Recommendations for Method Development and Validation of qPCR and dPCR Assays in Support of Cell and Gene Therapy Drug Development.

The emerging use of qPCR and dPCR in regulated bioanalysis and absence of regulatory guidance on assay validations for these platforms has resulted in discussions on lack of harmonization on assay design and appropriate acceptance criteria for these assays. Both qPCR and dPCR are extensively used to answer bioanalytical questions for novel modalities such as cell and gene therapies. Following cross-industry conversations on the lack of information and guidelines for these assays, an American Association of Pharmaceutical Scientists working group was formed to address these gaps by bringing together 37 industry experts from 24 organizations to discuss best practices to gain a better understanding in the industry and facilitate filings to health authorities.

Chemerin Reactivates PTEN and Suppresses PD-L1 in Tumor Cells via Modulation of a Novel CMKLR1-mediated Signaling Cascade.

Purpose: Chemerin (retinoic acid receptor responder 2, ) is an endogenous leukocyte chemoattractant that recruits innate immune cells through its receptor, ChemR23. is widely expressed in nonhematopoietic tissues and often downregulated across multiple tumor types compared with normal tissue. Recent studies show that augmenting chemerin in the tumor microenvironment significantly suppresses tumor growth, in part, by immune effector cells recruitment.

Chemerin Suppresses Breast Cancer Growth by Recruiting Immune Effector Cells Into the Tumor Microenvironment.

Infiltration of immune cells into the tumor microenvironment (TME) can regulate growth and survival of neoplastic cells, impacting tumorigenesis and tumor progression. Correlations between the number of effector immune cells present in a tumor and clinical outcomes in many human tumors, including breast, have been widely described. Current immunotherapies utilizing checkpoint inhibitors or co-stimulatory molecule agonists aim to activate effector immune cells.

Shear stress attenuates apoptosis due to TNFα, oxidative stress, and serum depletion via death-associated protein kinase (DAPK) expression.

Background: Misdirected apoptosis in endothelial cells participates in the development of pathological conditions such as atherosclerosis. Tight regulation of apoptosis is necessary to ensure normal cell function. The rate of cell turnover is increased at sites prone to lesion development.

The role of death-associated protein kinase (DAPK) in endothelial apoptosis under fluid shear stress.

Endothelial cells are the interface between hemodynamic fluid flow and vascular tissue contact. They actively translate physical and chemical stimuli into intracellular signaling cascades which in turn regulate cell function, and endothelial dysfunction leads to inflammation and diseased conditions. For example, atherosclerosis, a chronic vascular disease, favorably develops in regions of disturbed fluid flow and low shear stress.

Effect of shear stress and substrate on endothelial DAPK expression, caspase activity, and apoptosis.

Background: In the vasculature, misdirected apoptosis in endothelial cells leads to pathological conditions such as inflammation. Along with biochemical and molecular signals, the hemodynamic forces that the cells experience are also important regulators of endothelial functions such as proliferation and apoptosis. Laminar shear stress inhibits apoptosis induced by serum depletion, oxidative stress, and tumor necrosis factor α (TNFα).

Shear stress regulates expression of death-associated protein kinase in suppressing TNFα-induced endothelial apoptosis.

Death associated protein kinase (DAPK) is a positive regulator in tumor necrosis factor α (TNFα)-induced apoptotic pathway, and DAPK expression is lost in cancer cells. In the vasculature, misdirected apoptosis in endothelial cells leads to pathological conditions such as inflammation and physiological shear stress is protective against apoptosis. Using bovine aortic endothelial cells, we found that DAPK expression increased, while the auto-inhibitory phosphorylation of serine 308 decreased with shear stress at 12 dynes/cm(2) for 6 h.