatory stimulation (LPS) triggers B-1 progenitors in spleen to expand and initiate development along this B-1 developmental pathway.

Basophil CD203c levels are increased at baseline and can be used to monitor omalizumab treatment in subjects with nut allergy.

RATIONALE: Basophils contribute to anaphylaxis and allergies. We examined the utility of assessing basophil-associated surface antigens (CD11b/CD63/CD123/CD203c/CD294) in characterizing and monitoring subjects with nut allergy. METHODS: We used flow cytometry to analyze basophils at baseline (without any activation) and after ex vivo stimulation of whole blood by addition of nut or other allergens for 2, 10, and 30 min. We also evaluated whether basophil expression of CD11b/CD63/CD123/CD203c/CD294 was altered in subjects treated with anti-IgE monoclonal antibody (omalizumab) to reduce plasma levels of IgE. RESULTS: We demonstrate that basophil CD203c levels are increased at baseline in subjects with nut allergy compared to healthy controls (13 subjects in each group, p < 0.0001). Furthermore, we confirm that significantly increased expression of CD203c occurs on subject basophils when stimulated with the allergen to which the subject is sensitive and can be detected rapidly (10 min of stimulation, n = 11, p < 0.0008). In 5 subjects with severe peanut allergy, basophil CD203c expression following stimulation with peanut allergen was significantly decreased (p < 0.05) after 4 and 8 weeks of omalizumab treatment but returned toward pretreatment levels after treatment cessation. CONCLUSIONS: Subjects with nut allergy show an increase of basophil CD203c levels at baseline and following rapid ex vivo stimulation with nut allergen. Both can be reduced by omalizumab therapy. These results highlight the potential of using basophil CD203c levels for baseline diagnosis and therapeutic monitoring in subjects with nut allergy.

Genetic immunization in the lung induces potent local and systemic immune responses.

Successful vaccination against respiratory infections requires elicitation of high levels of potent and durable humoral and cellular responses in the lower airways. To accomplish this goal, we used a fine aerosol that targets the entire lung surface through normal respiration to deliver replication-incompetent recombinant adenoviral vectors expressing gene products from several infectious pathogens. We show that this regimen induced remarkably high and stable lung T-cell responses in nonhuman primates and that it also generated systemic and respiratory tract humoral responses of both IgA and IgG isotypes. Moreover, strong immunogenicity was achieved even in animals with preexisting antiadenoviral immunity, overcoming a critical hurdle to the use of these vectors in humans, who commonly are immune to adenoviruses. The immunogenicity profile elicited with this regimen, which is distinct from either intramuscular or intranasal delivery, has highly desirable properties for protection against respiratory pathogens. We show that it can be used repeatedly to generate mucosal humoral, CD4, and CD8 T-cell responses and as such may be applicable to other mucosally transmitted pathogens such as HIV. Indeed, in a lethal challenge model, we show that aerosolized recombinant adenoviral immunization completely protects ferrets against H5N1 highly pathogenic avian influenza virus. Thus, genetic immunization in the lung offers a powerful platform approach to generating protective immune responses against respiratory pathogens.

Two physically, functionally, and developmentally distinct peritoneal macrophage subsets.

The peritoneal cavity (PerC) is a unique compartment within which a variety of immune cells reside, and from which macrophages (MØ) are commonly drawn for functional studies. Here we define two MØ subsets that coexist in PerC in adult mice. One, provisionally called the large peritoneal MØ (LPM), contains approximately 90% of the PerC MØ in unstimulated animals but disappears rapidly from PerC following lipopolysaccharide (LPS) or thioglycolate stimulation. These cells express high levels of the canonical MØ surface markers, CD11b and F4/80. The second subset, referred to as small peritoneal MØ (SPM), expresses substantially lower levels of CD11b and F4/80 but expresses high levels of MHC-II, which is not expressed on LPM. SPM, which predominates in PerC after LPS or thioglycolate stimulation, does not derive from LPM. Instead, it derives from blood monocytes that rapidly enter the PerC after stimulation and differentiate to mature SPM within 2 to 4 d. Both subsets show clear phagocytic activity and both produce nitric oxide (NO) in response to LPS stimulation in vivo. However, their responses to LPS show key differences: in vitro, LPS stimulates LPM, but not SPM, to produce NO; in vivo, LPS stimulates both subsets to produce NO, albeit with different response patterns. These findings extend current models of MØ heterogeneity and shed new light on PerC MØ diversity, development, and function. Thus, they introduce a new context for interpreting (and reinterpreting) data from ex vivo studies with PerC MØ.

Antigen-specific B-1a antibodies induced by Francisella tularensis LPS provide long-term protection against F. tularensis LVS challenge.

Francisella tularensis (Ft), a gram-negative intracellular bacterium, is the etiologic agent of tularemia. Infection of mice with <10 Ft Live Vaccine Strain (Ft LVS) organisms i.p. causes a lethal infection that resembles human tularemia. Here, we show that immunization with as little as 0.1 ng Ft LVS lipopolysaccharide (Ft-LPS), but not Ft lipid A, generates a rapid antibody response that protects wild-type (WT) mice against lethal Ft LVS challenge. Protection is not induced in Ft-LPS-immunized B cell-deficient mice (muMT or JhD), male xid mice, or Ig transgenic mice that produce a single IgH (not reactive with Ft-LPS). Focusing on the cellular mechanisms that underlie this protective response, we show that Ft-LPS specifically stimulates proliferation of B-1a lymphocytes that bind fluorochrome-labeled Ft-LPS and the differentiation of these cells to plasma cells that secrete antibodies specific for Ft-LPS. This exclusively B-1a antibody response is equivalent in WT, T-deficient (TCRalphabeta(-/-), TCRgammadelta(-/-)), and Toll-like receptor 4 (TLR4)-deficient (TLR4(-/-)) mice and thus is not dependent on T cells or typical inflammatory processes. Serum antibody levels peak approximately 5 days after Ft-LPS immunization and persist at low levels for months. Thus, immunization with Ft-LPS activates a rare population of antigen-specific B-1a cells to produce a persistent T-independent antibody response that provides long-term protection against lethal Ft LVS infection. These data support the possibility of creating effective, minimally invasive vaccines that can provide effective protection against pathogen invasion.