Flow cytometry and pharmacokinetics.

Multiparametric flow cytometry is a powerful cellular analysis tool used in various stages of drug development. In adoptive cell therapies, the flow cytometry methods are used for the evaluation of advanced cellular products during manufacturing and to monitor cellular kinetics after infusion. In this report, we discussed the bioanalytical method development challenges to monitor cellular kinetics in CAR-T cell therapies.

Critical reagents in flow cytometry, instrumentation and application in drug discovery development.

Flow cytometer is a powerful cellular analysis tool consists of three main components; fluidics, optics and electronics. Flow cytometry methods have been used in all stages of drug development as like ligand binding assays (LBA). Both LBA and flow cytometry methods require specific interaction between the critical reagents and the analytes.

Evaluation of sample stability for cellular kinetics and pharmacodynamic flow cytometry methods.

We evaluated the sample stability for a cellular kinetics and a pharmacodynamic flow cytometry methods. First, the blood collection tubes were compared for the enumeration of chimeric antigen receptor-T cells in human whole blood. Blood samples with chimeric antigen receptor-T cells were stable up to 3 days at room temperature in both conventional EDTA and Cyto-Chex blood collection tubes (Streck Laboratories, NE, USA), but with better consistency in Cyto-Chex-BCT than conventional EDTA tubes.

12th GCC Closed Forum: critical reagents; oligonucleotides; CoA; method transfer; HRMS; flow cytometry; regulatory findings; stability and immunogenicity.

The 12th GCC Closed Forum was held in Philadelphia, PA, USA, on 9 April 2018. Representatives from international bioanalytical Contract Research Organizations were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at the meeting included: critical reagents; oligonucleotides; certificates of analysis; method transfer; high resolution mass spectrometry; flow cytometry; recent regulatory findings and case studies involving stability and nonclinical immunogenicity.

Recommendations for classification of commercial LBA kits for biomarkers in drug development from the GCC for bioanalysis.

Over the last decade, the use of biomarker data has become integral to drug development. Biomarkers are not only utilized for internal decision-making by sponsors; they are increasingly utilized to make critical decisions for drug safety and efficacy. As the regulatory agencies are routinely making decisions based on biomarker data, there has been significant scrutiny on the validation of biomarker methods.

Implementation of highly sophisticated flow cytometry assays in multicenter clinical studies: considerations and guidance.

Flow cytometry is increasingly becoming an important technology for biomarkers used in drug discovery and development. Within clinical development flow cytometry is used for the determination of PD biomarkers, disease or efficacy biomarkers or patient stratification biomarkers. Significant differences exist between flow cytometry methodology and other widely used technologies measuring soluble biomarkers including ligand binding and mass spectrometry.

Hypoestoxide, a natural nonmutagenic diterpenoid with antiangiogenic and antitumor activity: possible mechanisms of action.

We have shown previously that hypoestoxide (HE), a natural diterpenoid [a bicyclo (9, 3, 1) pentadecane], is a potent nonsteroidal anti-inflammatory drug. In this report, we demonstrate that HE also inhibits the growth of a variety of human and murine tumor cell lines in vitro at concentrations ranging from 0.3 to 10 microM and was inactive as a mutagen in the Ames test.

Hypoestoxide, a novel anti-inflammatory natural diterpene, inhibits the activity of IkappaB kinase.

Most inflammatory agents activate nuclear factor-kappaB (NF-kappaB), resulting in induction of genes coding for cytokines, chemokines, and enzymes involved in amplification and perpetuation of inflammation. Hypoestoxide (a bicyclo [9,3,1] pentadecane) is a diterpene from Hypoestes rosea, a tropical shrub in the family Acanthacea, several members of which are used in folk medicine in Nigeria. Here, we demonstrate that hypoestoxide (HE) abrogates the production of pro-inflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) in lipopolysaccharide (LPS)-activated normal human peripheral blood mononuclear cells.

Differential control of autoantibodies and lymphoproliferation by Fas ligand expression on CD4+ and CD8+ T cells in vivo.

We have previously shown that the gld autoimmune syndrome is suppressed in lethally irradiated gld mice reconstituted with a mixture of normal and gld bone marrow (BM). Furthermore, in vivo depletion of normal Thy-1+ cells restores lymphoproliferation and autoantibody production in such chimeras, suggesting that T cells bearing Fas ligand are responsible for correcting the gld defect. In this study, mixed-BM chimeras lacking either normal CD4+ (B6CD4KO-B6gld) or normal CD8+ T cells (B6CD8KO-B6gld) were generated to determine the contribution of the normal T cell subsets to disease suppression.

The role of environmental antigens in the spontaneous development of autoimmunity in MRL-lpr mice.

It has been proposed that the "normal" stimulation of the immune system that occurs from interactions with environmental stimuli, whether infectious or dietary, is necessary for the initiation and/or continuation of autoimmunity. We tested this hypothesis by deriving a group of MRL-lpr mice into a germfree (GF) environment. At 5 mo of age, no differences between GF and conventional MRL-lpr mice were noted in lymphoproliferation, flow cytometric analysis of lymph node cells (LN), or histologic analysis of the kidneys.

Concentrations of circulating beta-chemokines do not correlate with viral load in human immunodeficiency virus-infected individuals.

The CC or beta-chemokines MIP-1alpha, MIP-1beta, and RANTES are the primary components of human immunodeficiency virus type 1 (HIV-1)-suppressive soluble factors in vitro. We studied the relationship between the concentrations of MIP-1alpha, MIP-1beta, and RANTES in plasma and HIV viral load in HIV-infected subjects. The HIV-positive patient group (n = 140) had significantly lower concentrations of all three beta-chemokines (MIP-1alpha, P < 0.

Association of low concentrations of serum mannose-binding protein with recurrent infections in adults.

Low concentrations of mannose-binding protein (MBP; also known as mannose-binding lectin) are associated with common opsonic defect in immunodeficient children. We compared the concentrations of MBP in the sera of 47 adults with non-human immunodeficiency virus-related recurrent infections (group I) and 50 healthy adult controls. Mean serum MBP concentrations in the patient group did not differ significantly from those in the control group (P < 0.

Novel immunoregulatory B cell pathways revealed by lpr-+ mixed chimeras.

lpr, a murine mutation of the Fas apoptosis receptor, causes lymphadenopathy and autoantibody production, with lymphadenopathy primarily due to a population of CD4-CD8-B220+ T cells. Previous in vivo experiments, in which lpr and normal bone marrow cells were coinfused into lpr hosts, have demonstrated that only T cells of lpr origin accumulated abnormally and only B cells of lpr origin produced autoantibodies. Moreover, in these chimeras, B cells of normal origin were unable to respond to conventional, T cell-dependent exogenous Ag.

B cell genotype determines the fine specificity of autoantibody in lpr mice.

Anti-Sm Abs are specific markers of human systemic lupus erythematosus (SLE) and of murine models of this disease. In humans, anti-Sm Abs are mostly IgG1, and in MRL/lpr mice, IgG2a; both are T-dependent isotypes. Other lpr strains, such as B6/lpr, do not produce anti-Sm Ab spontaneously.

Suppression and reversal of gld disease by parabiosis with normal mice.

The disruption of the Fas receptor or Fas ligand by the lpr or gld mutations, respectively, results in severe autoimmune and lymphoproliferative disease due to the failure of Fas-mediated deletion of self-reactive lymphocytes. Recently, we have shown in mixed chimeras that gld-induced autoimmunity could be corrected by normal bone marrow, in particular by normal T cells. In contrast, lpr-mediated autoimmunity could not be influenced by normal bone marrow-derived cells.

The abnormal lpr double-negative T cell fails to proliferate in vivo.

Mice homozygous for the autosomal recessive gene lpr develop marked lymphadenopathy and a systemic autoimmune disease resembling human systemic lupus erythematosus. The enlarged nodes are dominated by T cells with an unusual surface phenotype: dull Thy-1+, dull CD3+, CD4-, CD8-, B220+ (double-negative T cells or DNTs). Despite their massive accumulation in vivo, these cells fail to proliferate in response to conventional T-cell mitogens in vitro.

Co-infusion of normal bone marrow partially corrects the gld T-cell defect. Evidence for an intrinsic and extrinsic role for Fas ligand.

Ipr and gld mice develop systemic autoimmune diseases with nearly indistinguishable manifestations, including the accumulation of massive numbers of CD4-CD8- T lymphocytes. In vivo chimera experiments have shown that the Ipr mutation is functionally expressed in both T and B cells. When lethally irradiated Ipr mice were given a combination of normal and Ipr bone marrow, only Ipr-derived B cells produced autoantibodies and only Ipr-derived T cells hyperproliferated.

In vivo depletion of Thy-1-positive cells originating from normal bone marrow abrogates the suppression of gld disease in normal-gld mixed bone marrow chimeras.

Mice homozygous for gld develop an autoimmune syndrome characterized by hypergammaglobulinemia, massive accumulation of abnormal T cells and the production of autoantibodies. Previous studies in our laboratory have shown that reconstitution of lethally irradiated B6/gld recipients with a mixture of normal and gld bone marrow (BM) suppresses the gld-induced syndrome. In this report we extend this observation by demonstrating that the depletion of normal Thy-1+ cells, but not normal B cells, restores gld disease in mixed BM chimeras congenic for Thy-1 and IgH alleles.

T-B collaboration for autoantibody production in lpr mice is cognate and MHC-restricted.

A central question in autoimmunity is the mechanism of T cell help for autoantibody production. For responses to exogenous Ag, T-B collaboration is restricted by MHC class II molecules. To determine whether T cell help that leads to autoantibodies in murine SLE is also MHC-restricted, we have constructed bone marrow chimeras with Ig heavy chain (lgh) allotype- and I-A-congenic donor B6/lpr mice and I-A-congenic recipients.

Selection of the T cell receptor repertoire in Lpr mice.

The development of double-negative (CD4-, CD8-) T cells and other T cells subsets in lymphoproliferation (lpr) mice continues to be poorly defined. Recent studies indicate that lpr is a mutation of a receptor mediating apoptosis. It has thus been hypothesized that T cell development in the thymus should be abnormally affected.

Correction of gld autoimmunity by co-infusion of normal bone marrow suggests that gld is a mutation of the Fas ligand gene.

lpr and gld mice develop phenotypically indistinguishable systemic autoimmune diseases and marked lymphadenopathy dominated by CD4-CD8- T cells. In vivo chimera experiments have demonstrated that both lpr T and lpr B cells are intrinsically defective. Analogous experiments were conducted using gld mice.

Murine lymphocytes exhibit heterogeneity in their proliferative responsiveness to calcium ionophore.

The calcium ionophore, A23187, when used alone was found to induce proliferation of murine T cells, at concentrations of 0.5-1 mM. This response required the presence of syngeneic splenic adherant cells (SAC) as a source of accessory cells.