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Several studies have now described instances where G-rich sequences in promoters and enhancers regulate gene expression through forming G-quadruplex (G4) structures. Relatedly, our group recently identified 301 long genomic stretches significantly enriched for minimal G4 motifs (LG4s) in humans and found the majority of these overlap annotated enhancers, and furthermore, that the promoters regulated by these LG4 enhancers are similarly enriched with G4-capable sequences. While the generally accepted model for enhancer:promoter specificity maintains that interactions are dictated by enhancer- and promoter-bound transcriptional activator proteins, the current study tested an alternative hypothesis: that LG4 enhancers interact with cognate promoters via a direct G4:G4 DNA-based mechanism. This work establishes the nuclear proximity of LG4 enhancer:promoter pairs, biochemically demonstrates the ability of individual LG4 single-stranded DNAs (ssDNAs) to directly interact target promoter ssDNAs, and confirms that these interactions, as well as the ability of LG4 enhancers to activate target promoters in culture, are mediated by G4 DNA., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Whether pediatric dialysis is morally obligatory is an ethical issue. The study's aim was to understand neonatal and pediatric intensive care unit (ICU) nurses' beliefs regarding the ethical use of pediatric dialysis. A single center study was conducted using theoretical and case-based surveys. Sixty-three (63) registered nurses completed at least part of the survey. The most important factors influencing nurses' beliefs on the appropriateness of dialysis were patient quality of life, prognosis, and severity of comorbidities. Nurses with experience vs. nurses without experience caring for patients on dialysis ranked family wishes as more important (p < 0.05) and were more likely to express their beliefs when they disagreed with the decision (p < 0.005). The data suggest that beliefs of nurses vary based on demographic and situational factors. Consideration of these factors may improve care team communication., Competing Interests: The authors reported no actual or potential conflict of interest in relation to this nursing continuing professional development (NCPD) activity., (Copyright© by the American Nephrology Nurses Association.)

Immune checkpoint inhibitors can stimulate antitumor immunity but can also induce toxicities termed immune-related adverse events (irAEs). Colitis is a common and severe irAE that can lead to treatment discontinuation. Mechanistic understanding of gut irAEs has been hampered because robust colitis is not observed in laboratory mice treated with checkpoint inhibitors. We report here that this limitation can be overcome by using mice harboring the microbiota of wild-caught mice, which develop overt colitis following treatment with anti-CTLA-4 antibodies. Intestinal inflammation is driven by unrestrained activation of IFNγ-producing CD4 + T cells and depletion of peripherally induced regulatory T cells through Fcγ receptor signaling. Accordingly, anti-CTLA-4 nanobodies that lack an Fc domain can promote antitumor responses without triggering colitis. This work suggests a strategy for mitigating gut irAEs while preserving antitumor stimulating effects of CTLA-4 blockade.

Targeted cytokine delivery has been gaining popularity in cancer immunotherapy since systemic recombinant cytokine treatment has not been successful due to low response rate and systemic toxicities in the clinical studies. In order to address these issues, we propose a new concept that cytokine signal is specifically activated at tumor-micro-environment (TME) by delivering two protein subunits of heterodimeric cytokine fused with a tumor targeting antibody respectively to TME and by bridging the two subunits into active heterodimeric form.Interleukin-12 (IL-12) is one of the major cytokines which can induce immune activation. IL-12 consists of two protein subunits which are p35 and p40. IL-12 signaling is initiated when it forms as the heterodimeric protein and binds to IL-12 receptor complex. We made fusion proteins of both IL-12p35 and IL-12p40 targeting specific tumor associated antigens (TAAs) and demonstrated the formation of bioactive IL12p70 with TME targeting antibody toward both p35 and p40 to form as the active molecule. We describe our concept validation in an in vitro based functional assay., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mike Shaw reports financial support was provided by Takeda Pharmaceutical Company Limited., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Residents are commonly targets and bystanders of workplace discrimination, yet little is known about how best to train residents to manage these incidents. We sought to train residents to respond effectively to being a target or bystander of discrimination., Approach: We used a novel, 75-min theatrical role-playing intervention called Theatre for Healthcare Equity (T.H.E.) to teach 71 internal medicine residents between December 2017 and February 2018. In T.H.E. residents took turns acting as either a 'resident' target or a 'student' bystander in a simulated scenario of discrimination. A facilitator led follow-up discussions including group reflection and development of learning scripts to help with difficult situations. A post-graduation survey was sent in November 2021 to assess residents' retention of knowledge, attitudes and potential application in practice., Evaluation: T.H.E. was well received by residents, though survey response rates were low. All respondents to a post-session survey reported having acquired knowledge and skills to help them respond to incidents of bias and discrimination. Most respondents to the post-graduation survey nearly 4 years later remembered T.H.E.; seven wrote reflective narrative responses indicating that T.H.E. had raised awareness of these issues, empowered them to speak up on behalf of colleagues and validated their emotional reactions to hurtful speech from patients. We describe an incident in which a former resident attributed his ability to serve as an effective bystander ally to participating in T.H.E. years earlier., Implications: T.H.E. was an efficient, well-received intervention that some of our residents found to have been helpful years later. We continue to use T.H.E. as the basis for periodic ongoing allyship training for residents and teaching faculty to improve the inclusiveness of our clinical learning environment., (© 2023 Association for the Study of Medical Education and John Wiley & Sons Ltd.)

In organ transplantation, there is a lack of ethical discussion about the recipient's right not to receive a transplant. Using the current situation of living organ transplantation and deceased organ transplantation in Japan as an example, we prospectively discussed to what extent the recipient's right not to receive a transplant is ethically acceptable. In directed transplantation from a living donor, a recipient may refuse organ donation from a particular donor. It is preferable that a recipient's request for organ donation from a donor occurs as part of a transparent process. In nondirected transplantation from a deceased donor, refusal of transplantation from a particular type of donor appears potentially justifiable. There are both moral and pragmatic considerations. Certain refusals based solely on belief are morally unacceptable, and refusal to transplant a recipient based on the donor's age jeopardizes the entire transplant system. When religious beliefs affect mental and physical health, individualized measures are required for transplant rejection. We also deductively developed a prospective argument based on the current status of donor-recipient communication in living organ transplantation in Japan and the 2010 amendment of the law allowing relatives to be given priority in organ transplantation from deceased donors.

AbstractWe explore the various ethical challenges that arise during the practical implementation of an emergency resource allocation protocol. We argue that to implement an allocation plan in a crisis, a hospital system must complete five tasks: (1) formulate a set of general principles for allocation, (2) apply those principles to the disease at hand to create a concrete protocol, (3) collect the data required to apply the protocol, (4) construct a system to implement triage decisions with those data, and (5) create a system for managing the consequences of implementing the protocol, including the effects on those who must carry out the plan, the medical staff, and the general public. Here we illustrate the complexities of each task and provide tentative solutions, by describing the experiences of the Coronavirus Ethics Response Group, an interdisciplinary team formed to address the ethical issues in pandemic resource planning at the University of Rochester Medical Center. While the plan was never put into operation, the process of preparing for emergency implementation exposed ethical issues that require attention.

Ethical discourse on prognosis disclosure is not yet well established. The core of the problem continues to be the dilemma between the right of self-determination and non-maleficence of patients. The prognosis disclosure policy based on Kantian autonomy provides a good solution for the problem. The policy includes demand for strict truth telling and its compatibility with patients' best interest. However, there remains a discrepancy between theory and practice, especially when prognosis is disclosed just prior to their death. Kantian theory of prognosis is supplemented by a moralistic perspective. The moralistic perspective places high importance on temporality and relationships with others, which all human beings inherently possess. From the moralistic viewpoint, decisions about prognosis disclosure at the final stages of life must be individualized in order to be authentically autonomous. The decision to disclose a prognosis or not can only be determined by the relationships fostered over time with patients.

Ipilimumab, a monoclonal antibody that recognizes cytotoxic T-lymphocyte associated protein 4 (CTLA-4), was the first immune checkpoint inhibitor approved by the FDA to treat metastatic melanoma patients. Multiple preclinical studies have proposed that Fc effector functions of anti-CTLA-4 therapy are required for anti-tumor efficacy, in part, through the depletion of intratumoral regulatory T cells (Tregs). However, the contribution of the Fc-independent functions of anti-CTLA-4 antibodies to the observed efficacy is not fully understood. H11, a non-Fc-containing single-domain antibody (VHH) against CTLA-4, has previously been demonstrated to block CTLA-4-ligand interaction. However, in vivo studies demonstrated lack of anti-tumor efficacy with H11 treatment. Here, we show that a half-life extended H11 (H11-HLE), despite the lack of Fc effector functions, induced potent anti-tumor efficacy in mouse syngeneic tumor models. In addition, a non-Fc receptor binding version of ipilimumab (Ipi-LALAPG) also demonstrated anti-tumor activity in the absence of Treg depletion. Thus, we demonstrate that Fc-independent functions of anti-CTLA-4 antibodies contributed to anti-tumor efficacy, which may indicate that non-Treg depleting activity of anti-CTLA-4 therapy could benefit cancer patients in the clinic., (© 2022. The Author(s).)

Oncology therapies targeting the immune system have improved patient outcomes across a wide range of tumor types, but resistance due to an inadequate T-cell response in a suppressive tumor microenvironment (TME) remains a significant problem. New therapies that activate an innate immune response and relieve this suppression may be beneficial to overcome this hurdle. TAK-676 is a synthetic novel stimulator of interferon genes (STING) agonist designed for intravenous administration. Here we demonstrate that TAK-676 dose-dependently triggers activation of the STING signaling pathway and activation of type I interferons. Furthermore, we show that TAK-676 is a highly potent modulator of both the innate and adaptive immune system and that it promotes the activation of dendritic cells, natural killer cells, and T cells in preclinical models. In syngeneic murine tumor models in vivo, TAK-676 induces dose-dependent cytokine responses and increases the activation and proliferation of immune cells within the TME and tumor-associated lymphoid tissue. We also demonstrate that TAK-676 dosing results in significant STING-dependent antitumor activity, including complete regressions and durable memory T-cell immunity. We show that TAK-676 is well tolerated, exhibits dose-proportional pharmacokinetics in plasma, and exhibits higher exposure in tumor. The intravenous administration of TAK-676 provides potential treatment benefit in a broad range of tumor types. Further study of TAK-676 in first-in-human phase I trials is ongoing., Significance: TAK-676 is a novel systemic STING agonist demonstrating robust activation of innate and adaptive immune activity resulting in durable antitumor responses within multiple syngeneic tumor models. Clinical investigation of TAK-676 is ongoing., Competing Interests: V.A. Appleman reports personal fees from Takeda Development Centers of America, Inc during the conduct of the study; personal fees from Takeda and other from Takeda outside the submitted work. V. Kolev reports other from Takeda during the conduct of the study. M. Mochizuki reports patent application WO2018/100558. S. Haridas reports personal fees from Takeda during the conduct of the study; personal fees from Takeda outside the submitted work. T.D. Thelen reports other from Takeda outside the submitted work. A.O. Abu-Yousif reports other from Takeda during the conduct of the study. M. Okaniwa reports patent application WO2018/100558. No other disclosures were reported., (© 2022 The Authors; Published by the American Association for Cancer Research.)

Background: Immune checkpoint blockade (ICB) therapies have changed the paradigm of cancer therapies. However, anti-tumor response of the ICB is insufficient for many patients and limited to specific tumor types. Despite many preclinical and clinical studies to understand the mechanism of anti-tumor efficacy of ICB, the mechanism is not completely understood. Harnessing preclinical tumor models is one way to understand the mechanism of treatment response., Methods: In order to delineate the mechanisms of anti-tumor activity of ICB in preclinical syngeneic tumor models, we selected two syngeneic murine colorectal cancer models based on in vivo screening for sensitivity with anti-PD-1 therapy. We performed tumor-immune profiling of the two models to identify the potential mechanism for anti-PD-1 response., Results: We performed in vivo screening for anti-PD-1 therapy across 23 syngeneic tumor models and found that CT-26 and Colon 26, which are murine colorectal carcinoma derived from BALB/c mice, showed different sensitivity to anti-PD-1. CT-26 tumor mice were more sensitive to the anti-PD-1 antibody than Colon 26, while both models show similarly sensitivity to anti-CTLA4 antibody. Immune-profiling showed that CT-26 tumor tissue was infiltrated with more immune cells than Colon 26. Genomic/transcriptomic analyses highlighted thatWnt pathway was one of the potential differences between CT-26 and Colon 26, showing Wnt activity was higher in Colon 26 than CT-26. ., Conclusions: CT-26 and Colon 26 syngeneic tumor models showed different sensitivity to anti-PD-1 therapy, although both tumor cells are murine colorectal carcinoma cell lines from BALB/c strain. By characterizing the mouse cells lines and tumor-immune context in the tumor tissues with comprehensive analysis approaches, we found that CT-26 showed "hot tumor" profile with more infiltrated immune cells than Colon 26. Further pathway analyses enable us to propose a hypothesis that Wnt pathway could be one of the major factors to differentiate CT-26 from Colon 26 model and link to anti-PD-1 response. Our approach to focus on preclinical tumor models with similar genetic background but different sensitivity to anti-PD-1 therapy would contribute to illustrating the potential mechanism of anti-PD-1 response and to generating a novel concept to synergize current anti-PD-1 therapies for cancer patients., (© 2021. The Author(s).)

This case reports an ethical dilemma in which a mother who had recently delivered a baby through cesarean section after sustaining life-threatening injuries in a car accident did not have documented wishes whether she wanted to breastfeed. Her medical condition rendered her temporarily mentally incapacitated and critically ill, and the lactation medicine team was consulted about whether lactation choice should be preserved by pumping. Complicating considerations in this case were (1) lack of family or designated decision-makers available at the time of injury and emergent delivery, (2) lack of prenatal care, and (3) complex social situation, including prior history of illicit substance use, and state removal of other children into foster care. This case raises important ethical considerations regarding breastfeeding decision-making when a mother is incapable of making the decision, and if there is an intrinsic right for an infant to be breastfed in situations where maternal choice to lactate or to formula feed is unknown. Ultimately, the mother chose to discontinue breastfeeding once she was able to voice her own opinion. The issues discussed in this study may be relevant for future cases when providing guidance on the ethical argument to preserve maternal choice when a mother is critically ill and mentally incapacitated.

Introduction: Health care professionals who identify as members of underrepresented and racial minority groups may experience bias from patients and patient families. These occurrences disrupt the educational and therapeutic environments, distress the targeted individuals and allies, and create potential legal liability. Yet there are few educational opportunities for individuals to brainstorm and implement strategies for responding professionally during such instances., Methods: Presented first as a grand rounds, then an invited workshop, and finally an invited series, this educational activity was developed in a stepwise manner over the course of a year. Each format was sequentially modified based on feedback from participants-more than 200 physicians and other health care professionals-using evaluation forms that were voluntary and anonymous. The educational activity used an adaptation of forum theater, in which participants role-played an instance of oppression with a goal of altering the ultimate outcome. This approach provided participants with the opportunity to develop and rehearse responses to workplace bias in a way that preserved the provider-patient relationship., Results: Feedback for these educational sessions was overwhelmingly positive. Participants noted the importance of acknowledging and addressing bias in the workplace and encouraged facilitators to expand the sessions in length, frequency, and scope., Discussion: Forum theater is a methodology that can be employed in health care to teach appropriate and authentic responses to expressed patient bias while maintaining the therapeutic relationship. The positive reception from participants in our preliminary sessions established a strong foundation for future improvements to this work., (© 2020 Rizk et al.)

An ene-reductase (ERED 36) with broad substrate specificity was identified, and optimization studies led to the development of an enzymatic protocol for the reduction of α,β-unsaturated acids under mild, aqueous conditions. The substrate scope includes aromatic- and aliphatic-substituted acrylic acids, as well as cyclic α,β-substituted acrylic acids, yielding chiral α-substituted acids with exquisite levels of enantioselectivity (>99% ee).

In this article, we perform a thought experiment about living donor kidney transplantation. If a living kidney donor becomes in need of renal replacement treatment due to dysfunction of the remaining kidney after donation, can the donor ask the recipient to give back the kidney that had been donated? We call this problem organ restitution and discussed it from the ethical viewpoint. Living organ transplantation is a kind of 'designated donation' and subsequently has a contract-like character. First, assuming a case in which original donor (A) wishes the return of the organ which had been transplanted into B, and the original recipient (B) agrees, organ restitution will be permissible based on contract-like agreement. However, careful and detailed consideration is necessary to determine whether this leaves no room to question the authenticity of B's consent. Second, if B offers to give back the organ to A, then B's act is a supererogatory act, and is praiseworthy and meritorious. Such an offer is a matter of virtue, not obligation. Third, if A wishes B to return the organ, but B does not wish/allow this to happen, it is likely difficult to justify returning the organ to A by violating B's right to bodily integrity. But B's refusal to return the donated organ cannot be deemed praiseworthy, because B forgets the great kindness once received from A. Rather than calling this an obligation, we encourage B to consider such virtuous conduct., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Dietary amino acids play an important role in maintaining health. Branched chain amino acids can adversely increase blood pressure whereas arginine and citrulline can reduce it. D-amino acids play important roles in several cell types including testis, the nervous system and adrenal glands. Several amino acids also can have dramatic effects on diabetes; branched chain amino acids, phenylalanine and tyrosine have been implicated while others, namely arginine and citrulline can improve outcomes. Leucine has been shown to play important roles in muscle primarily through the mTOR pathway though this effect does not translate across every population. Glutamine, arginine and D-aspartate also exert their muscle effects through mTOR. Relationships between amino acids and endocrine function include that of glucocorticoids, thyroid function, glucagon-like peptide 1 (GLP-1), ghrelin, insulin-like growth factor-1 (IGF-1) and leptin. Leucine, for example, can alleviate the effect of dexamethasone on muscle protein accretion. Interestingly, amino acid transporters play an important role in thyroid function. Several amino acids have been shown to increase GLP-1 levels in non-diabetics when administered orally. Similarly, several amino acids increase ghrelin levels in different species while cysteine can decrease it in mice. There is evidence to suggest that the arginine/NO pathway may be involved in modulating some of the effects of ghrelin on cells. In regard to IGF-1, branched chain amino acids can increase levels in adults while tryptophan and phenylalanine have been shown to increase levels in infants. Finally, leptin levels can be elevated by branched chain amino acids while restricting leucine in high fat diets can increase leptin sensitivity.

Background: Moral distress (MD) is an emotional and psychological response to morally challenging dilemmas. Moral distress is experienced frequently by nurses in the intensive care unit (ICU) and can result in emotional anguish, work dissatisfaction, poor patient outcomes, and high levels of nurse turnover. Opportunities to discuss ethically challenging situations may lessen MD and its associated sequela., Objective: The purpose of this project was to develop, implement, and evaluate the impact of nursing ethics huddles on participants' MD, clinical ethics knowledge, work satisfaction, and patient care among ICU nurses., Sample and Setting: The sample, 32 nurses from three ICU settings in an 800-bed tertiary academic medical center, participated in six nursing ethics huddles over a two-month period., Methods: Alvita K. Nathaniel's Theory of Moral Reckoning guided development of the nursing ethics huddle process. The Moral Distress Thermometer was administered at three data points: baseline level of MD, and pre- and post-huddle to determine changes in the subjects' level of MD. Focused content analysis was used to analyze qualitative responses from questionnaires about the subjects' perception of the effect of the huddles on work satisfaction and patient care. Knowledge attainment was evaluated via open-ended short-answer questions., Results: Overall, use of nurse-ethicist-led nursing ethics huddles was associated with improved quality of work life, patient care, and clinical ethics knowledge. The change in pre- and post-nursing ethics huddles MD scores was statistically significant., (Copyright 2018 The Journal of Clinical Ethics. All rights reserved.)

Background: This paper examines the ethical aspects of organ transplant surgery in which a donor heart is transplanted from a first recipient, following determination of death by neurologic criteria, to a second recipient. Retransplantation in this sense differs from that in which one recipient undergoes repeat heart transplantation of a newly donated organ, and is thus referred to here as "reuse cardiac organ transplantation.", Methods: Medical, legal, and ethical analysis, with a main focus on ethical analysis., Results: From the medical perspective, it is critical to ensure the quality and safety of reused organs, but we lack sufficient empirical data pertaining to medical risk. From the legal perspective, a comparative examination of laws in the United States and Japan affirms no illegality, but legal scholars disagree on the appropriate analysis of the issues, including whether or not property rights apply to transplanted organs. Ethical arguments supporting the reuse of organs include the analogous nature of donation to gifts, the value of donations as inheritance property, and the public property theory as it pertains to organs. Meanwhile, ethical arguments such as those that address organ recycling and identity issues challenge organ reuse., Conclusion: We conclude that organ reuse is not only ethically permissible, but even ethically desirable. Furthermore, we suggest changes to be implemented in the informed consent process prior to organ transplantation. The organ transplant community worldwide should engage in wider and deeper discussions, in hopes that such efforts will lead to the timely preparation of guidelines to implement reuse cardiac organ transplantation as well as reuse transplantation of other organs such as kidney and liver.

Qualitative and quantitative research on the impact of medical and health humanities teaching in baccalaureate education is sparse. This paper reviews recent studies of the impact of medical and health humanities coursework in pre-health professions education and describes a pilot study of baccalaureate students who completed semester-long medical humanities courses in the Division of Medical Humanities & Bioethics at the University of Rochester. The study format was an email survey. All participants were current or former baccalaureate students who had taken one or more courses in literature and narrative in medicine, bioethics, history of medicine, and/or visual arts and healthcare during the past four years. The survey gathered numerical data in several areas: demographic information, career plans, self-reported influence of coursework on educational and career plans, and self-reported influence of coursework on intellectual skills and abilities. It also gathered narrative commentary that elaborated on students' responses to the numerically-based questions. Notable findings from preliminary analysis of the data include higher scores of self-reported impact of the coursework on specific habits of mind and on preparedness for intended career rather than on gaining admission to future educational programs. Discussion of the results focuses on several potential future directions this type of study might take, including multi-center, longitudinal, and sequential approaches.

Under carbonylative conditions, neutral Rh(I)-systems modified with weak donor ligands (AsPh 3 or 1,4-oxathiane) undergo N-Cbz, N-benzoyl, or N-Ts directed insertion into the proximal C-C bond of aminomethylcyclopropanes to generate rhodacyclopentanone intermediates. These are trapped by N-tethered alkenes to provide complex perhydroisoindoles.

Rhodacyclopentanones, an "sp(3)-rich" class of metallacycle, underpin an emerging range of catalytic methodologies for the direct generation of complex scaffolds. This review highlights strategies for accessing rhodacyclopentanones (and related species) by C-C bond activation of cyclobutanones or cyclopropanes. The scope and mechanism of methodologies that exploit these activation modes is outlined.

In recent years, photoredox catalysis has come to the forefront in organic chemistry as a powerful strategy for the activation of small molecules. In a general sense, these approaches rely on the ability of metal complexes and organic dyes to convert visible light into chemical energy by engaging in single-electron transfer with organic substrates, thereby generating reactive intermediates. In this Perspective, we highlight the unique ability of photoredox catalysis to expedite the development of completely new reaction mechanisms, with particular emphasis placed on multicatalytic strategies that enable the construction of challenging carbon-carbon and carbon-heteroatom bonds.

The use of sp(3) C-H bonds--which are ubiquitous in organic molecules--as latent nucleophile equivalents for transition metal-catalyzed cross-coupling reactions has the potential to substantially streamline synthetic efforts in organic chemistry while bypassing substrate activation steps. Through the combination of photoredox-mediated hydrogen atom transfer (HAT) and nickel catalysis, we have developed a highly selective and general C-H arylation protocol that activates a wide array of C-H bonds as native functional handles for cross-coupling. This mild approach takes advantage of a tunable HAT catalyst that exhibits predictable reactivity patterns based on enthalpic and bond polarity considerations to selectively functionalize α-amino and α-oxy sp(3) C-H bonds in both cyclic and acyclic systems., (Copyright © 2016, American Association for the Advancement of Science.)

Conventional and plasmacytoid dendritic cells (cDCs and pDCs) are the two populations of DCs that can be readily identified in human blood. Conventional DCs have been subdivided into CD1c(+), or blood dendritic cells antigen (BDCA) 1 and CD141(+), or BDCA-3, DCs, each having both unique gene expression profiles and functions. BDCA-3 DCs express high levels of toll-like receptor 3 and upon stimulation with Poly I:C secrete IFN-β, CXCL10, and IL-12p70. In this article, we show that activation of human BDCA-3 DCs with Poly I:C induces the expression of activation markers (CD40, CD80, and CD86) and immunoglobulin-like transcript (ILT) 3 and 4. This Poly I:C stimulation results in four populations identifiable by flow cytometry based on their expression of ILT3 and ILT4. We focused our efforts on profiling the ILT4(-) and ILT4(+) DCs. These ILT-expressing BDCA-3 populations exhibit similar levels of activation as measured by CD40, CD80, and CD86; however, they exhibit differential cytokine secretion profiles, unique gene signatures, and vary in their ability to prime allogenic naïve T cells. Taken together, these data illustrate that within a pool of BDCA-3 DCs, there are cells poised to respond differently to a given input stimulus with unique output of immune functions.

Pd-catalyzed cyclizations of oxime esters with pendant alkenes are subject to an unusual ligand controlled mechanistic divergence. Pd-systems modified with electron deficient phosphines ( e.g. P(3,5-(CF 3 ) 2 C 6 H 3 ) 3 ) promote efficient aza-Heck cyclization, wherein C-N bond formation occurs via alkene imino-palladation. Conversely, electron rich ligands, such as P( t -Bu) 3 , cause deviation to a SET pathway and, in these cases, C-N bond formation occurs via cyclization of an iminyl radical. A series of mechanistic experiments differentiate the two pathways and the scope of the hybrid organometallic radical cyclization is outlined. This study represents a rare example in Pd-catalysis where selection between dichotomous mechanistic manifolds is facilitated solely by choice of phosphine ligand.

A short entry to substituted azocanes by a Rh-catalyzed cycloaddition-fragmentation process is described. Specifically, exposure of diverse N-cyclopropylacrylamides to phosphine-ligated cationic Rh(I) catalyst systems under a CO atmosphere enables the directed generation of rhodacyclopentanone intermediates. Subsequent insertion of the alkene component is followed by fragmentation to give the heterocyclic target. Stereochemical studies show, for the first time, that alkene insertion into rhodacyclopentanones can be reversible.

Upon exposure to neutral or cationic Rh(I)-catalyst systems, amino-substituted cyclopropanes undergo carbonylative cycloaddition with tethered alkenes to provide stereochemically complex N-heterocyclic scaffolds. These processes rely upon the generation and trapping of rhodacyclopentanone intermediates, which arise by regioselective, Cbz-directed insertion of Rh and CO into one of the two proximal aminocyclopropane C-C bonds. For cyclizations using cationic Rh(I)-systems, synthetic and mechanistic studies indicate that rhodacyclopentanone formation is reversible and that the alkene insertion step determines product diastereoselectivity. This regime facilitates high levels of stereocontrol with respect to substituents on the alkene tether. The option of generating rhodacyclopentanones dynamically provides a new facet to a growing area of catalysis and may find use as a (stereo)control strategy in other processes.

Aminocyclopropanes equipped with suitable N-directing groups undergo efficient and regioselective Rh-catalyzed carbonylative C-C bond activation. Trapping of the resultant metallacycles with tethered alkynes provides an atom-economic entry to diverse N-heterobicyclic enones. These studies provide a blueprint for myriad N-heterocyclic methodologies.

Intestinal phagocytes transport oral antigens and promote immune tolerance, but their role in innate immune responses remains unclear. Here we found that intestinal phagocytes were anergic to ligands for Toll-like receptors (TLRs) or commensals but constitutively expressed the precursor to interleukin 1β (pro-IL-1β). After infection with pathogenic Salmonella or Pseudomonas, intestinal phagocytes produced mature IL-1β through the NLRC4 inflammasome but did not produce tumor necrosis factor (TNF) or IL-6. BALB/c mice deficient in NLRC4 or the IL-1 receptor were highly susceptible to orogastric but not intraperitoneal infection with Salmonella. That enhanced lethality was preceded by impaired expression of endothelial adhesion molecules, lower neutrophil recruitment and poor intestinal pathogen clearance. Thus, NLRC4-dependent production of IL-1β by intestinal phagocytes represents a specific response that discriminates pathogenic bacteria from commensal bacteria and contributes to host defense in the intestine.

T(H)17 cells are a lineage of CD4(+) T cells that are critical for host defense and autoimmunity by expressing the cytokines IL-17A, IL-17F, and IL-22. A feature of T(H)17 cells at steady state is their ubiquitous presence in the lamina propria of the small intestine. The induction of these steady-state intestinal T(H)17 (sT(H)17) cells is dependent on the presence of the microbiota. However, the signaling pathway linking the microbiota to the development of intestinal sT(H)17 cells remains unclear. In this study, we show that IL-1β, but not IL-6, is induced by the presence of the microbiota in intestinal macrophages and is required for the induction of sT(H)17 cells. In the absence of IL-1β-IL-1R or MyD88 signaling, there is a selective reduction in the frequency of intestinal sT(H)17 cells and impaired production of IL-17 and IL-22. Myeloid differentiation factor 88-deficient (MyD88(-/-)) and germ-free (GF) mice, but not IL-1R(-/-) mice, exhibit impairment in IL-1β induction. Microbiota-induced IL-1β acts directly on IL-1R-expressing T cells to drive the generation of sT(H)17 cells. Furthermore, administration of IL-1β into GF mice induces the development of retinoic acid receptor-related orphan receptor γt-expressing sT(H)17 cells in the small intestine, but not in the spleen. Thus, commensal-induced IL-1β production is a critical step for sT(H)17 differentiation in the intestine, which may have therapeutic implications for T(H)17-mediated pathologies.

Nucleotide-binding oligomerization domain 2 (Nod2) mutations including L1007fsinsC are associated with the development of Crohn's disease (CD). These CD-associated Nod2 mutations are common in healthy white populations, suggesting that they may confer some protective function, but experimental evidence is lacking. Using a mouse strain that expresses Nod2(2939iCstop), the equivalent of the L1007fsinsC mutation, we found that macrophages homozygous for Nod2(2939iCstop) are impaired in the recognition of muramyl dipeptide and Enterococcus faecalis, a commensal bacterium that is a common cause of sepsis-associated lethality in humans. Notably, Nod2 deficiency and homozygocity for Nod2(2939iCstop) were associated with reduced production of TNF-α and IL-6 and lethality after systemic infection with E. faecalis despite normal bacteria loads. Consistently, inhibition of TNF-α signaling protected wild-type mice from E. faecalis-induced lethality. These results suggest that the same Nod2 mutation can increase the susceptibility to CD, but also protect the host from systemic infection by a common enteric bacterium.

The intracellular sensor Nod2 is activated in response to bacteria, and the impairment of this response is linked to Crohn's disease. However, the function of Nod2 in host defense remains poorly understood. We found that Nod2-/- mice exhibited impaired intestinal clearance of Citrobacter rodentium, an enteric bacterium that models human infection by pathogenic Escherichia coli. The increased bacterial burden was preceded by reduced CCL2 chemokine production, inflammatory monocyte recruitment, and Th1 cell responses in the intestine. Colonic stromal cells, but not epithelial cells or resident CD11b+ phagocytic cells, produced CCL2 in response to C. rodentium in a Nod2-dependent manner. Unlike resident phagocytic cells, inflammatory monocytes produced IL-12, a cytokine that induces adaptive immunity required for pathogen clearance. Adoptive transfer of Ly6C(hi) monocytes restored the clearance of the pathogen in infected Ccr2-/- mice. Thus, Nod2 mediates CCL2-CCR2-dependent recruitment of inflammatory monocytes, which is important in promoting bacterial eradication in the intestine., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The identification of several families of innate pattern recognition receptors has greatly enhanced our understanding of the host innate immune response against a variety of pathogens. One such family of innate receptors is the nucleotide-binding domain and leucine rich repeat containing receptors (NLRs). NOD2 has been characterized as a cytosolic sensor of bacteria peptidoglycan (PGN). For almost 10 years, NOD2 was assigned with the function of mediating the RICK- and nuclear factor-κB induced proinflammatory response triggered by PGN. Recent studies have extended the biological activity of NOD2 to include the induction of autophagy and antiviral responses, as well as mediating direct T cell activation. Here, we highlight and discuss these new findings in the context of immune activation and pathogen detection., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Cerebral malaria is the most severe complication of Plasmodium falciparum infection in humans and the pathogenesis is still unclear. Using the P. berghei ANKA infection model of mice, we investigated a potential involvement of Nlrp3 and the inflammasome in the pathogenesis of cerebral malaria. Nlrp3 mRNA expression was upregulated in brain endothelial cells after exposure to P. berghei ANKA. Although beta-hematin, a synthetic compound of the parasites heme polymer hemozoin, induced the release of IL-1beta in macrophages through Nlrp3, we did not obtain evidence for a role of IL-1beta in vivo. Nlrp3 knock-out mice displayed a delayed onset of cerebral malaria; however, mice deficient in caspase-1, the adaptor protein ASC or the IL-1 receptor succumbed as WT mice. These results indicate that the role of Nlrp3 in experimental cerebral malaria is independent of the inflammasome and the IL-1 receptor pathway.

Nod2 belongs to the nucleotide-binding oligomerization domain receptor (NLR) family of proteins, which function as intracellular pathogen sensors in innate immune cells. Nod2 deficiency results in an impaired immune response to bacterial pathogens. However, how this protein promotes host defense against intracellular parasites is unknown. Here we found that Nod2(-/-) mice had less clearance of Toxoplasma gondii and lower interferon-gamma (IFN-gamma) production. Reconstitution of T cell-deficient mice with Nod2(-/-) T cells followed by T. gondii infection demonstrated a T cell-intrinsic defect. Nod2(-/-) CD4(+) T cells had poor helper T cell differentiation, which was associated with impaired production of interleukin 2 (IL-2) and nuclear accumulation of the transcription factor subunit c-Rel. Our data demonstrate a T cell-intrinsic role for Nod2 signaling that is critical for host defense against T. gondii.

The NOD-like receptors (NLRs) are a specialized group of intracellular receptors that represent a key component of the host innate immune system. Since the discovery of the first NLR almost 10 years ago, the study of this special class of microbial sensors has burgeoned; consequently, a better understanding of the mechanism by which these receptors recognize microbes and other danger signals and of how they activate inflammatory signaling pathways has emerged. Moreover, in addition to their primary role in host defense against invading pathogens, their ability to regulate nuclear factor-kappa B (NF-kappaB) signaling, interleukin-1-beta (IL-1beta) production, and cell death indicates that they are crucial to the pathogenesis of a variety of inflammatory human diseases.

There is growing evidence that the host innate immune system has a critical role in regulating carcinogenesis, but the specific receptors involved and the importance of their interaction with commensal bacteria need to be elucidated. Two major classes of innate immune receptors, the Toll-like receptors and Nod-like receptors, many of which are upstream of nuclear factor-kappaB, are involved in the detection of intestinal bacteria. The Toll-like receptors have been implicated in promoting colon tumorigenesis, but the role of Nod-like receptors in regulating tumorigenesis remains unclear. Using an established mouse model system of colitis-associated colon tumorigenesis, we show that Nod1 deficiency results in the increased development of both colitis-associated and Apc tumor suppressor-related colon tumors. In the absence of Nod1 signaling, there is a greater disruption of the intestinal epithelial cell barrier due to chemically induced injury as manifested by increased surface epithelial apoptosis early on during chemically induced colitis and increased intestinal permeability. The increased intestinal permeability is associated with enhanced inflammatory cytokine production and epithelial cell proliferation in Nod1-deficient mice as compared with wild-type mice. Depletion of the gut microbiota suppressed tumor development in Nod1-deficient mice, thus highlighting a link between the commensal bacteria within the intestine and the host innate immune Nod1 signaling pathway in the regulation inflammation-mediated colon cancer development.

The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) (nucleotide-binding domain leucine-rich repeat containing) family of proteins has been demonstrated to function as regulators of innate immune response against microbial pathogens. Stimulation of NOD1 and NOD2, two prototypic NLRs, results in the activation of MAPK and NF-kappaB. On the other hand, a different set of NLRs induces caspase-1 activation through the assembly of an inflammasome. This review discusses recent findings regarding the signaling pathways utilized by NLR proteins in the control of caspase-1 and NF-kappaB activation, as well as the nonredundant role of NLRs in pathogen clearance. The review also covers advances regarding the cellular localization of these proteins and the implications this may have on pathogen sensing and signal transduction.

The cytosolic sensors Nod1 and Nod2 and Toll-like receptors (TLRs) activate defense signaling pathways in response to microbial stimuli. However, the role of Nod1 and Nod2 and their interplay with TLRs during systemic bacterial infection remains poorly understood. Here, we report that macrophages or mice made insensitive to TLRs by previous exposure to microbial ligands remained responsive to Nod1 and Nod2 stimulation. Furthermore, Nod1- and Nod2-mediated signaling and gene expression are enhanced in TLR-tolerant macrophages. Further analyses revealed that innate immune responses induced by bacterial infection relied on Nod1 and Nod2 and their adaptor RICK in macrophages pretreated with TLR ligands but not in naive macrophages. In addition, bacterial clearance upon systemic infection with L. monocytogenes was critically dependent on Nod1 and Nod2 when mice were previously stimulated with lipopolysaccharide or E. coli. Thus, Nod1 and Nod2 are important for microbial recognition and host defense after TLR stimulation.

The innate immune system comprises several classes of pattern-recognition receptors, including Toll-like receptors (TLRs) and nucleotide binding and oligomerization domain-like receptors (NLRs). TLRs recognize microbes on the cell surface and in endosomes, whereas NLRs sense microbial molecules in the cytosol. In this review, we focus on the role of NLRs in host defence against bacterial pathogens. Nod1 and Nod2 sense the cytosolic presence of molecules containing meso-diaminopimelic acid and muramyl dipeptide respectively, and drive the activation of mitogen-activated protein kinase and NF-kappaB. In contrast, Ipaf, Nalp1b and Cryopyrin/Nalp3 promote the assembly of inflammasomes that are required for the activation of caspase-1. Mutation in several NLR members, including NOD2 and Cryopyrin, is associated with the development of inflammatory disorders. Further understanding of NLRs should provide new insights into the mechanisms of host defence and the pathogenesis of inflammatory diseases.

Apicomplexan protozoan pathogens avoid destruction and establish a replicative niche within host cells by forming a nonfusogenic parasitophorous vacuole (PV). Here we present evidence for lysosome-mediated degradation of Toxoplasma gondii after invasion of macrophages activated in vivo. Pathogen elimination was dependent on the interferon gamma inducible-p47 GTPase, IGTP, required PI3K activity, and was preceded by PV membrane indentation, vesiculation, disruption, and, surprisingly, stripping of the parasite plasma membrane. Denuded parasites were enveloped in autophagosome-like vacuoles, which ultimately fused with lysosomes. These observations outline a series of mechanisms used by effector cells to redirect the fate of a classically nonfusogenic intracellular pathogen toward a path of immune elimination.

The Jak, Tyk2, is activated in response to IL-12 and IFN-alphabeta and promotes IFN-gamma production by Th1-type CD4 cells. Mice deficient in Tyk2 function have been previously shown to be resistant to autoimmune arthritis and septic shock but are acutely susceptible to opportunistic pathogens such as Toxoplasma gondii. In this study, we show that Tyk2, in addition to mediating the biological effects of IL-12 and IFN-alphabeta, is an important regulator for the signaling and expression of the immunosuppressive cytokine IL-10. In the absence of Tyk2, Ag-reactive CD4 cells exhibit impaired IL-10 synthesis following rechallenge of T. gondii vaccine-primed mice. The impaired IL-10 reactivation leads to unopposed antimicrobial effector mechanisms which results in a paradoxically superior protection of immune Tyk2(-/-) mice against virulent T. gondii challenge. We further demonstrate that Tyk2 indirectly controls CD4 IL-10 reactivation by signaling for maximal IFN-gamma secretion. The unexpected role of IFN-gamma in mediating IL-10 reactivation by Th1 cells provides compelling evidence that conditions driving Th1 responses establish a negative feedback loop, which will ultimately lead to its autoregulation. Thus, Tyk2 can be viewed as a dual-function Jak, mediating both pro and anti-inflammatory cytokine responses.

Cell-mediated immunity to Toxoplasma gondii establishes and maintains a balanced host-pathogen relationship. Recent analyses using spontaneous and genetically engineered mouse mutants have yielded a clearer picture of factors positively and negatively regulating the host immune response and a better understanding of cytokine-inducible intracytoplasmic mechanisms that lead to intracellular pathogen suppression and demise.

The B10.Q/J strain of mice was serendipitously discovered to be highly susceptible to infection by the intracellular protozoan parasite, Toxoplasma gondii but markedly resistant to induction of autoimmune arthritis. We have previously shown that the B10.Q/J phenotype is controlled by a single recessive locus and is associated with lymphocyte hyporesponsiveness to IL-12. Using genetic approaches, we have now localized the B10.Q/J locus to chromosome 9 and established its identity as Tyk2, a Janus kinase essential for IL-12 and IFN-alpha/beta cytokine signaling. The B10.Q/J Tyk2 gene contained a single missense mutation resulting in a nonconservative amino acid substitution (E775K) in an invariant motif of the pseudokinase (Janus kinase homology 2) domain. This mutation appeared to result in the absence of the B10.Q/J-encoded Tyk2 protein, despite presence of Tyk2-specific transcripts. Phenotypically, B10.Q/J cells were indistinguishable from Tyk2-deficient cells, showing impaired signaling and biologic responses to IL-12, IL-23, and type I IFNs. The analogous E782K mutant of human Tyk2 failed to restore IFN-alpha responsiveness in Tyk2 null 11,1 cells. Our results indicate a crucial role for Tyk2 in T helper 1-mediated protective and pathogenic immune responses. An additional implication of our findings is that naturally occurring mutations in the Tyk2 gene may underlie altered susceptibilities to infectious or autoimmune diseases in human and animal populations.

Cross-sectional data on 19 long-distance runners (LD), 17 aerobic dancers (AD), 19 recreational joggers (RJ), and 15 inactive controls (IC) were examined for cardiovascular endurance and determination of plasma lipoproteins. Subjects included premenopausal eumenorrheic females who were non-smokers and presently not using oral contraceptives. Results indicated that all groups were similar in age, height and weight. Only the AD group had a significantly lower percent body fat (p less than .001) than the other groups. The LD, AD and RJ groups had a significantly higher VO2max than the IC group (p less than .05), and the LD and AD groups had a significantly higher VO2max than the RJ group (p less than .05). Analysis of a one-day food log indicated that the only difference in diet among the groups was a significantly lower intake of total and monounsaturated fat in the AD group (p less than .05) and a significantly lower intake of carbohydrates in the IC group than the AD group (p less than .05). Analysis of plasma lipids revealed no significant differences in any lipid variables among the groups. These findings indicate that healthy premenopausal eumenorrheic females with similar physical characteristics also have similar plasma lipid profiles regardless of their physical activity level.