Contribution of enhanced engagement of antigen presentation machinery to the clinical immunogenicity of a human interleukin (IL)‐21 receptor‐blocking therapeutic antibody

Reliable risk assessment for biotherapeutics requires accurate evaluation of risk factors associated with immunogenicity. Immunogenicity risk assessment tools were developed and applied to investigate the immunogenicity of a fully human therapeutic monoclonal antibody, ATR‐107 [anti‐interleukin (IL)‐21 receptor] that elicited anti‐drug antibodies (ADA) in 76% of healthy subjects in a Phase 1 study. Because the ATR‐107 target is expressed on dendritic cells (DCs), the immunogenicity risk related to engagement with DC and antigen presentation pathways was studied. Despite the presence of IL‐21R on DCs, ATR‐107 did not bind to the DCs more extensively than the control therapeutic antibody (PF‐1) that had elicited low clinical ADA incidence. However, ATR‐107, but not the control therapeutic antibody, was translocated to the DC late endosomes, co‐localized with intracellular antigen‐D related (HLA‐DR) molecules and presented a dominant T cell epitope overlapping the complementarity determining region 2 (CDR2) of the light chain. ATR‐107 induced increased DC activation exemplified by up‐regulation of DC surface expression of CD86, CD274 (PD‐L1) and CD40, increased expansion of activated DC populations expressing CD86^hi, CD40^hi, CD83^hi, programmed death ligand 1 (PD‐L1)^hi, HLA‐DR^hi or CCR7^hi, as well as elevated secretion of tumour necrosis factor (TNF)‐α by DCs. DCs exposed to ATR‐107 stimulated an autologous T cell proliferative response in human donor cells, in concert with the detection of immunoglobulin (Ig)G‐type anti‐ATR‐107 antibody response in clinical samples. Collectively, the enhanced engagement of antigen presentation machinery by ATR‐107 was suggested. The approaches and findings described in this study may be relevant to identifying lower immunogenicity risk targets and therapeutic molecules.     

Rapid array-based genomic characterization of a subtle structural abnormality: a patient with psychosis and der(18)t(5;18)(p14.1;p11.23)

Array-based copy number analysis has recently emerged as a rapid means of mapping complex and/or subtle chromosomal abnormalities. We have compared two such techniques, using bacterial artificial chromosome (BAC) and single nucleotide polymorphism (SNP) arrays in the evaluation of a 45-year-old woman with dysmorphic features, mental retardation, psychosis, and an unbalanced derivative chromosome 18, (46,XX, der(18)t(18;?)(p12;?)). Both array-based methods demonstrated that the additional material on chromosome 18 was of 5p origin. The 5p duplication mapped telomeric to 25.320 Mb (BAC array) and 25.607 Mb (SNP array), corresponding to the band 5p14.1. Both BAC and SNP arrays also showed a deletion involving chromosome 18p extending telomeric from 8.437 Mb (BAC array) and 8.352 Mb (SNP array), corresponding to the band 18p11.23. Molecular cytogenetic mapping using fluorescence in situ hybridization (FISH) supported the array findings and further refined the breakpoint regions, confirming that the BAC and SNP chips were both useful in this regard. Both case reports and linkage analyses have implicated these chromosomal intervals in psychosis. The array-based experiments were completed over the course of several days. While these methods do not eliminate the requirement for traditional fine-mapping, they provide an efficient approach to identifying the origin and extent of deleted and duplicated material in chromosomal rearrangements.     

Evidence that orexins A and B stimulate insulin secretion from rat pancreatic islets via both receptor subtypes

Orexins are recently identified neuropeptides that appear to play a role in the regulation of energy homeostasis and arousal. They bind to and activate two closely related G protein-coupled receptors (OXR1 and OXR2), previously described as orphans. In this study we examined involvement of orexins in regulation of insulin secretion from rat pancreatic islets utilizing an in situ perfused pancreas and isolated pancreatic islet models. By means of RT-PCR we found that both OXR1 and OXR2 are expressed in rat pancreatic islets. Furthermore, the expression levels of OXR1 were higher than OXR2. In both experimental models applied, orexins A and B (1, 10 and 100 nmol/l) concentration dependently stimulated insulin secretion at two different glucose concentrations (6.66 or 26.4 mmol/l), with orexin A being more potent than orexin B. This study demonstrates that orexins A and B modulate insulin secretion in vitro.     

Genome-wide aberrations in pancreatic adenocarcinoma

Chromosomal instability, manifesting as copy number alterations (CNAs), is characteristic of pancreatic adenocarcinoma. We used bacterial artificial chromosome (BAC) array-based comparative genomic hybridization (aCGH) to examine the pancreatic adenocarcinoma genome for submicroscopic amplifications and deletions. Profiles of 33 samples (17 first-passage xenografts and 16 cell lines) identified numerous chromosomal regions with CNAs, including losses at 1p36.33 approximately p34.3, 1p13.3 approximately p13.2, 3p26, 3p25.2 approximately p22.3, 3p22.1 approximately p14.1, 4q28.3, 4q31, 4q35.1, 5q14.3, 6p, 6q, 8p23.3 approximately p12, 9p, 9q22.32 approximately q31.1, 13q33.2, 15q11.2, 16p13.3, 17p, 18q11.21 approximately q23 , 19p13.3 approximately p13.12, 19q13.2, 21p, 21q, and 22p, 22q and gains at 7p21.1 approximately p11.2, 7q31.32, 7q33, 8q11.1 approximately q24, 11p13, 14q22.2, 20p12.2, and 20q11.23 approximately q13.33. Novel regions containing CNAs were identified and refined by combining the increased resolution of our BAC CGH array with a statistical algorithm developed for assigning significance values to altered BACs across samples. A subset of array-based CNAs was validated using polymerase chain reaction-based techniques, immunohistochemistry and fluorescence in situ hybridization. BAC aCGH proved to be a powerful genome-wide strategy to identify molecular alterations in pancreatic cancer and to distinguish differences between cell line and xenograft aberration profiles. These findings should greatly facilitate further research in understanding the pathogenesis of this lethal disease, and could lead to the identification of novel therapeutic targets and biomarkers for early detection.     

Treatment with continuous positive airway pressure may affect blood glucose levels in nondiabetic patients with obstructive sleep apnea syndrome

STUDY OBJECTIVES: Obstructive sleep apnea syndrome (OSAS) is often associated with impaired glucose metabolism. Data on the effects of OSAS treatment with continuous positive airway pressure (CPAP) on blood glucose and insulin resistance are conflicting. The study aimed at assessing the immediate effect of CPAP on glucose control measured with a continuous glucose monitoring system (CGMS). PARTICIPANTS AND MEASUREMENTS: Nine non-diabetes subjects with OSAS (mean age 53.0 +/- 8.0 years; body mass index 34.8 +/- 5.3 kg/m2) underwent 2 overnight polysomnographic examinations: a diagnostic study and one with CPAP treatment. Continuous glucose monitoring system (CGMS) was applied overnight on both occasions. Glucose metabolism was assessed with a 75-g oral glucose tolerance test, plasma insulin and homeostatic model assessment of insulin resistance (HOMA-IR) index. RESULTS: The mean (+/- SD) apnoea-hypopnea index (AHI) at diagnostic polysomnography was 54.3 +/- 29.3 (range 16-81). Fasting plasma insulin levels in patients with OSAS was 84.3 +/- 43.4 pM at baseline, and the HOMA-IR was 3.6 +/- 2.2. CPAP treatment in the subjects with OSAS resulted in a significant reduction in the AHI to 4.5 +/- 7.1. All of the major saturation parameters improved significantly on CPAP. CGMS showed mean glucose values significantly higher during the CPAP night than during the diagnostic night: 80 +/- 11 mg/dL versus 63 +/- 7 mg/dL (P < .01). Fasting insulin and HOMA-IR measured after the CPAP night tended to be higher than at baseline (98.4 +/- 51.0 pmol vs 84.3 +/- 43.4 pmol and 3.9 pmol +/- 2.6 vs 3.6 +/- 2.2 pmol, respectively, P > .05). CONCLUSION: CPAP treatment in nondiabetic obese patients with OSAS may have an immediate elevating effect on blood glucose.     

Microbial exposure of rural school children, as assessed by levels of N-acetyl-muramic acid in mattress dust, and its association with respiratory health

BACKGROUND: Endotoxin exposure has been shown to be associated with a decreased prevalence of atopic sensitization and symptoms. Yet endotoxin represents only a part of the indoor microbial exposure. Muramic acid, a constituent of peptidoglycan, is present in gram-negative and gram-positive bacteria in the environment and may therefore serve as an additional marker of microbial exposure. OBJECTIVE: To study the factors determining the level of indoor exposure to muramic acid/peptidoglycan, as well as its potential association with respiratory health. METHODS: In 553 farm and nonfarm school children from Austria, Switzerland, and Germany, mattress dust muramic acid concentrations were determined, and health was assessed by using IgE measurements and questionnaire information. RESULTS: The muramic acid concentration was found to be significantly higher in dust from farm children's mattresses than in dust from nonfarm children's mattresses (157 vs 131 ng/mg). Children with higher mattress dust muramic acid concentrations had a significantly lower prevalence of wheezing (odds ratio of highest vs lowest tertile of muramic acid concentration, 0.3; 95% CI, 0.1-0.9), regardless of farming status and endotoxin exposure. The association for asthma was similar, and no association was found with atopic sensitization. CONCLUSION: Next to endotoxin, muramic acid provides us with an independent marker of microbial exposure. Unlike endotoxin, muramic acid was inversely associated with wheezing rather than with atopic sensitization.     

Temperature can prime human peripheral blood neutrophils in a p38MAPKalpha-dependent manner

INTRODUCTION: Previous ex vivo experiments by others suggest that elevated body temperature can prime the respiratory burst of human neutrophils. The mechanism of the priming phenomenon induced by temperature has not been addressed so far. Furthermore, the priming temperature range was not defined. MATERIAL/METHODS: In the present study we explored ,under in vitro conditions, the influence of febrile-range temperatures on reactive oxygen species (ROS)generation by human peripheral blood neutrophils. ROS production was measured using whole.blood luminol-dependent chemiluminescence. Two elements of signal transduction pathways, calcium and p38 mitogen.activated protein kinase alpha (p38MAPKalpha) ,frequently underlying neutrophil priming were also examined.Calcium levels in the cytosol of resting and fMLP. stimulated isolated neutrophils were measured with the Fura-2AM spectrofluorimetric method. The activity of p38MAPKalpha was assessed indirectly with a specific inhibitor of the kinase, SB 203580. RESULTS: The study revealed a priming effect at 38 degrees C toward human peripheral blood neutrophil ROS production. Any concomitant effect on calcium response was not observed. Instead, experiments with SB 203580,a specific inhibitor of p38MAPKalpha, pointed to an increased activity of the kinase as a molecular background of temperature-induced priming. However, the priming effect of temperature was confined to 38 degrees C, while higher temperatures proved to exert no effect (39 and 40 degrees C)or even inhibited ROS generation by neutrophils (43 degrees C). CONCLUSIONS: Our study suggests a heterogeneous influence of temperature on human neutrophil functioning, including the priming of the cells by a low-febrile-range temperature. It also suggests a p38MAPKalpha-dependent molecular background of the priming phenomenon.     

Proteolytic activities in cultures of selected white-rot fungi

The presence of multiple intracellular and extracellular proteolytic activities in trophophasic (nutrientrich) and idiophasic (carbon-or nitrogen-starved) cultures of the white-rot fungi Trametes versicolor and Phlebia radiata was demonstrated by electrophoresis on polyacrylamide gels containing denatured haemoglobin as a substrate. In the trophophasic cultures of T. versicolor, seven electrophoretically distinguishable proteases were defined using mycelial extracts and six (three clear and three less intensive) of secreted proteases. For P. radiata eight bands of intracellular and five bands (one distinct and four less active) of extracellular proteolytic activities were detected. Gel electrophoresis revealed changes in patterns of secreted and mycelial proteinases upon carbon or nitrogen deprivation. The changes were seen both as an increase in activity of certain bands and as the appearance of new proteolytic bands. Specific activities of extracellular proteinases, assayed under idiophasic (—C or —N) conditions, increased 2—3 fold as compared to those upon nutrient sufficiency. These changes accompanied a shift to secondary metabolism and onset of ligninolytic activity.