Upfront acid treatment with low final pH: the development and testing of a novel method to improve drug tolerance in antidrug antibody assays.

Background: The drug tolerance of an antidrug antibody (ADA) assay for a therapeutic monoclonal antibody was insufficient to meet the level of biotherapeutic expected in sera, and a typical acid dissociation method was inadequate. Other strategies were investigated to dissociate ADA-drug complexes and thereby improve drug tolerance. Results: Having a lower final pH of samples after acid dissociation was shown to greatly improve drug tolerance. This method was shown to improve drug tolerance in the ADA assays for four additional monoclonal antibodies and to better detect ADAs in clinical samples. Conclusion: These findings provide a novel alternative method for improving drug tolerance when other methods are not sufficient.

Assessment and Clinical Relevance of Serum IL-19 Levels in Psoriasis and Atopic Dermatitis Using a Sensitive and Specific Novel Immunoassay.

Because development of reliable biomarkers in psoriasis and atopic dermatitis has lagged behind therapeutic progress, we created a blood-based test to fill the void in objective methods available for dermatological assessments. Our novel interleukin-19 (IL-19) immunoassay was initially tested to determine concentrations of IL-19 serum levels, then correlated with the psoriasis activity and severity index (PASI) in psoriasis, and the eczema area and severity index (EASI) in atopic dermatitis. Not only was IL-19 increased in psoriasis and correlated to PASI, but ixekizumab administration led to rapid, sustained IL-19 decreases to normal levels, with decreases at 2-weeks correlating with PASI improvement at 16-weeks. IL-19 increased upon ixekizumab withdraw, prior to relapse, and decreased following re-treatment. In baricitinib- and etanercept-treated psoriasis patients, IL-19 decreases also correlated with improvement. Many patients with limited skin disease, including genital psoriasis and psoriatic arthritis patients, also had increased IL-19, which was reduced to normal levels upon ixekizumab treatment, correlating with PASI improvement. We also measured IL-19 in baricitinib-treated atopic dermatitis patients. In atopic dermatitis, IL-19 was significantly elevated, correlated with EASI scores, and decreased with skin improvement. Therefore, measurement of serum IL-19 provides clinicians with an objective disease-activity assessment tool for psoriasis and atopic dermatitis patients.

Genome-Wide Mapping and Interrogation of the Nmp4 Antianabolic Bone Axis.

PTH is an osteoanabolic for treating osteoporosis but its potency wanes. Disabling the transcription factor nuclear matrix protein 4 (Nmp4) in healthy, ovary-intact mice enhances bone response to PTH and bone morphogenetic protein 2 and protects from unloading-induced osteopenia. These Nmp4(-/-) mice exhibit expanded bone marrow populations of osteoprogenitors and supporting CD8(+) T cells. To determine whether the Nmp4(-/-) phenotype persists in an osteoporosis model we compared PTH response in ovariectomized (ovx) wild-type (WT) and Nmp4(-/-) mice. To identify potential Nmp4 target genes, we performed bioinformatic/pathway profiling on Nmp4 chromatin immunoprecipitation sequencing (ChIP-seq) data. Mice (12 w) were ovx or sham operated 4 weeks before the initiation of PTH therapy. Skeletal phenotype analysis included microcomputed tomography, histomorphometry, serum profiles, fluorescence-activated cell sorting and the growth/mineralization of cultured WT and Nmp4(-/-) bone marrow mesenchymal stem progenitor cells (MSPCs). ChIP-seq data were derived using MC3T3-E1 preosteoblasts, murine embryonic stem cells, and 2 blood cell lines. Ovx Nmp4(-/-) mice exhibited an improved response to PTH coupled with elevated numbers of osteoprogenitors and CD8(+) T cells, but were not protected from ovx-induced bone loss. Cultured Nmp4(-/-) MSPCs displayed enhanced proliferation and accelerated mineralization. ChIP-seq/gene ontology analyses identified target genes likely under Nmp4 control as enriched for negative regulators of biosynthetic processes. Interrogation of mRNA transcripts in nondifferentiating and osteogenic differentiating WT and Nmp4(-/-) MSPCs was performed on 90 Nmp4 target genes and differentiation markers. These data suggest that Nmp4 suppresses bone anabolism, in part, by regulating IGF-binding protein expression. Changes in Nmp4 status may lead to improvements in osteoprogenitor response to therapeutic cues.

TGFß-Mediated induction of SphK1 as a potential determinant in human MDA-MB-231 breast cancer cell bone metastasis.

Mechanistic understanding of the preferential homing of circulating tumor cells to bone and their perturbation on bone metabolism within the tumor-bone microenvironment remains poorly understood. Alteration in both transforming growth factor ß (TGFß) signaling and sphingolipid metabolism results in the promotion of tumor growth and metastasis. Previous studies using MDA-MB-231 human breast cancer-derived cell lines of variable metastatic potential were queried for changes in sphingolipid metabolism genes to explore correlations between TGFß dependence and bone metastatic behavior. Of these genes, only sphingosine kinase-1 (SPHK1) was identified to be significantly increased following TGFß treatment. Induction of SPHK1 expression correlated to the degree of metastatic capacity in these MDA-MB-231-derived cell lines. We demonstrate that TGFß mediates the regulation of SPHK1 gene expression, protein kinase activity and is critical to MDA-MB-231 cell viability. Furthermore, a bioinformatic analysis of human breast cancer gene expression supports SPHK1 as a hallmark TGFß target gene that also bears the genetic fingerprint of the basal-like/triple-negative breast cancer molecular subtype. These data suggest a potential new signaling axis between TGFß/SphK1 that may have a role in the development, prognosis or the clinical phenotype associated with tumor-bone metastasis.

Differential flux of macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant from the lung after intrapulmonary delivery.

Previously we showed that cytokine-induced neutrophil chemoattractant (CINC), but not macrophage inflammatory protein-2 (MIP-2), is detected in plasma after intratracheal challenge with LPS or the particular chemokines. To further understand the differences between CINC and MIP-2 flux from the lung, we attempted to detect the two chemokines in isolated erythrocytes and leukocytes in rats after intratracheal LPS challenge. In response to intratracheal LPS, we found both CINC and MIP-2 in isolated erythrocytes and leukocytes, suggesting that MIP-2 produced in the LPS-challenged lung entered the circulation like CINC. To assess the relative flux of CINC and MIP-2 from the intra-alveolar compartment into the blood, experiments were performed in rats implanted with vascular catheters in which both chemokines were either injected intratracheally (5 µg) or infused intravenously (20 ng/min) and subsequently measured in plasma or with the cellular elements. Both chemokines appeared in the blood following intratracheal injection, with CINC detected in plasma and cells but MIP-2 only detected in the cellular fraction of blood. Infusion of both chemokines allowed detection of MIP-2 and CINC in plasma and with the cellular elements, which allowed us to calculate clearance for each chemokine and to assess CINC and MIP-2 rates of appearance (Ra) following intratracheal injection. On the basis of plasma and whole blood clearance, CINC Ra was more than sevenfold and fourfold higher, respectively, than MIP-2 Ra. This analysis indicates that differences exist in the rate of flux of CINC and MIP-2 across the epithelial/endothelial barrier of the lung, despite similar molecular size.

The SISTA pilot project: understanding the training and technical assistance needs of community-based organizations implementing HIV prevention interventions for African American women--implications for a capacity building strategy.

The disproportionate rates of HIV/AIDS among African American women in the U.S. signify the ongoing need for targeted HIV prevention interventions. Additionally, building the capacity of service providers to sustain prevention efforts is a major concern. The Centers for Disease Control and Prevention (CDC) conducted a pilot project to disseminate the Sisters Informing Sisters about Topics on AIDS (SISTA), an HIV prevention intervention designed for African American women. The project was to inform the diffusion process and examine the training and technical assistance needs of participating community-based organizations. Results demonstrated a need for extensive pre-planning and skills-building prior to implementation.