Small amounts of sub-visible aggregates enhance the immunogenic potential of monoclonal antibody therapeutics.

PURPOSE: Determine the effect of minute quantities of sub-visible aggregates on the in vitro immunogenicity of clinically relevant protein therapeutics. METHODS: Monoclonal chimeric (rituximab) and humanized (trastuzumab) antibodies were subjected to fine-tuned stress conditions to achieve low levels (<3% of total protein) of sub-visible aggregates. The effect of stimulating human dendritic cells (DC) and CD4(+) T cells with the aggregates was measured in vitro using cytokine secretion, proliferation and confocal microscopy. RESULTS: Due to its intrinsic high clinical immunogenicity, aggregation of rituximab had minimal effects on DC activation and T cell responses compared to monomeric rituximab. However, in the case of trastuzumab (low clinical immunogenicity) small quantities of aggregates led to potent CD4(+) T cell proliferation as a result of strong cytokine and co-stimulatory signals derived from DC. Consistent with this, confocal studies showed that stir-stressed rituximab was rapidly internalised and associated with late endosomes of DC. CONCLUSIONS: These data link minute amounts of aggregates with activation of the innate immune response, involving DC, resulting in T cell activation. Thus, when protein therapeutics with little or no clinical immunogenicity, such as trastuzumab, contain minute amounts of sub-visible aggregates, they are associated with significantly increased potential risk of clinical immunogenicity.

Hovering or invalidating? Examining nuances in the associations between controlling parents and problematic outcomes for college students.

Objective: The purpose of these studies was to examine whether college students' beliefs about themselves (i.e., self-compassion and beliefs about emotions) could be mechanisms explaining the relationship between problematic parenting behaviors (helicopter parenting and parental invalidation) and outcomes including perfectionism, affective distress, locus of control, and distress tolerance. Participants: Respondents included 255 (Study 1) and 277 (Study 2) college undergraduates. Methods: Simultaneous regressions and separate path analyses with helicopter parenting and parental invalidation as predictors, with self-compassion and emotion beliefs as mediators. Results: Across both studies, parental invalidation predicted perfectionism, affective distress, distress tolerance, and locus of control, and these links were often mediated by self-compassion. Self-compassion emerged as the most consistent and strongest link between parental invalidation and negative outcomes. Conclusion: People who internalize their parents' criticism and invalidation such that they hold negative beliefs about themselves (i.e., low self-compassion) may be vulnerable to negative psychosocial outcomes.

Characterization of fentanyl HCl powder prior to and after systematic degradation.

Fentanyl HCl is of particular interest in forensic cases but there is a notable gap in literature regarding its analysis. This study utilized a multi-method approach to characterize fentanyl HCl powder, both fresh and following a forced degradation process. Using sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) and direct injection gas chromatography-mass spectrometry (GC-MS), five compounds were identified in fresh fentanyl HCl powder. The identified compounds were: N-phenylpropanamide, 1-phenethyl-4-propionyloxypiperidine (1-P-4-POP), 4-anilino-N-phenethylpiperidine (4-ANPP), acetylfentanyl, and fentanyl; all identified compounds but acetylfentanyl and fentanyl decreased in quantity as the sample was degraded. Fresh headspace samples analyzed with solid phase microextraction (SPME)-GC-MS identified four compounds in common with the powder analyses: N-phenylpropanamide,1-P-4-POP, 4-ANPP, and fentanyl. Acetylfentanyl was not present in the headspace samples, although two additional compounds were: N-phenylacetamide and N-phenethyl-4-piperidinone (NPP). Where direct analysis of degraded fentanyl HCl showed decreased quantities of the identified compounds, headspace samples of the degraded fentanyl HCl resulted in higher quantities, implying that the degradation process drove those compounds to volatilize. Notably, fentanyl was identified in the headspace, implying that this could be an appropriate target for standoff detection. Finally, thermogravimetric analysis (TGA) and differential scanning calorimetry (DCS) confirmed that the forced degradation process had little permanent effect on the powder.

The effect of deleting p110delta on the phenotype and function of PTEN-deficient B cells.

Control of the intracellular levels of phosphatidylinositol-(3, 4, 5)-trisphosphate by PI3K and phosphatase and tensin homolog (PTEN) is essential for B cell development and differentiation. Deletion of the PI3K catalytic subunit p110delta leads to a severe reduction in B1 and marginal zone (MZ) B cells, whereas deletion of PTEN results in their expansion. We have examined the relationship between these two molecules by generating mice with a B cell-specific deletion of PTEN (PTENB) and a concurrent germline deletion of p110delta. The expanded B1 cell population of PTENB mice was reduced to normal levels in PTENB/p110delta mutant mice, indicating a critical role for the p110delta isoform in the expansion of B1 cells. However, numbers of MZ B cells in the PTENB/p110delta mutants was intermediate between wild-type and PTENB-deficient mice, suggesting an additional role for other PI3K catalytic isoforms in MZ differentiation. Furthermore, the defective class switch recombination in PTENB B cells was only partially reversed in PTENB/p110delta double mutant B cells. These results demonstrate an epistatic relationship between p110delta and PTEN. In addition, they also suggest that additional PI3K catalytic subunits contribute to B cell development and function.

Alloantigen-enhanced accumulation of CCR5+ 'effector' regulatory T cells in the gravid uterus.

Regulatory T cells play an essential role in preventing fetal rejection by the maternal immune system. Here we show that, based on the expression of CCR5, regulatory T cells can be divided into a highly suppressive CCR5+ and a far less suppressive CCR5- subpopulation, suggesting that the former represent the effector arm of regulatory T cells. Although regulatory T cells from CCR5-/- gene deletion mutants still suppress, they are less effective mediators of maternal-fetal tolerance. The accumulation of CCR5+ regulatory T cells at this site appears to be enhanced by alloantigen. This finding is in stark contrast to the systemic expansion of regulatory T cells during pregnancy, which appears to be alloantigen-independent. The fact that CCR5+ regulatory T cells preferentially accumulate in the gravid uterus and that expression of CCR5 on regulatory T cells can be induced by activation lead us to propose that CCR5 is responsible for the accumulation of those regulatory T cells that have been activated by paternal antigens.