Differential IRF8 Transcription Factor Requirement Defines Two Pathways of Dendritic Cell Development in Humans.

The formation of mammalian dendritic cells (DCs) is controlled by multiple hematopoietic transcription factors, including IRF8. Loss of IRF8 exerts a differential effect on DC subsets, including plasmacytoid DCs (pDCs) and the classical DC lineages cDC1 and cDC2. In humans, cDC2-related subsets have been described including AXL+SIGLEC6+ pre-DC, DC2 and DC3. The origin of this heterogeneity is unknown. Using high-dimensional analysis, in vitro differentiation, and an allelic series of human IRF8 deficiency, we demonstrated that cDC2 (CD1c+DC) heterogeneity originates from two distinct pathways of development. The lymphoid-primed IRF8hi pathway, marked by CD123 and BTLA, carried pDC, cDC1, and DC2 trajectories, while the common myeloid IRF8lo pathway, expressing SIRPA, formed DC3s and monocytes. We traced distinct trajectories through the granulocyte-macrophage progenitor (GMP) compartment showing that AXL+SIGLEC6+ pre-DCs mapped exclusively to the DC2 pathway. In keeping with their lower requirement for IRF8, DC3s expand to replace DC2s in human partial IRF8 deficiency.

Allogeneic stem cell transplantation compared to conservative management in adults with inborn errors of immunity.

Allogeneic hematopoietic stem cell transplantation (alloSCT) is curative for severe inborn errors of immunity (IEIs), with recent data suggesting alloSCT in adulthood is safe and effective in selected patients. However, questions remain regarding the indications for and optimal timing of transplant. We retrospectively compared outcomes of transplanted vs matched nontransplanted adults with severe IEIs. Seventy-nine patients (aged ≥ 15 years) underwent alloSCT between 2008 and 2018 for IEIs such as chronic granulomatous disease (n = 20) and various combined immune deficiencies (n = 59). A cohort of nontransplanted patients from the French Centre de Référence Déficits Immunitaires Héréditaires registry was identified blindly for case-control analysis, with ≤3 matched controls per index patient, without replacement. The nontransplanted patients were matched for birth decade, age at last review greater than index patient age at alloSCT, chronic granulomatous disease or combined immune deficiencies, and autoimmune/lymphoproliferative complications. A total of 281 patients were included (79 transplanted, 202 nontransplanted). Median age at transplant was 21 years. Transplant indications were mainly lymphoproliferative disease (n = 23) or colitis (n = 15). Median follow-up was 4.8 years (interquartile range, 2.5-7.2). One-year transplant-related mortality rate was 13%. Estimated disease-free survival at 5 years was higher in transplanted patients (58% vs 33%; P = .007). Nontransplanted patients had an ongoing risk of severe events, with an increased mean cumulative number of recurrent events compared with transplanted patients. Sensitivity analyses removing patients with common variable immune deficiency and their matched transplanted patients confirm these results. AlloSCT prevents progressive morbidity associated with IEIs in adults, which may outweigh the negative impact of transplant-related mortality.

International retrospective study of allogeneic hematopoietic cell transplantation for activated PI3K-delta syndrome.

BACKGROUND: Activated phosphoinositide 3-kinase delta syndrome (APDS) is a combined immunodeficiency with a heterogeneous phenotype considered reversible by allogeneic hematopoietic cell transplantation (HCT). OBJECTIVES: This study sought to characterize HCT outcomes in APDS. METHODS: Retrospective data were collected on 57 patients with APDS1/2 (median age, 13 years; range, 2-66 years) who underwent HCT. RESULTS: Pre-HCT comorbidities such as lung, gastrointestinal, and liver pathology were common, with hematologic malignancy in 26%. With median follow-up of 2.3 years, 2-year overall and graft failure-free survival probabilities were 86% and 68%, respectively, and did not differ significantly by APDS1 versus APDS2, donor type, or conditioning intensity. The 2-year cumulative incidence of graft failure following first HCT was 17% overall but 42% if mammalian target of rapamycin inhibitor(s) (mTORi) were used in the first year post-HCT, compared with 9% without mTORi. Similarly, 2-year cumulative incidence of unplanned donor cell infusion was overall 28%, but 65% in the context of mTORi receipt and 23% without. Phenotype reversal occurred in 96% of evaluable patients, of whom 17% had mixed chimerism. Vulnerability to renal complications continued post-HCT, adding new insights into potential nonimmunologic roles of phosphoinositide 3-kinase not correctable through HCT. CONCLUSIONS: Graft failure, graft instability, and poor graft function requiring unplanned donor cell infusion were major barriers to successful HCT. Post-HCT mTORi use may confer an advantage to residual host cells, promoting graft instability. Longer-term post-HCT follow-up of more patients is needed to elucidate the kinetics of immune reconstitution and donor chimerism, establish approaches that reduce graft instability, and assess the completeness of phenotype reversal over time.

The human IL-15 superagonist N-803 promotes migration of virus-specific CD8+ T and NK cells to B cell follicles but does not reverse latency in ART-suppressed, SHIV-infected macaques.

Despite the success of antiretroviral therapy (ART) to halt viral replication and slow disease progression, this treatment is not curative and there remains an urgent need to develop approaches to clear the latent HIV reservoir. The human IL-15 superagonist N-803 (formerly ALT-803) is a promising anti-cancer biologic with potent immunostimulatory properties that has been extended into the field of HIV as a potential "shock and kill" therapeutic for HIV cure. However, the ability of N-803 to reactivate latent virus and modulate anti-viral immunity in vivo under the cover of ART remains undefined. Here, we show that in ART-suppressed, simian-human immunodeficiency virus (SHIV)SF162P3-infected rhesus macaques, subcutaneous administration of N-803 activates and mobilizes both NK cells and SHIV-specific CD8+ T cells from the peripheral blood to lymph node B cell follicles, a sanctuary site for latent virus that normally excludes such effector cells. We observed minimal activation of memory CD4+ T cells and no increase in viral RNA content in lymph node resident CD4+ T cells post N-803 administration. Accordingly, we found no difference in the number or magnitude of plasma viremia timepoints between treated and untreated animals during the N-803 administration period, and no difference in the size of the viral DNA cell-associated reservoir post N-803 treatment. These results substantiate N-803 as a potent immunotherapeutic candidate capable of activating and directing effector CD8+ T and NK cells to the B cell follicle during full ART suppression, and suggest N-803 must be paired with a bona fide latency reversing agent in vivo to facilitate immune-mediated modulation of the latent viral reservoir.

Development of "Imprint-and-Report" Dynamic Combinatorial Libraries for Differential Sensing Applications.

Sensor arrays using synthetic receptors have found great utility in analyte detection, resulting from their ability to distinguish analytes based on differential signals via indicator displacement. However, synthesis and characterization of receptors for an array remain a bottleneck in the field. Receptor discovery has been streamlined using dynamic combinatorial libraries (DCLs), but the resulting receptors have primarily been utilized in isolation rather than as part of the entire library, with only a few examples that make use of the complexity of a library of receptors. Herein, we demonstrate a unique sensor array approach using "imprint-and-report" DCLs that obviates the need for receptor synthesis and isolation. This strategy leverages information stored in DCLs in the form of differential library speciation to provide a high-throughput method for both developing a sensor array and analyzing data for analyte differentiation. First, each DCL is templated with analyte to give an imprinted library, followed by in situ fluorescent indicator displacement analysis. We further demonstrate that the reverse strategy, imprinting with the fluorescent reporter followed by displacement with each analyte, provides a more sensitive method for differentiating analytes. We describe the development of this differential sensing system using the methylated Arg and Lys post-translational modifications (PTMs). Altogether, 19 combinations of 3-5 DCL data sets that discriminate all 7 PTMs were identified. Thus, a comparable sensor array workflow results in a larger payoff due to the immense information stored within multiple noncovalent networks.

Successful outcome following allogeneic hematopoietic stem cell transplantation in adults with primary immunodeficiency.

The primary immunodeficiencies (PIDs), rare inherited diseases characterized by severe dysfunction of immunity, have been successfully treated by allogeneic hematopoietic stem cell transplantation (Allo-HSCT) in childhood. Controversy exists regarding optimal timing and use of Allo-HSCT in adults, due to lack of experience and previous poor outcomes. Twenty-nine consecutive adult patients, with a mean age at transplant of 24 years (range, 17-50 years), underwent Allo-HSCT. Reduced-intensity conditioning (RIC) included fludarabine (Flu)/melphalan/alemtuzumab (n = 20), Flu/busulfan (Bu)/alemtuzumab (n = 8), and Flu/Bu/antithymocyte globulin (n = 1). Stem cell donors were matched unrelated donors or mismatched unrelated donors (n = 18) and matched related donors (n = 11). Overall survival (OS), event-free survival, transplant-related mortality (TRM), acute and chronic graft-versus-host disease incidence and severity, time to engraftment, lineage-specific chimerism, immune reconstitution, and discontinuation of immunoglobulin replacement therapy were recorded. OS at 3 years for the whole cohort was 85.2%. The rarer PID patients without chronic granulomatous disease (CGD) achieved an OS at 3 years of 88.9% (n = 18), compared with 81.8% for CGD patients (n = 11). TRM was low with only 4 deaths observed at a median follow-up of 3.5 years. There were no cases of early or late rejection. In all surviving patients, either stable mixed chimerism or full donor chimerism were observed. At last follow-up, 87% of the surviving patients had no evidence of persistent or recurrent infections. Allo-HSCT is safe and effective in young adult patients with severe PID and should be considered the treatment of choice where an appropriate donor is available.

Comparison of Gas Treatments of High Oxygen, Carbon Monoxide, and Nitric Oxide on Ground Beef Color in Modified Atmosphere Packaging.

The objective of this study was to evaluate the viability and performance of nitric oxide modified-atmosphere packaging (MAP) as a novel alternative to high oxygen and carbon monoxide MAP for ground beef. Packages of ground beef under high oxygen (HI-OX), carbon monoxide (CO), and nitric oxide (NO) atmospheres were evaluated for descriptive and instrumental color every 12 h during a 120 h display period. Surface myoglobin percentages, internal cooked color, thiobarbituric acid reactive substances (TBARS), and residual nitrite and nitrate were also evaluated. There were gas × time interactions for descriptive color, discoloration, a* values, b* values, deoxymyoglobin percentages, and metmyoglobin percentages (p < 0.05). There were also gas-type main effects for cooked color and TBARS (p < 0.05). Carbon monoxide maintained the most redness and least discoloration throughout the display period, while HI-OX started with a bright red color but rapidly browned (p < 0.05). Nitric oxide started as dark red to tannish-red but transitioned to a dull red (p < 0.05). However, NO had increased redness and a* values for internal cooked color (p < 0.05). Although CO outperformed NO packages, NO exhibited a unique color cycle warranting further research to optimize its use.

Mismatched covalent and noncovalent templating leads to large coiled coil-templated macrocycles.

Herein we describe the use of dynamic combinatorial chemistry to self-assemble complex coiled coil motifs. We amide-coupled a series of peptides designed to form homodimeric coiled coils with 3,5-dithiobenzoic acid (B) at the N-terminus and then allowed each B-peptide to undergo disulfide exchange. In the absence of peptide, monomer B forms cyclic trimers and tetramers, and thus we expected that addition of the peptide to monomer B would shift the equilibrium towards the tetramer to maximize coiled coil formation. Unexpectedly, we found that internal templation of the B-peptide through coiled coil formation shifts the equilibrium towards larger macrocycles up to 13 B-peptide subunits, with a preference for 4, 7, and 10-membered macrocycles. These macrocyclic assemblies display greater helicity and thermal stability relative to intermolecular coiled coil homodimer controls. The preference for large macrocycles is driven by the strength of the coiled coil, as increasing the coiled coil affinity increases the fraction of larger macrocycles. This system represents a new approach towards the development of complex peptide and protein assemblies.

Nonsurgical Treatment of Delayed-Onset Brachial Plexopathy Due to Hypertrophic Clavicular Callus: A Case Report.

Clavicular fractures are common injuries that traditionally are managed nonsurgically without clinically significant sequelae. However, they may develop hypertrophic callus formation that compresses the brachial plexus. These cases may present months to years after initial injury with varying degrees of pain, paresthesia, and weakness on the affected side and usually are treated by surgical resection of the hypertrophic callus. We present a case of brachial plexopathy due to hypertrophic clavicular callus causing weakness and paresthesia. The plexopathy was confirmed with imaging and electrodiagnostic studies. This case was unusual in that resolution of symptoms was achieved nonsurgically. LEVEL OF EVIDENCE: V.

Training the next generation of surgeons in robotic surgery.

CONTEXT: Robotic surgery has been used with rapidly increasing frequency within urology and across many other surgical specialties. A standardized curriculum for the training and credentialing of robotic surgeons has unfortunately trailed far behind the adoption of this surgical technology. OBJECTIVE: To review the current available surgical skills training models, assessments, and curricula for the purpose of training resident, fellow, and practicing surgeons in an effort to promote surgical skill proficiency and mastery and to minimize the risk of patient harm. EVIDENCE ACQUISITION: We performed a thorough review of available literature through a PubMed database search in February 2015. EVIDENCE SYNTHESIS: In this article, we compiled and scrutinized the available relevant literature regarding past and present robotic surgical training techniques and credentialing criteria. This review details the basic surgical skills (both technical and nontechnical) that are necessary for individuals and teams to be successful in the operative setting. We go on to discuss the role of current robotic surgical training techniques including dry lab and virtual simulators. Finally, we offer current validated training curricula, the Global Evaluative Assessment of Robotic Skills and Fundamentals of Robotic Surgery models, which have laid the groundwork for a future standardized model that could be applied on a national and international level and across several surgical subspecialties. The ultimate goal of the review is to provide a foundation from which a future standardized training and credentialing curriculum could be based. CONCLUSION: There is currently a great need for a standardized curriculum to be developed and employed for the use of training and credentialing future and current robotic surgeons.