Modulation of in vivo IgG crystallization in the secretory pathway by heavy chain isotype class switching and N-linked glycosylation.

Crystalline bodies (CBs) can develop in the endoplasmic reticulum (ER) of antibody-producing cells. Although this phenotype is often reported in association with plasma cell dyscrasias and other hematological disorders, the details of CB biogenesis and CB's roles in pathophysiology remain poorly understood. Using an imaging-based screening method, we identified a secretion-competent human IgG2/λ clone that develops spindle-shaped intracellular crystals in transiently-transfected HEK293 cells upon Brefeldin A treatment. When stably overexpressed from CHO cells, the IgG2/λ clone spontaneously produced spindle-shaped CBs in the ER. Some CBs were released to the extracellular space while remaining enclosed by the membranes of secretory pathway origin. Structural modeling on the variable-region did not uncover prominent surface characteristics such as charge clusters. In contrast, alterations to the constant domain-encoded properties revealed their modulatory roles in CB-inducing propensities and CB morphology. For example, deletion of the entire Fc domain changed the morphology of CBs into thin filaments. Elimination of an N-linked glycan by a N297A mutation promoted Russell body biogenesis accompanied by marked reduction in IgG secretion. Isotype class switching from the original IgG2 to IgG1 and IgG4 changed the crystal morphology from spindle-shaped to long needle and acicular shaped, respectively. The IgG3 version, in contrast, suppressed the CB formation. Either the HC or LC alone or the Fc-domain alone did not trigger CB biogenesis. An IgG's in vivo crystal morphology and crystallization propensity can thus be modulated by the properties genetically and biochemically encoded in the HC constant region.

IL-22 regulates iron availability in vivo through the induction of hepcidin.

Iron is a trace element important for the proper folding and function of various proteins. Physiological regulation of iron stores is of critical importance for RBC production and antimicrobial defense. Hepcidin is a key regulator of iron levels within the body. Under conditions of iron deficiency, hepcidin expression is reduced to promote increased iron uptake from the diet and release from cells, whereas during conditions of iron excess, induction of hepcidin restricts iron uptake and movement within the body. The cytokine IL-6 is well established as an important inducer of hepcidin. The presence of this cytokine during inflammatory states can induce hepcidin production, iron deficiency, and anemia. In this study, we show that IL-22 also influences hepcidin production in vivo. Injection of mice with exogenous mouse IgG1 Fc fused to the N terminus of mouse IL-22 (Fc-IL-22), an IL-22R agonist with prolonged and enhanced functional potency, induced hepcidin production, with a subsequent decrease in circulating serum iron and hemoglobin levels and a concomitant increase in iron accumulation within the spleen. This response was independent of IL-6 and was attenuated in the absence of the IL-22R-associated signaling kinase, Tyk2. Ab-mediated blockade of hepcidin partially reversed the effects on iron biology caused by IL-22R stimulation. Taken together, these data suggest that exogenous IL-22 regulates hepcidin production to physiologically influence iron usage.

Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG.

Naturally occurring IgG antibodies are bivalent and monospecific. Bispecific antibodies having binding specificities for two different antigens can be produced using recombinant technologies and are projected to have broad clinical applications. However, co-expression of multiple light and heavy chains often leads to contaminants and pose purification challenges. In this work, we have modified the CH3 domain interface of the antibody Fc region with selected mutations so that the engineered Fc proteins preferentially form heterodimers. These novel mutations create altered charge polarity across the Fc dimer interface such that coexpression of electrostatically matched Fc chains support favorable attractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation. This new Fc heterodimer format was used to produce bispecific single chain antibody fusions and monovalent IgGs with minimal homodimer contaminants. The strategy proposed here demonstrates the feasibility of robust production of novel Fc-based heterodimeric molecules and hence broadens the scope of bispecific molecules for therapeutic applications.

Identification of dietary patterns in urban population of Argentina: study on diet-obesity relation in population-based prevalence study.

BACKGROUND/OBJECTIVES: In Argentina, obesity prevalence rose from 14.6% in 2005 to 20.8% in 2013. Although the number of studies on noncommunicable diseases and dietary patterns as a unique dietary exposure measure has increased, information on this topic remains scarce in developing countries. This is the first population-based study investigating the association between diet and obesity using a dietary pattern approach in Argentina. We aimed (a) to identify current dietary patterns of the population of Córdoba city, (b) to investigate its association with obesity prevalence, and (c) to identify and describe dietary patterns from the subgroup of people with obesity. SUBJECTS/METHODS: The Córdoba Obesity and Diet Study (CODIES) was conducted in Córdoba city by using a random sample of n = 4,327 subjects between 2005 and 2012. Empirically derived dietary patterns were identified through principal component factor analysis. A multiple logistic regression analysis was used to investigate the association of dietary patterns with obesity. RESULTS: Four dietary patterns were identified, called "Starchy-Sugar", "Prudent", "Western", and "Sugary drinks". High scores for the "Western" pattern (with strongest factor loading on meats/eggs, processed meats, and alcohol) showed a positive association with obesity (OR: 1.33, 95% CI: 1.06-1.67, for third versus first tertile of factor score). "Meats/Cheeses" and "Snacks/Alcohol" patterns emerged in people with obesity. CONCLUSIONS: The findings suggest that high adherence to the "Western" pattern promoted obesity in this urban population. In addition, people with obesity showed characteristic dietary patterns that differ from those identified in the overall population.

Identification of potent human anti-IL-1RI antagonist antibodies.

Interleukin-1 (IL-1) blockade by IL-1 receptor antagonist benefits some arthritis patients by reducing joint damage. This fact inspired us to develop antagonist human therapeutic antibodies against IL-1R(I) using phage libraries that display single-chain variable fragment (scFv) antibody fragments. Panning libraries against human IL-1R(I) generated 39 unique scFv-phage whose binding to IL-1R(I) was competed by IL-1 ligands. Fifteen of these scFv-phage, identified using IL-1R(I)-binding assays and dissociation rate ranking, were reformatted as scFv-Fc and IgG(4) molecules. The ease of producing antibodies in the scFv-Fc format permitted rapid identification of four lead clones (C10, C13, C14, C15) that inhibit NF-kappaB nuclear translocation induced by IL-1. Reformatting these clones as IgG(4) molecules increased their inhibition potency by

Performance of formulae based estimates of glomerular filtration rate for carboplatin dosing in stage 1 seminoma.

BACKGROUND: Single cycle carboplatin, dosed by glomerular filtration rate (GFR), is standard adjuvant therapy for stage 1 seminoma. Accurate measurement of GFR is essential for correct dosing. Isotopic methods remain the gold standard for the determination of GFR. Formulae to estimate GFR have improved the assessment of renal function in non-oncological settings. We assessed the utility of these formulae for carboplatin dosing. METHODS: We studied consecutive subjects receiving adjuvant carboplatin for stage 1 seminoma at our institution between 2007 and 2012. Subjects underwent 51Cr-ethylene diamine tetra-acetic acid (EDTA) measurement of GFR with carboplatin dose calculated using the Calvert formula. Theoretical carboplatin doses were calculated from estimated GFR using Chronic Kidney Disease-Epidemiology (CKD-EPI), Management of Diet in Renal Disease (MDRD) and Cockcroft-Gault (CG) formulae with additional correction for actual body surface area (BSA). Carboplatin doses calculated by formulae were compared with dose calculated by isotopic GFR; a difference <10% was considered acceptable. RESULTS: 115 patients were identified. Mean isotopic GFR was 96.9 ml/min/1.73 m(2). CG and CKD-EPI tended to overestimate GFR whereas MDRD tended to underestimate GFR. The CKD-EPI formula had greatest accuracy. The CKD-EPI formula, corrected for actual BSA, performed best; 45.9% of patients received within 10% of correct carboplatin dose. Patients predicted as underdosed (13.5%) by CKD-EPI were more likely to be obese (p=0.013); there were no predictors of the 40.5% receiving an excess dose. CONCLUSIONS: Our data support further evaluation of the CKD-EPI formula in this patient population but clinically significant variances in carboplatin dosing occur using non-isotopic methods of GFR estimation. Isotopic determination of GFR should remain the recommended standard for carboplatin dosing when accuracy is essential.

Biomarkers for non-human primate type-I hypersensitivity: antigen-specific immunoglobulin E assays.

Immunoglobulin E (IgE) is the least abundant immunoglobulin in serum. However, development of an IgE immune response can induce IgE receptor-expressing cells to carry out potent effector functions. A reliable antigen-specific IgE biomarker method for use in non-human primate studies would facilitate (i) confirmation of Type-I hypersensitivity reactions during safety toxicology testing, and (ii) a better understanding of non-human primate models of allergic disease. We cloned and expressed a recombinant cynomolgus monkey IgE molecule in order to screen a panel of commercially available detection reagents raised against human IgE for cross-reactivity. The reagent most reactive to cynomolgus IgE was confirmed to be specific for IgE and did not bind recombinant cynomolgus monkey IgG1-4. A drug-specific IgE assay was developed on the MSD electrochemiluminescent (ECL) platform. The assay is capable of detecting 10 ng/mL drug-specific IgE. Importantly, the assay is able to detect IgE in the presence of excess IgG, the scenario likely to be present in a safety toxicology study. Using our ECL assay, we were able to confirm that serum from cynomolgus monkeys that had experienced clinical symptoms consistent with hypersensitivity responses contained IgE specific for a candidate therapeutic antibody. In addition, a bioassay for mast cell activation was developed using CD34(+)-derived cynomolgus monkey mast cells. This assay confirmed that plasma from animals identified as positive in the drug-specific IgE immunoassay contained biologically active IgE (i.e. could sensitize cultured mast cells), resulting in histamine release after exposure to the therapeutic antibody. These sensitive assays for Type-I hypersensitivity in the NHP can confirm that secondary events are downstream of immunogenicity.

Síndrome de realimentación: el diagnóstico pendiente / Refeeding syndrome: the pending diagnosis

Introducción: El síndrome de realimentación (SR) puede definirse como el conjunto de alteraciones metabólicas desencadenadas tras la rápida reintroducción del soporte nutricional en pacientes severamente desnutridos o con ayuno prolongado. Puede tener repercusiones clínicas, neurológicas y cardiológicas. La hipofosfatemia es el fenómeno predominantemente asociado con el SR. Paciente masculino de 68 años con antecedentes de tabaquismo, EPOC y depresión. Ingresa a UTI por sepsis a foco respiratorio severamente desnutrido (por Valoración Global Subjetiva), con IMC 14.6 kg/m². Inicia nutrición enteral (NE) al 30% de sus requerimientos por riesgo de SR con P sérico basal de 3,9 mg/dl. Al 2do día no se dosa P ni Mg séricos, pero sí se observa K dentro de parámetros normales. Se progresa NE al 50%, y se observa al 3ro°día una disminución significativa del P a 2,1 mg/dl, llegando a 1,9 mg/dl el 4to°día, sin haber progresado aportes (K y Mg en descenso pero dentro de parámetros normales). Se carga al 4to°día con una ampolla de fosfato de potasio, evolucionando favorablemente. Objetivo: Destacar la importancia de prevenir el SR. Discusión: Se observó la repercusión bioquímica característica del SR. El momento de detección de la hipofosfatemia significativa (3ro°día) coincide con el promedio general visto en otros estudios. No hubieron signos clínicos, probablemente porque el P no tuvo un valor crítico. Como terapéutica, se utilizó la lenta progresión de aportes y la corrección de P fue tardía. Podría haber sido adecuada la suplementación de tiamina previo inicio de NE

Introduction: The refeeding syndrome (SR) can be defined as the metabolic alterations developed after a rapid nutrition repletion (oral, enteral, as well as parenteral feeding) of severely malnourished patients. It can have clinical, neurological and cardiological effects. Hypophosphatemia is the predominantl phenomenon associated with SR. A 68-year-old male patient with a history of smoking, COPD, and depression, who is admitted in ICU severely malnourished (Subjetive Global Assesment) due to sepsis at a respiratory focus, with a BMI of 14.6 kg / m². The patient initiates enteral nutrition (NE) at 30% of its requirements due to risk of SR, with baseline serum P value of 3.9 mg/dl. At day 2, no serum P or Mg is given, but K is observed within normal parameters. NE is progressed up to 50%, and a significant decrease of P at 2.1 mg/dl is observed at day 3, reaching 1.9 mg/dl at day 4, with no progress (K and Mg in decline but within normal parameters). He is loaded at day 4 with a potassium phosphate ampoule, evolving favorably. Objective: Highlighting the importance of preventing SR. Discussion: The characteristic biochemical repercussion of SR was observed. The moment of detection of significant hypophosphatemia (day 3) coincides with the general average seen in other studies. There were no clinical signs, probably because P didn´t have a critical value. As therapy, the slow progression of caloric contributions was used, and the correction of P was late. Thiamine supplementation may have been adequate prior to initiation of NE.

Loperamida en el manejo de ileostomía de alto débito. A propósito de un caso / Loperamide in the management of high-debit ileostomy. About a case

Introducción: La Ostomía de Alto Débito es una complicación frecuente y poco identificada. Se recomienda la utilización de loperamida. Sin embargo, es una indicación off label.Paciente femenino de 78 años con antecedente de una ileostomía por cáncer de colon derecho, internada por sepsis en terapia intensiva. Presenta abundante débito por ileostomía permeable.Se acuerda con médico tratante y equipo de soporte nutricional iniciar nutrición enteral y loperamida. La paciente evoluciona favorablemente y normaliza el débito.Algunos profesionales, deciden suspender la loperamida y otros disminuir la frecuencia. La paciente aumenta nuevamente la producción del débito.Se justifica el propósito de la intervención con evidencia científica y se optimiza la comunicación interdisciplinaria para asegurar la dosificación que optimiza el tratamiento. Evoluciona de forma favorable y es dada de alta. Objetivo: Destacar la importancia del trabajo interdisciplinario para el abordaje de complicaciones, dando a conocer un tratamiento efectivo en ileostomías de alto débito.Discusión:La información publicada en relación con el uso de loperamida para esta indicación es escasa. El tratamiento sigue siendo basado en la observación y la experiencia de los expertos en los centros especializados.En cuanto a la seguridad de instaurar el tratamiento, evaluando que los efectos adversos cardíacos no han sido reportados en éstos pacientes, y que éstos presentan mayor riesgo de tener eventos cardíacos secundarios a los trastornos hidroelectrolíticos concomitantes de la misma patología de base, se decidió avalar laconducta de la indicación de loperamida que mostró mejor riesgo-beneficio con respecto a la no indicación. La comunicación interdisciplinaria impacta en la mejoría clínica del paciente y en la calidad de atención brindada

ntroduction: High-output Ostomy is a frequent and poorly identified complication. The use of loperamide is recommended. However, it is an off label indication.A 78-year-old female patient with a history of an ileostomy due to right colon cancer hospitalized for sepsis in intensive care. It has an abundant flow rate due to permeable ileostomy.It is agreed with treating physician and nutritional support team to initiate enteral nutrition and loperamide. The patient progresses favorably and normalizes th flow rate.Some professionals, decide to suspend loperamide and others decrease the frequency. The patient again increases the output of the flow.The purpose of the intervention with scientific evidence is justified and the interdisciplinary communication is optimized to ensure the dosage that optimizes the treatment. Evolves favorably and is discharged.Objective: Highlight the importance of interdisciplinary work in the management of complications, revealing an effective treatment in high-output ileostomies.Discussion: The published information regarding the use of loperamide for this indication is scarce. Treatment is still based on the observation and experience of experts in specialized centers.As regard the safety of establishing the treatment, evaluating that cardiac adverse effects have not been reported in these patients, and that they are at increased risk of having cardiac events secondary to the concomitant hydroelectrolytic disorders of the same underlying disease, it was decided to endorse The behavior of the indication of loperamide that showed better risk-benefit with respect to no indication.Interdisciplinary communication impacts on the clinical improvement of the patient and on the quality of care provided

Kinetic analysis of a high-affinity antibody/antigen interaction performed by multiple Biacore users.

To explore the reliability of Biacore-based assays, 22 study participants measured the binding of prostate-specific antigen (PSA) to a monoclonal antibody (mAb). Each participant was provided with the same reagents and a detailed experimental protocol. The mAb was immobilized on the sensor chip at three different densities and a two-step assay was used to determine the kinetic and affinity parameters of the PSA/mAb complex. First, PSA was tested over a concentration range of 2.5-600 nM to obtain k(a) information. Second, to define the k(d) of this stable antigen/antibody complex accurately, the highest PSA concentration was retested with the dissociation phase of each binding cycle monitored for 1h. All participants collected data that could be analyzed to obtain kinetic parameters for the interaction. The association and the extended-dissociation data derived from the three antibody surfaces were globally fit using a simple 1:1 interaction model. The average k(a) and k(d) for the PSA/mAb interaction as calculated from the 22 analyses were (4.1+/-0.6) x 10(4) M(-1) s(-1) and (4.5+/-0.6) x 10(-5) s(-1), respectively. Overall, the experimental standard errors in the rate constants were only approximately 14%. Based on the kinetic rate constants, the affinity (K(D)) of the PSA/mAb interaction was 1.1+/-0.2 nM.