Preclinical Analysis of Candidate Anti-Human CD79 Therapeutic Antibodies Using a Humanized CD79 Mouse Model.

The BCR comprises a membrane-bound Ig that is noncovalently associated with a heterodimer of CD79A and CD79B. While the BCR Ig component functions to sense extracellular Ag, CD79 subunits contain cytoplasmic ITAMs that mediate intracellular propagation of BCR signals critical for B cell development, survival, and Ag-induced activation. CD79 is therefore an attractive target for Ab and chimeric Ag receptor T cell therapies for autoimmunity and B cell neoplasia. Although the mouse is an attractive model for preclinical testing, due to its well-defined immune system, an obstacle is the lack of cross-reactivity of candidate therapeutic anti-human mAbs with mouse CD79. To overcome this problem, we generated knockin mice in which the extracellular Ig-like domains of CD79A and CD79B were replaced with human equivalents. In this study, we describe the generation and characterization of mice expressing chimeric CD79 and report studies that demonstrate their utility in preclinical analysis of anti-human CD79 therapy. We demonstrate that human and mouse CD79 extracellular domains are functionally interchangeable, and that anti-human CD79 lacking Fc region effector function does not cause significant B cell depletion, but induces 1) decreased expression of plasma membrane-associated IgM and IgD, 2) uncoupling of BCR-induced tyrosine phosphorylation and calcium mobilization, and 3) increased expression of PTEN, consistent with the levels observed in anergic B cells. Finally, anti-human CD79 treatment prevents disease development in two mouse models of autoimmunity. We also present evidence that anti-human CD79 treatment may inhibit Ab secretion by terminally differentiated plasmablasts and plasma cells in vitro.

Capacitively coupled nonthermal plasma synthesis of aluminum nanocrystals for enhanced yield and size control.

Uniform-size, non-native oxide-passivated metallic aluminum nanoparticles (Al NPs) have desirable properties for fuel applications, battery components, plasmonics, and hydrogen catalysis. Nonthermal plasma-assisted synthesis of Al NPs was previously achieved with an inductively coupled plasma (ICP) reactor, but the low production rate and limited tunability of particle size were key barriers to the applications of this material. This work focuses on the application of capacitively coupled plasma (CCP) to achieve improved control over Al NP size and a ten-fold increase in yield. In contrast with many other materials, where NP size is controlled via the gas residence time in the reactor, the Al NP size appeared to depend on the power input to the CCP system. The results indicate that the CCP reactor assembly, with a hydrogen-rich argon/hydrogen plasma, was able to produce Al NPs with diameters that were tunable between 8 and 21 nm at a rate up ∼ 100 mg h-1. X-ray diffraction indicates that a hydrogen-rich environment results in crystalline metal Al particles. The improved synthesis control of the CCP system compared to the ICP system is interpreted in terms of the CCP's lower plasma density, as determined by double Langmuir probe measurements, leading to reduced NP heating in the CCP that is more amenable to NP nucleation and growth.

Lrp4 is a receptor for Agrin and forms a complex with MuSK.

Neuromuscular synapse formation requires a complex exchange of signals between motor neurons and skeletal muscle fibers, leading to the accumulation of postsynaptic proteins, including acetylcholine receptors in the muscle membrane and specialized release sites, or active zones in the presynaptic nerve terminal. MuSK, a receptor tyrosine kinase that is expressed in skeletal muscle, and Agrin, a motor neuron-derived ligand that stimulates MuSK phosphorylation, play critical roles in synaptic differentiation, as synapses do not form in their absence, and mutations in MuSK or downstream effectors are a major cause of a group of neuromuscular disorders, termed congenital myasthenic syndromes (CMS). How Agrin activates MuSK and stimulates synaptic differentiation is not known and remains a fundamental gap in our understanding of signaling at neuromuscular synapses. Here, we report that Lrp4, a member of the LDLR family, is a receptor for Agrin, forms a complex with MuSK, and mediates MuSK activation by Agrin.

Integrating sphere port error in diffuse reflectance measurements.

The impact of a finite thickness integrating sphere port on the measurement of diffuse reflectance is addressed in a combined numerical and experimental study. It is shown that for a finite thickness port, additional light losses occur due to scattering between the sphere port wall and the test sample, causing the sample reflectance to be underestimated. Monte Carlo ray tracing is applied to obtain quantitative estimates of the resulting measurement error for the case of a diffusely reflecting sample. The effects of sample reflectance, port geometry, and illumination beam size on the measurement error are explored. Experimental data collected with a pair of integrating sphere reflectometers with different port geometries support the validity of the numerical results. It is argued that finite port thickness may be an important source of measurement error, even for a well-designed integrating sphere port, and a strategy for minimizing this error is discussed.

MOG autoantibodies trigger a tightly-controlled FcR and BTK-driven microglia proliferative response.

Autoantibodies are a hallmark of numerous neurological disorders, including multiple sclerosis, autoimmune encephalitides and neuromyelitis optica. Whilst well understood in peripheral myeloid cells, the pathophysiological significance of autoantibody-induced Fc receptor signalling in microglia remains unknown, in part due to the lack of a robust in vivo model. Moreover, the application of therapeutic antibodies for neurodegenerative disease also highlights the importance of understanding Fc receptor signalling in microglia. Here, we describe a novel in vivo experimental paradigm that allows for selective engagement of Fc receptors within the CNS by peripherally injecting anti-myelin oligodendrocyte glycoprotein (MOG) monoclonal antibodies into normal wild-type mice. MOG antigen-bound immunoglobulins were detected throughout the CNS and triggered a rapid and tightly regulated proliferative response in both brain and spinal cord microglia. This microglial response was abrogated when anti-MOG antibodies were deprived of Fc receptor effector function or injected into Fcγ receptor knockout mice and was associated with the downregulation of Fc receptors in microglia, but not peripheral myeloid cells, establishing that this response was dependent on central Fc receptor engagement. Downstream of the Fc receptors, BTK was a required signalling node for this response, as microglia proliferation was amplified in BtkE41K knock-in mice expressing a constitutively active form of the enzyme and blunted in mice treated with a CNS-penetrant small molecule inhibitor of BTK. Finally, this response was associated with transient and stringently regulated changes in gene expression predominantly related to cellular proliferation, which markedly differed from transcriptional programs typically associated with Fc receptor engagement in peripheral myeloid cells. Together, these results establish a physiologically-meaningful functional response to Fc receptor and BTK signalling in microglia, while providing a novel in vivo tool to further dissect the roles of microglia-specific Fc receptor and BTK-driven responses to both pathogenic and therapeutic antibodies in CNS homeostasis and disease.

Kinetics of early T cell receptor signaling regulate the pathway of lytic granule delivery to the secretory domain.

Cytolytic granules mediate killing of virus-infected cells by cytotoxic T lymphocytes. We show here that the granules can take long or short paths to the secretory domain. Both paths utilized the same intracellular molecular events, which have different spatial and temporal arrangements and are regulated by the kinetics of Ca(2+)-mediated signaling. Rapid signaling caused swift granule concentration near the microtubule-organizing center (MTOC) and subsequent delivery by the polarized MTOC directly to the secretory domain-the shortest path. Indolent signaling led to late recruitment of granules that moved along microtubules to the periphery of the synapse and then moved tangentially to fuse at the outer edge of the secretory domain-a longer path. The short pathway is associated with faster granule release and more efficient killing than the long pathway. Thus, the kinetics of early signaling regulates the quality of the T cell cytolytic response.

A Prodomain Fragment from the Proteolytic Activation of Growth Differentiation Factor 11 Remains Associated with the Mature Growth Factor and Keeps It Soluble.

Growth differentiation factor 11 (GDF11), a member of the transforming growth factor ß (TGF-ß) family, plays diverse roles in mammalian development. It is synthesized as a large, inactive precursor protein containing a prodomain, pro-GDF11, and exists as a homodimer. Activation requires two proteolytic processing steps that release the prodomains and transform latent pro-GDF11 into active mature GDF11. In studying proteolytic activation in vitro, we discovered that a 6-kDa prodomain peptide containing residues 60-114, PDP60-114, remained associated with the mature growth factor. Whereas the full-length prodomain of GDF11 is a functional antagonist, PDP60-114 had no impact on activity. The specific activity of the GDF11/PDP60-114 complex (EC50 = 1 nM) in a SMAD2/3 reporter assay was identical to that of mature GDF11 alone. PDP60-114 improved the solubility of mature GDF11 at neutral pH. As the growth factor normally aggregates/precipitates at neutral pH, PDP60-114 can be used as a solubility-enhancing formulation. Expression of two engineered constructs with PDP60-114 genetically fused to the mature domain of GDF11 through a 2x or 3x G4S linker produced soluble monomeric products that could be dimerized through redox reactions. The construct with a 3x G4S linker retained 10% activity (EC50 = 10 nM), whereas the construct connected with a 2x G4S linker could only be activated (EC50 = 2 nM) by protease treatment. Complex formation with PDP60-114 represents a new strategy for stabilizing GDF11 in an active state that may translate to other members of the TGF-ß family that form latent pro/mature domain complexes.

Interference with Ca(2+) release activated Ca(2+) (CRAC) channel function delays T-cell arrest in vivo.

Entry of lymphocytes into secondary lymphoid organs (SLOs) involves intravascular arrest and intracellular calcium ion ([Ca(2+)]i) elevation. TCR activation triggers increased [Ca(2+)]i and can arrest T-cell motility in vitro. However, the requirement for [Ca(2+)]i elevation in arresting T cells in vivo has not been tested. Here, we have manipulated the Ca(2+) release-activated Ca(2+) (CRAC) channel pathway required for [Ca(2+)]i elevation in T cells through genetic deletion of stromal interaction molecule (STIM) 1 or by expression of a dominant-negative ORAI1 channel subunit (ORAI1-DN). Interestingly, the absence of CRAC did not interfere with homing of naïve CD4(+) T cells to SLOs and only moderately reduced crawling speeds in vivo. T cells expressing ORAI1-DN lacked TCR activation induced [Ca(2+)]i elevation, yet arrested motility similar to control T cells in vitro. In contrast, antigen-specific ORAI1-DN T cells had a twofold delayed onset of arrest following injection of OVA peptide in vivo. CRAC channel function is not required for homing to SLOs, but enhances spatiotemporal coordination of TCR signaling and motility arrest.

Comparison of a Fully Weight-Based Protocol with a Non-Weight-Based Dosage Titration Protocol for IV Unfractionated Heparin: A Before-and-After Study.

Background: Unfractionated heparin (UFH) is used for the prevention and treatment of arterial or venous thromboembolism. The dosage for IV infusion of UFH is generally based on the patient's weight, with adjustment to a specific target for activated partial thromboplastin time (aPTT). In May 2019, the UFH protocols at the study institution were changed from being fully weight-based (i.e., for both initial dosing and subsequent dosage titrations) to weight-based initial dosing and non-weight-based dosage titrations, but the relative effectiveness of these 2 approaches was not known. Objectives: The primary objective was to compare the effectiveness in achieving therapeutic aPTT with the fully weight-based and non-weight-based dosage titration protocols. The secondary objective was to compare the effectiveness of the non-weight-based dosage titration protocol with that of the previous fully weight-based one for patients with low-target aPTT. Methods: A single-centre, retrospective, observational before-and-after study was conducted for patients receiving therapeutic UFH for any indication. Patients in the "before" group (fully weight-based protocol) were treated from January 2015 to October 2016, and those in the "after" group (non-weight-based titration) from January to October 2020. Results: From a total of 1969 charts screened, 137 patients treated according to the fully weight-based protocols and 130 patients treated according to the non-weight-based titration protocols were included. In terms of the co-primary objective, the median number of dosage adjustments to achieve therapeutic anticoagulation was 1 in both groups (p = 0.48), and the proportion of patients with therapeutic anticoagulation at 24 h was similar (96.2% [125/130] with the non-weight-based titration protocols versus 99.3% [136/137] with the fully weight-based protocols; p = 0.09). Among patients treated according to the low-target UFH protocols, those with the non-weight-based titration protocol were less likely to have therapeutic anticoagulation at first measurement of aPTT than those with the fully weight-based protocol (37.9% [25/66] versus 44.6% [41/92], p = 0.033). Conclusions: This retrospective, observational, before-and-after study showed that the effectiveness of the non-weight-based dosage titration protocols in achieving therapeutic aPTT was similar to that of fully weight-based UFH protocols.

Contexte: L'héparine non fractionnée (HNF) est utilisée pour la prévention et le traitement de la thromboembolie artérielle ou veineuse. La posologie de la perfusion par IV d'HNF se base généralement sur le poids du patient, avec un ajustement à un objectif précis du temps moyen de céphaline activée (TCA). En mai 2019, les protocoles d'HNF de l'établissement à l'étude sont passés d'une approche entièrement basée sur le poids (à la fois pour la posologie initiale et les titrages posologiques ultérieurs) à une posologie initiale basée sur le poids, et à des titrages posologiques non basés sur le poids. Cependant, l'efficacité relative de ces 2 approches était inconnue. Objectifs: L'objectif principal de l'étude consistait à comparer dans quelle mesure les protocoles entièrement basés sur le poids et les protocoles de titrage non basés sur le poids étaient efficaces pour atteindre le TCA thérapeutique. L'objectif secondaire consistait quant à lui à comparer l'efficacité du protocole de titrage de dose non basé sur le poids au protocole précédent entièrement basé sur le poids chez les patients ayant une faible cible de TCA. Méthodes: Une étude monocentrique, rétrospective, observationnelle avant-après a été menée chez des patients recevant de l'HNF thérapeutique, toutes indications confondues. Les patients du groupe « Avant ¼ (protocole entièrement basé sur le poids) ont été traités de janvier 2015 à octobre 2016, et ceux du groupe « Après ¼ (protocole de titrage de dose non basé sur le poids) de janvier à octobre 2020. Résultats: À partir de 1969 dossiers examinés, 137 patients traités selon les protocoles entièrement basés sur le poids et 130 patients traités selon les protocoles d'ajustement posologique non basés sur le poids ont été inclus. En ce qui concerne l'objectif co-principal, le nombre médian d'ajustements posologiques pour obtenir une anticoagulation thérapeutique était de 1 dans les deux groupes (p = 0,48), et la part de patients ayant une anticoagulation thérapeutique à 24 h était similaire (96,2 % [125/130] avec les protocoles non basés sur le poids contre 99,3 % [136/137] avec ceux entièrement basés sur le poids [p = 0,09]). Parmi les patients traités selon les protocoles HNF à faible cible, ceux avec le protocole de titrage non basé sur le poids étaient moins susceptibles de connaître une anticoagulation thérapeutique à la première mesure du TCA que ceux avec le protocole entièrement basé sur le poids (37,9 % [25/66] contre 44,6 % [41/92], p = 0,033). Conclusions: Cette étude rétrospective et observationnelle avant-après a montré que l'efficacité des protocoles d'ajustement posologique non basés sur le poids pour obtenir un TCA thérapeutique était similaire à celle des protocoles d'HNF entièrement basés sur le poids.

Cephalic arch stenosis in the arteriovenous fistula: A retrospective analysis of predisposing factors.

BACKGROUND: Cephalic Arch Stenosis (CAS) is a frequently observed complication in brachiocephalic and radiocephalic arteriovenous fistulae (AVF) associated with high morbidity and healthcare expenditure. The predisposing factors and preventative strategies for CAS remain unclear. Our aim was to examine predisposing factors for CAS development in the AVF. METHODS: A retrospective case-control study was performed at Gold University Coast Hospital on patients with AVFs created from 2009 to 2018 with ⩾18 months follow-up. CAS was defined as a >50% narrowing on angiographic assessment with clinically significant symptoms (dialysis dysfunction, arm swelling, prolonged bleeding after access). RESULTS: About 187 patients with AVF were included in the analysis (36 brachiocephalic, 151 radiocephalic). CAS developed in 22 of 36 (61%) of brachiocephalic AVF and 9 of 151 (6%) of radiocephalic AVFs. Brachiocephalic AVF were ⩾12 times more likely to develop CAS than radiocephalic AVF (Hazard Ratio (HR) 12.7, 95% CI [5.6-28.3], p < 0.001). Each 1 mL/min increase in flow rate through the AVF, correlated with a 0.07% increase in the probability of development of CAS (HR 1.0007, 95% CI [1.0001-1.0012], p = 0.011). Brachiocephalic AVFs with CAS were associated with a higher number of interventional procedures per access-year compared with their non-CAS counterparts (Median [Interquartile range]: 1.76 [0.74, 3.97] vs 0.41 [0.27, 0.67], p = 0.003). CONCLUSION: Brachiocephalic AVF with higher access flow rates are more likely to develop CAS and earlier than radiocephalic AVF, and in a dose dependent fashion. AVF flow rate is a major factor in CAS development within brachiocephalic AVF and has potential utility in surveillance thresholds for prophylactic blood flow reduction procedures. AVFs with CAS are associated with a greater number of interventional procedures per access-year, heralding higher patient morbidity and healthcare expenditure. Further prospective studies will help define an AVF access flow rate threshold in the implementation of prophylactic strategies for CAS.

Clinical Team Response to the Impact of COVID-19 on Diabetes Self-Management: Findings From a Qualitative Study.

The aims of this study were to explore providers' perceptions of how COVID-19 affected patients' psychological wellbeing and diabetes self-care and discover how providers responded to sustain and improve patients' psychological health and diabetes management during the pandemic. Twenty-four semi-structured interviews were completed with primary care providers (n=14) and endocrine specialty clinicians (n=10) across sixteen clinics in North Carolina. Interview topics included: (1) current glucose monitoring approaches and diabetes management strategies for people with diabetes (2) barriers and unintended consequences encountered with respect to diabetes self-management, and (3) innovative strategies developed to overcome barriers. Interview transcripts were coded using qualitative analysis software and analyzed to identify cross-cutting themes and differences between participants. Primary care providers and endocrine specialty clinicians reported that people with diabetes experienced increased mental health symptoms, increased financial challenges and positive and negative changes in self-care routines due to COVID-19. To offer support, primary care providers and endocrine specialty providers focused discussions on lifestyle management and utilized telemedicine to connect with patients. Additionally, endocrine specialty clinicians helped patients access financial assistance programs. Findings indicate that people with diabetes experienced unique challenges to self-management during the pandemic and providers responded with targeted support strategies. Future research should explore the effectiveness of these provider interventions as the pandemic continues to evolve.

Medical Waste Due to Intravitreal Injection Procedures in a Retina Clinic.

Purpose: Medical waste contributes to health care costs and has a direct negative impact on the environment. The goals of this study are to quantify and categorize the medical waste generated by intravitreal injection procedures and identify opportunities to reduce waste. Methods: This is a prospective observational series. Medical waste from intravitreal injections was collected from 337 consecutive intravitreal injections by a retina specialist over 2 weeks. The waste was sorted, photographed, weighed, and recorded. Results: A total of 65.6 kg of waste was collected across 3 broad categories: (1) shipping waste (cardboard boxes, foam coolers, cold packs, and bubble wrap); (2) waste from administering the intravitreal injection (nitrile gloves, tissues, wipes, and plastic or paper packaging); and (3) biohazard waste (used syringes and needles). Shipping waste contributed 83% of the overall waste, by mass, and varied greatly based on the size of the order and how efficiently shipments were packed. Cold packs, foam coolers, cardboard/paper, and nitrile gloves were the greatest contributors to carbon emissions and landfill. Conclusions: Waste due to shipping of medication is a major opportunity for reducing the environmental impact of intravitreal injections. Buying in bulk is a simple way for retina practices to reduce waste. Manufacturers should consider less bulky packaging for branded intravitreal injections; distributors and outsourcing facilities should consider take-back programs to reuse coolers and cold packs. Improved sustainability in the treatment of retinal disease is achievable but requires awareness and optimization of a clinic's routine.

Protein kinase C theta regulates stability of the peripheral adhesion ring junction and contributes to the sensitivity of target cell lysis by CTL.

Destruction of virus-infected cells by CTL is an extremely sensitive and efficient process. Our previous data suggest that LFA-1-ICAM-1 interactions in the peripheral supramolecular activation cluster (pSMAC) of the immunological synapse mediate formation of a tight adhesion junction that might contribute to the sensitivity of target cell lysis by CTL. Herein, we compared more (CD8(+)) and less (CD4(+)) effective CTL to understand the molecular events that promote efficient target cell lysis. We found that abrogation of the pSMAC formation significantly impaired the ability of CD8(+) but not CD4(+) CTL to lyse target cells despite having no effect of the amount of released granules by both CD8(+) and CD4(+) CTL. Consistent with this, CD4(+) CTL break their synapses more often than do CD8(+) CTL, which leads to the escape of the cytolytic molecules from the interface. CD4(+) CTL treatment with a protein kinase Ctheta inhibitor increases synapse stability and sensitivity of specific target cell lysis. Thus, formation of a stable pSMAC, which is partially controlled by protein kinase Ctheta, functions to confine the released lytic molecules at the synaptic interface and to enhance the effectiveness of target cell lysis.

Radiation-induced CXCL16 release by breast cancer cells attracts effector T cells.

Recruitment of effector T cells to inflamed peripheral tissues is regulated by chemokines and their receptors, but the factors regulating recruitment to tumors remain largely undefined. Ionizing radiation (IR) therapy is a common treatment modality for breast and other cancers. Used as a cytocidal agent for proliferating cancer cells, IR in combination with immunotherapy has been shown to promote immune-mediated tumor destruction in preclinical studies. In this study we demonstrate that IR markedly enhanced the secretion by mouse and human breast cancer cells of CXCL16, a chemokine that binds to CXCR6 on Th1 and activated CD8 effector T cells, and plays an important role in their recruitment to sites of inflammation. Using a poorly immunogenic mouse model of breast cancer, we found that irradiation increased the migration of CD8(+)CXCR6(+) activated T cells to tumors in vitro and in vivo. CXCR6-deficient mice showed reduced infiltration of tumors by activated CD8 T cells and impaired tumor regression following treatment with local IR to the tumor and Abs blocking the negative regulator of T cell activation, CTLA-4. These results provide the first evidence that IR can induce the secretion by cancer cells of proinflammatory chemotactic factors that recruit antitumor effector T cells. The ability of IR to convert tumors into "inflamed" peripheral tissues could be exploited to overcome obstacles at the effector phase of the antitumor immune response and improve the therapeutic efficacy of immunotherapy.

Human immunodeficiency virus type 1 envelope gp120 induces a stop signal and virological synapse formation in noninfected CD4+ T cells.

Human immunodeficiency virus type 1 (HIV-1)-infected T cells form a virological synapse with noninfected CD4(+) T cells in order to efficiently transfer HIV-1 virions from cell to cell. The virological synapse is a specialized cellular junction that is similar in some respects to the immunological synapse involved in T-cell activation and effector functions mediated by the T-cell antigen receptor. The immunological synapse stops T-cell migration to allow a sustained interaction between T-cells and antigen-presenting cells. Here, we have asked whether HIV-1 envelope gp120 presented on a surface to mimic an HIV-1-infected cell also delivers a stop signal and if this is sufficient to induce a virological synapse. We demonstrate that HIV-1 gp120-presenting surfaces arrested the migration of primary activated CD4 T cells that occurs spontaneously in the presence of ICAM-1 and induced the formation of a virological synapse, which was characterized by segregated supramolecular structures with a central cluster of envelope surrounded by a ring of ICAM-1. The virological synapse was formed transiently, with the initiation of migration within 30 min. Thus, HIV-1 gp120-presenting surfaces induce a transient stop signal and supramolecular segregation in noninfected CD4(+) T cells.

Intravascular immune surveillance by CXCR6+ NKT cells patrolling liver sinusoids.

We examined the in vivo behavior of liver natural killer T cells (NKT cells) by intravital fluorescence microscopic imaging of mice in which a green fluorescent protein cDNA was used to replace the gene encoding the chemokine receptor CXCR6. NKT cells, which account for most CXCR6(+) cells in liver, were found to crawl within hepatic sinusoids at 10-20 microm/min and to stop upon T cell antigen receptor activation. CXCR6-deficient mice exhibited a selective and severe reduction of CD1d-reactive NKT cells in the liver and decreased susceptibility to T-cell-dependent hepatitis. CXCL16, the cell surface ligand for CXCR6, is expressed on sinusoidal endothelial cells, and CXCR6 deficiency resulted in reduced survival, but not in altered speed or pattern of patrolling of NKT cells. Thus, NKT cells patrol liver sinusoids to provide intravascular immune surveillance, and CXCR6 contributes to liver-based immune responses by regulating their abundance.

Structural and kinetic basis for the selectivity of aducanumab for aggregated forms of amyloid-ß.

Aducanumab, a human-derived antibody targeting amyloid-ß (Aß), is in Phase 3 clinical trials for the treatment of Alzheimer's disease. Biochemical and structural analyses show that aducanumab binds a linear epitope formed by amino acids 3-7 of the Aß peptide. Aducanumab discriminates between monomers and oligomeric or fibrillar aggregates based on weak monovalent affinity, fast binding kinetics and strong avidity for epitope-rich aggregates. Direct comparative studies with analogs of gantenerumab, bapineuzumab and solanezumab demonstrate clear differentiation in the binding properties of these antibodies. The crystal structure of the Fab fragment of aducanumab bound to its epitope peptide reveals that aducanumab binds to the N terminus of Aß in an extended conformation, distinct from those seen in structures with other antibodies that target this immunodominant epitope. Aducanumab recognizes a compact epitope that sits in a shallow pocket on the antibody surface. In silico analyses suggest that aducanumab interacts weakly with the Aß monomer and may accommodate a variety of peptide conformations, further supporting its selectivity for Aß aggregates. Our studies provide a structural rationale for the low affinity of aducanumab for non-pathogenic monomers and its greater selectivity for aggregated forms than is seen for other Aß-targeting antibodies.

Antagonism of HIV-specific CD4+ T cells by C-terminal truncation of a minimum epitope.

Antagonism of T cell responses by variants of the cognate peptide is a potential mechanism of viral escape from immune responses and may play a role in the ability of HIV to evade immune control. We show here a rarely described mechanism of antagonism by a peptide shorter than the minimum length epitope for an HIV p24-specific CD4+ T cell clone. The shorter antagonist peptide-MHC complex bound the T cell receptor (TCR), albeit with lower affinity than the full-length agonist peptide. Prior work showing the crystal structure of the peptide-MHC complex revealed a unique glycine hinge near the C-terminus of the agonist peptide, allowing the generation of full-length antagonist peptide lacking the hinge. These results confirm the dependence of productive TCR engagement on residues spilling out from the C-terminus of the MHC binding groove and show that partial engagement of the TCR with a truncated, low-affinity ligand can result in T cell antagonism.

DNA testing in homicide investigations.

Objectives With the widespread use of DNA testing, police, death investigators, and attorneys need to be aware of the capabilities of this technology. This review provides an overview of scenarios where DNA evidence has played a major role in homicide investigations in order to highlight important educational issues for police, death investigators, forensic pathologists, and attorneys. Methods This was a nonrandom, observational, retrospective study. Data were obtained from the collective files of the authors from casework during a 15-year period, from 2000 through 2014. Results A series of nine scenarios, encompassing 11 deaths, is presented from the standpoint of the police and death investigation, the forensic pathology autopsy performance, the subsequent DNA testing of evidence, and, ultimately, the final adjudication of cases. Details of each case are presented, along with a discussion that focuses on important aspects of sample collection for potential DNA testing, especially at the crime scene and the autopsy. The presentation highlights the diversity of case and evidence types in which DNA testing played a valuable role in the successful prosecution of the case. Conclusions By highlighting homicides where DNA testing contributed to the successful adjudication of cases, police, death investigators, forensic pathologists, and attorneys will be better informed regarding the types of evidence and situations where such testing is of potential value.

Identification of Novel Fibrosis Modifiers by In Vivo siRNA Silencing.

Fibrotic diseases contribute to 45% of deaths in the industrialized world, and therefore a better understanding of the pathophysiological mechanisms underlying tissue fibrosis is sorely needed. We aimed to identify novel modifiers of tissue fibrosis expressed by myofibroblasts and their progenitors in their disease microenvironment through RNA silencing in vivo. We leveraged novel biology, targeting genes upregulated during liver and kidney fibrosis in this cell lineage, and employed small interfering RNA (siRNA)-formulated lipid nanoparticles technology to silence these genes in carbon-tetrachloride-induced liver fibrosis in mice. We identified five genes, Egr2, Atp1a2, Fkbp10, Fstl1, and Has2, which modified fibrogenesis based on their silencing, resulting in reduced Col1a1 mRNA levels and collagen accumulation in the liver. These genes fell into different groups based on the effects of their silencing on a transcriptional mini-array and histological outcomes. Silencing of Egr2 had the broadest effects in vivo and also reduced fibrogenic gene expression in a human fibroblast cell line. Prior to our study, Egr2, Atp1a2, and Fkbp10 had not been functionally validated in fibrosis in vivo. Thus, our results provide a major advance over the existing knowledge of fibrogenic pathways. Our study is the first example of a targeted siRNA assay to identify novel fibrosis modifiers in vivo.