Intra-donor Variability Research Articles

Inter- and Intra-donor variability in bone marrow–derived mesenchymal stromal cells: implications for clinical applications

Background aimsMesenchymal stromal cells (MSCs) are attractive as a therapeutic modality in multiple disease conditions characterized by inflammation and vascular compromise. Logistically they are advantageous because they can be isolated from adult tissue sources, such as bone marrow (BM). The phase 2a START clinical trial determined BM-MSCs to be safe in patients with moderate-to-severe acute respiratory distress syndrome (ARDS). Herein, we examine a subset of the clinical doses of MSCs generated for the phase 2a START trial from three unique donors (1–3), where one of the donors’ donated BM on two separate occasions (donor 3 and 3W). MethodsThe main objective of this study was to correlate properties of the cells from the four lots with plasma biomarkers from treated patients and relevant to ARDS outcomes. To do this we evaluated MSC donor lots for (i) post-thaw viability, (ii) growth kinetics, (iii) metabolism, (iv) surface marker expression, (v) protein expression, (vi) immunomodulatory ability and (vii) their functional effects on regulating endothelial cell permeability. ResultsMSC-specific marker expression and protection of thrombin-challenged endothelial barrier permeability was similar among all four donor lots. Inter and intra-donor variability was observed in all the other in vitro assays. Furthermore, patient plasma ANG-2 and protein C levels at 6 hours post-transfusion were correlated to cell viability in an inter- and intra-donor dependent manner. ConclusionsThese findings highlight the potential of donor dependent (inter-) and collection dependent (intra-) effects in patient biomarker expression.

Stability and heterogeneity in the antimicrobiota reactivity of human milk-derived immunoglobulin A.

Immunoglobulin A (IgA) is secreted into breast milk and is critical for both protecting against enteric pathogens and shaping the infant intestinal microbiota. The efficacy of breast milk-derived maternal IgA (BrmIgA) is dependent upon its specificity; however, heterogeneity in BrmIgA binding ability to the infant microbiota is not known. Using a flow cytometric array, we analyzed the reactivity of BrmIgA against bacteria common to the infant microbiota and discovered substantial heterogeneity between all donors, independent of preterm or term delivery. Surprisingly, we also observed intradonor variability in the BrmIgA response to closely related bacterial isolates. Conversely, longitudinal analysis showed that the antibacterial BrmIgA reactivity was relatively stable through time, even between sequential infants, indicating that mammary gland IgA responses are durable. Together, our study demonstrates that the antibacterial BrmIgA reactivity displays interindividual heterogeneity but intraindividual stability. These findings have important implications for how breast milk shapes the development of the preterm infant microbiota and protects against necrotizing enterocolitis.

Impact of Inter- and Intra-Donor Variability by Age on the Gel-to-Tissue Transition in MMP-Sensitive PEG Hydrogels for Cartilage Regeneration.

Matrix metalloproteinase (MMP)-sensitive hydrogels are promising for cartilage tissue engineering due to cell-mediated control over hydrogel degradation. However, any variability in MMP, tissue inhibitors of matrix metalloproteinase (TIMP), and/or extracellular matrix (ECM) production among donors will impact neotissue formation in the hydrogels. The goal for this study was to investigate the impact of inter- and intra-donor variability on the hydrogel-to-tissue transition. Transforming growth factor β3 was tethered into the hydrogel to maintain the chondrogenic phenotype and support neocartilage production, allowing the use of chemically defined medium. Bovine chondrocytes were isolated from two donor groups, skeletally immature juvenile and skeletally mature adult donors (inter-donor variability) and three donors within each group (intra-donor group variability). While the hydrogel supported neocartilaginous growth by all donors, donor age impacted MMP, TIMP, and ECM synthesis rates. Of the MMPs and TIMPs studied, MMP-1 and TIMP-1 were the most abundantly produced by all donors. Adult chondrocytes secreted higher levels of MMPs, which was accompanied by higher production of TIMPs. Juvenile chondrocytes exhibited more rapid ECM growth. By day 29, juvenile chondrocytes had surpassed the gel-to-tissue transition. On the contrary, the adult donors had a percolated polymer network indicating that despite higher levels of MMPs the gel-to-transition had not yet been achieved. The intra-donor group variability of MMP, TIMP, and ECM production was higher in adult chondrocytes but did not impact the extent of the gel-to-tissue transition. In summary, age-dependent inter-donor variations in MMPs and TIMPs significantly impact the timing of the gel-to-tissue transition in MMP-sensitive hydrogels.

Potential to Use Fingerprints for Monitoring Therapeutic Levels of Isoniazid and Treatment Adherence

A fingerprint offers a convenient, noninvasive sampling matrix for monitoring therapeutic drug use. However, a barrier to widespread adoption of fingerprint sampling is the fact that the sample volume is uncontrolled. Fingerprint samples (n = 140) were collected from patients receiving the antibiotic isoniazid as part of their treatment, as well as from a drug-naive control group (n = 50). The fingerprint samples were analyzed for isoniazid (INH) and acetylisoniazid (AcINH), using liquid chromatography high-resolution mass spectrometry. The data set was analyzed retrospectively for metabolites known to be present in eccrine sweat. INH or AcINH was detected in 89% of the fingerprints collected from patients and in 0% of the fingerprints collected from the control group. Metabolites lysine, ornithine, pyroglutamic acid, and taurine were concurrently detected alongside INH/AcINH and were used to determine whether the fingerprint sample was sufficient for testing. Given a sufficient sample volume, the fingerprint test for INH use has sensitivity, specificity, and accuracy of 100%. Normalization to taurine was found to reduce intradonor variability. Fingerprints are a novel and noninvasive approach to monitor INH therapy. Metabolites can be used as internal markers to demonstrate a sufficient sample volume for testing and reduce intradonor variability.

Integrating CRISPR Engineering and hiPSC-Derived 2D Disease Modeling Systems.

Human induced pluripotent stem cells (hiPSCs) have revolutionized research on human diseases, particularly neurodegenerative and psychiatric disorders, making it possible to study mechanisms of disease risk and initiation in otherwise inaccessible patient-specific cells. Today, the integration of CRISPR engineering approaches with hiPSC-based models permits precise isogenic comparisons of human neurons and glia. This review is intended as a guideline for neuroscientists and clinicians interested in translating their research to hiPSC-based studies. It offers state-of-the-art approaches to tackling the challenges that are unique to human in vitro disease models, particularly interdonor and intradonor variability, and limitations in neuronal maturity and circuit complexity. Finally, we provide a detailed overview of the immense possibilities the field has to offer, highlighting efficient neural differentiation and induction strategies for the major brain cell types and providing perspective into integrating CRISPR-based methods into study design. The combination of hiPSC-based disease modeling, CRISPR technology, and high-throughput approaches promises to advance our scientific knowledge and accelerate progress in drug discovery.Dual Perspectives Companion Paper: Studying Human Neurodevelopment and Diseases Using 3D Brain Organoids, by Ai Tian, Julien Muffat, and Yun Li.

Characterization of a novel role for the dynamin mechanoenzymes in the regulation of human sperm acrosomal exocytosis.

Does dynamin regulate human sperm acrosomal exocytosis? Our studies of dynamin localization and function have implicated this family of mechanoenzymes in the regulation of progesterone-induced acrosomal exocytosis in human spermatozoa. Completion of an acrosome reaction is a prerequisite for successful fertilization in all studied mammalian species. It follows that failure to complete this unique exocytotic event represents a common aetiology in the defective spermatozoa of male infertility patients that have failed IVF in a clinical setting. Recent studies have implicated the dynamin family of mechanoenzymes as important regulators of the acrosome reaction in murine spermatozoa. The biological basis of this activity appears to rest with the ability of dynamin to polymerize around newly formed membrane vesicles and subsequently regulate the rate of fusion pore expansion. To date, however, the dynamin family of GTPases have not been studied in the spermatozoa of non-rodent species. Here, we have sought to examine the presence and functional significance of dynamin in human spermatozoa. Dynamin expression was characterized in the testis and spermatozoa of several healthy normozoospermic individuals. In addition, we assessed the influence of selective dynamin inhibition on the competence of human spermatozoa to undergo a progesterone-induced acrosome reaction. A minimum of five biological and technical replicates were performed to investigate both inter- and intra-donor variability in dynamin expression and establish statistical significance in terms of the impact of dynamin inhibition. The expression and the localization of dynamin in the human testis, epididymis and mature spermatozoa were determined through the application of immunofluorescence, immunoblotting and/or electron microscopy. Human semen samples were fractionated via density gradient centrifugation and the resultant populations of good and poor quality spermatozoa were induced to capacitate and acrosome react in the presence or absence of selective dynamin inhibitors. The acrosome integrity of live spermatozoa was subsequently assessed via the use of fluorescently conjugated Arachis hypogea lectin (PNA). The influence of dynamin phosphorylation and the regulatory kinase(s) responsible for this modification in human spermatozoa were also assessed via the use of in situ proximity ligation assays and pharmacological inhibition. In all experiments, ≥100 spermatozoa were assessed/treatment group and all graphical data are presented as the mean values ± SEM, with statistical significance being determined by ANOVA. Dynamin 1 (DNM1) and DNM2, but not DNM3, were specifically localized to the acrosomal region of the head of human spermatozoa, an ideal position from which to regulate acrosomal exocytosis. In keeping with this notion, pharmacological inhibition of DNM1 and DNM2 was able to significantly suppress the rates of acrosomal exocytosis stimulated by progesterone. Furthermore, our comparison of dynamin expression in good and poor quality spermatozoa recovered from the same ejaculate, revealed a significant reduction in the amount of DNM2 in the latter subpopulation of cells. In contrast, DNM1 was detected at equivalent levels in both subpopulations of spermatozoa. Such findings are of potential significance given that the poor quality spermatozoa proved refractory to the induction of a progesterone stimulated acrosome reaction. In seeking to identify the regulatory influence of progesterone on DNM2 function, we were able to establish that the protein is a substrate for CDK1-dependent phosphorylation. The functional significance of DNM2 phosphorylation was illustrated by the fact that pharmacological inhibition of CDK1 elicited a concomitant suppression of both DNM2-Ser764 phosphorylation and the overall rates of progesterone-induced acrosomal exocytosis. N/A. This was an in vitro study performed mainly on ejaculated human spermatozoa. This experimental paradigm necessarily eliminates the physiological contributions of the female reproductive tract that would normally support capacitation and acrosomal responsiveness. This study identifies a novel causative link between dynamin activity and the ability of human spermatozoa to complete a progesterone-induced acrosome reaction. Such findings encourage a more detailed analysis of the contribution of dynamin dysregulation as an underlying aetiology in infertile males whose spermatozoa are unable to penetrate the zona pellucida. This research was supported by a National Health and Medical Research Council of Australia Project Grant (APP1103176) awarded to B.N. and E.A.M. The authors report no conflict of interest.

Variability in gut microbiota response to an inulin-type fructan prebiotic within an in vitro three-stage continuous colonic model system

Gut microbiota have a major influence on human health and disease. Dietary interventions have been shown to beneficially modulate the gut microbiota and improve health; however, it appears there is large inter-individual variability in gut microbiota responsiveness to dietary interventions. We aimed to determine whether different fermentable carbohydrate content media, mimicking Western and Prudent style dietary patterns, influence gut microbiota response to an inulin-type fructan prebiotic using an in vitro three-stage continuous colonic model system. We demonstrated that the addition of an inulin-type fructan prebiotic led to shifts in the organic acid concentrations and bacterial taxa in both the low (LF) and high fermentable carbohydrate medium (HF) gut models. The shifts that occurred after the addition of the prebiotic significantly differed between the LF and HF gut models. Acetate increased in the HF gut models but decreased in the LF gut models (p=0.021). The increases that occurred in Firmicutes (p=0.026), Lactobacillus (p=0.045) and Mitsuokella (p=0.012) were significantly greater in the HF gut models. Megasphaera (p=0.033) and an unknown genus of Enterobacteriaceae (p=0.011) decreased in the LF gut models but increased in the HF gut models. The reduction in an unknown genus of Lachnospiraceae (other) was significantly greater in LF gut models (p=0.040). Additionally, large inter- and intra-donor variability in gut microbiota responsiveness to the prebiotic were demonstrated. This study demonstrates that media with different fermentable carbohydrate contents caused variability in gut microbiota responsiveness to an inulin-type fructan prebiotic; however, these results will need to be replicated in an in vivo study.

CD11d β2 integrin expression on human NK, B, and γδ T cells.

The CD11d integrin is expressed on the cell surface of leukocytes that belong to the myeloid lineage, but its expression on lymphocytes remains unexplored. To test the hypothesis that CD11d is expressed on lymphocyte subsets, we employed a multicolor flow cytometry panel to identify CD11d expression on B, NK, CD4+ and CD8+ αβ T cells (αβTc), and γδ T cells (γδTc) in human PBMC samples. CD11d was highly expressed on NK cells, B cells, and γδTc, but not αβTc. CD11d expression was higher on freshly isolated γδTc compared with αβTc from healthy donors, yet both inter- and intradonor variability was evident. Over time in primary culture, we consistently observed higher CD11d levels on γδTc compared with αβTc from the same donor. Furthermore, CD11d expression on γδTc increased over time and correlated with levels of IL-2 supplementation. Of interest, a greater percentage of Vδ1 γδTc expressed CD11d than did Vδ2 γδTc, which suggested differential roles for this integrin that may segregate with γδTc subsets. These results expand the potential for CD11d to regulate lymphocyte migration and tissue retention, and illuminate the possibility of a previously unconsidered role for CD11d in leukocyte biology and disease.

Shear deformations of human articular cartilage: Certain mechanical anisotropies apparent at large but not small shear strains

Articular cartilage has pronounced through-the-thickness heterogeneity in both ultrastructure and mechanical function. The tissue undergoes a combination of large deformations in vivo, where shear is critical in both failure and chondrocyte death. Yet the microstructure mechanical response of cartilage to multi-axial large shear deformations is unknown. We harvested a total of 42 cartilage specimens from seven matched locations across the lateral femoral condyles and patellofemoral grooves of six human male donors (30.2±8.8yrs old, M±SD). With each specimen we applied a range of quasi-static, multi-axial large (simple) shear displacements both parallel and perpendicular to the local split-line direction (SLD). Shear stresses in cartilage specimens from the patellofemoral grooves were higher, and more energy was dissipated, at all applied strains under loading parallel to the local SLD versus perpendicular, while specimens from the lateral condyles were mechanically anisotropic only under larger strains of 20% and 25%. Cartilage also showed significant intra-donor variability at larger shear strains but no significant inter-donor variability. Overall, shear strain-energy dissipation was almost constant at 5% applied shear strain and increased nonlinearly with increasing shear magnitude. Our results suggest that full understanding of cartilage mechanics requires large-strain analyses to account for nonlinear, anisotropic and location-dependent effects not fully realized at small strains.

Paternal Age and Numerical Chromosome Abnormalities in Human Spermatozoa

This study explores the relationship between numerical chromosome abnormalities in sperm and age in healthy men. We performed FISH in the spermatozoa of 10 donors from the general population: 5 men younger than 40 years of age and 5 fertile men older than 60 years of age. For each chromosome, 1,000 sperm nuclei were analyzed, with a total of 15,000 sperm nuclei for each donor. We used a single sperm sample per donor, thus minimizing intra-donor variability and optimizing consistent analysis. FISH with a TelVysion assay, which provides data on aneuploidy of 19 chromosomes, was used in order to gain a more genome-wide perspective of the level of aneuploidy. Aneuploidy and diploidy rates observed in the younger and older groups were compared. There were no significant differences in the incidence of autosomal disomy, sex chromosome disomy, total chromosome disomy, diploidy, nor total numerical abnormalities between younger and older men. This work confirms that aneuploidy of the sex chromosomes is more common than that of autosomes and that this does not change with age. Our results suggest that some probe combinations have a tendency to indicate higher levels of diploidy, thus potentially affecting FISH results and highlighting the limitations of FISH.

Nondestructive Techniques to Evaluate the Characteristics and Development of Engineered Cartilage.

In this review, methods for evaluating the properties of tissue engineered (TE) cartilage are described. Many of these have been developed for evaluating properties of native and osteoarthritic articular cartilage. However, with the increasing interest in engineering cartilage, specialized methods are needed for nondestructive evaluation of tissue while it is developing and after it is implanted. Such methods are needed, in part, due to the large inter- and intra-donor variability in the performance of the cellular component of the tissue, which remains a barrier to delivering reliable TE cartilage for implantation. Using conventional destructive tests, such variability makes it near-impossible to predict the timing and outcome of the tissue engineering process at the level of a specific piece of engineered tissue and also makes it difficult to assess the impact of changing tissue engineering regimens. While it is clear that the true test of engineered cartilage is its performance after it is implanted, correlation of pre and post implantation properties determined non-destructively in vitro and/or in vivo with performance should lead to predictive methods to improve quality-control and to minimize the chances of implanting inferior tissue.

An improved cartilage digestion method for research and clinical applications.

Enzymatic isolation of chondrocytes from a cartilage biopsy is the first step to establish in vitro models of chondrogenesis or to generate cell-based grafts for cartilage repair. Such process is based on manually operated procedures and typically results in yields lower than 20% of the total available cells. In this study, we hypothesized that, as compared to conventionally used protocols, the enzymatic digestion of human articular cartilage in the presence of ascorbic acid 2-phosphate (AscA2P) or of sodium chloride (NaCl), in combination with the use of a perfusion bioreactor system, leads to a higher and more reproducible yield of cell populations with high proliferation and chondrogenic capacity. The addition of AscA2P within the enzymatic digestion medium did not significantly increase the cell yield, but resulted in a significant decrease of the intradonor variability in cell yield (-17.8% ± 10.7%, p = 0.0247) and in a significant increase of the proliferation rate of the isolated chondrocytes (+19.0% ± 1.4%, p < 0.05) with respect to the control group. The addition of NaCl during cartilage digestion did not modulate cell yield. When the cartilage digestion was further performed under direct perfusion flow, beneficial synergistic effects were achieved, with an overall increase of 34.7% ± 6.8% (p < 0.001) in the cell yield and an average decrease of 57.8% ± 11.2% (p < 0.01) in the coefficient of variation with respect to the control group. Importantly, by implementing this strategy it was possible to retrieve clonal subpopulations more efficiently capable of undergoing chondrogenesis, both in vitro and in vivo. Our findings bear relevance for the preparation of human chondrocytes for laboratory investigations, and in the perspective of efficient and streamlined manufacturing of cell/tissue grafts for articular cartilage repair.

Similar donors—similar blood?

Red blood cell (RBC) storage lesions have been suggested as contributing factors to suboptimal clinical outcomes. While undesirable effects of storage are well documented, their clinical relevance is still debated. Focus on storage time as the sole determinant of RBC quality ignores the variability in cell properties that may depend on factors other than age. Mechanical fragility (MF) aggregately reflects many storage-related functional and structural changes. This study evaluates interdonor versus intradonor variability, throughout storage, of both MF and autohemolysis (AH). Thirteen uniformly manufactured RBC units were collected initially as whole blood from nonsmoking, group A+, male Caucasian research donors. Mechanical stress was applied using a bead mill with oscillation at 50 Hz over durations varying from 0.5 to 60 minutes. MF profiles were described in terms of percent hemolysis after stresses of specified durations. Two months later, 11 of the 13 donors returned and assays were performed using the same protocol to allow comparison of intradonor versus interdonor variation. At 5 days postcollection, RBC MF profiles exhibited marked interdonor variability (up to twofold) overall. Both autolysis and MF across all units increased during storage-with rates of these increases varying by up to 10-fold for certain MF variables. Especially high AH and MF were observed for an outlier donor (with p < 0.05), for whom follow-up revealed previously undisclosed hereditary hypertriglyceridemia (levels exceeding approx. 1000 mg/dL). RBCs, even from similar donors, vary significantly in levels and changes of both AH and MF, the clinical significance of which must still be ascertained. While further study is needed, donors with severe hypertriglyceridemia may not be appropriate as blood donors due to the unacceptable level of hemolysis observed during storage of our affected study subject.

Age-related changes in human peripapillary scleral strain

To test the hypothesis that mechanical strain in the posterior human sclera is altered with age, 20 pairs of normal eyes from human donors aged 20 to 90 years old were inflation tested within 48-h postmortem. The intact posterior scleral shells were pressurized from 5 to 45 mmHg, while the full-field three-dimensional displacements of the scleral surface were measured using laser speckle interferometry. The full strain tensor of the outer scleral surface was calculated directly from the displacement field. Mean maximum principal (tensile) strain was computed for eight circumferential sectors (45° wide) within the peripapillary and mid-peripheral regions surrounding the optic nerve head (ONH). To estimate the age-related changes in scleral strain, results were fit using a functional mixed effects model that accounts for intradonor variability and spatial autocorrelation. Mechanical tensile strain in the peripapillary sclera is significantly higher than the strain in the sclera farther away from the ONH. Overall, strains in the peripapillary sclera decrease significantly with age. Sectorially, peripapillary scleral tensile strains in the nasal sectors are significantly higher than the temporal sectors at younger ages, but the sectorial strain pattern reverses with age, and the temporal sectors exhibited the highest tensile strains in the elderly. Overall, peripapillary scleral structural stiffness increases significantly with age. The sectorial pattern of peripapillary scleral strain reverses with age, which may predispose adjacent regions of the lamina cribrosa to biomechanical insult. The pattern and age-related changes in sectorial peripapillary scleral strain closely match those seen in disk hemorrhages and neuroretinal rim area measurement change rates reported in previous studies of normal human subjects.

Multimodal evaluation of tissue-engineered cartilage.

Tissue engineering (TE) has promise as a biological solution and a disease modifying treatment for arthritis. Although cartilage can be generated by TE, substantial inter- and intra-donor variability makes it impossible to guarantee optimal, reproducible results. TE cartilage must be able to perform the functions of native tissue, thus mechanical and biological properties approaching those of native cartilage are likely a pre-requisite for successful implantation. A quality-control assessment of these properties should be part of the implantation release criteria for TE cartilage. Release criteria should certify that selected tissue properties have reached certain target ranges, and should be predictive of the likelihood of success of an implant in vivo. Unfortunately, it is not currently known which properties are needed to establish release criteria, nor how close one has to be to the properties of native cartilage to achieve success. Achieving properties approaching those of native cartilage requires a clear understanding of the target properties and reproducible assessment methodology. Here, we review several main aspects of quality control as it applies to TE cartilage. This includes a look at known mechanical and biological properties of native cartilage, which should be the target in engineered tissues. We also present an overview of the state of the art of tissue assessment, focusing on native articular and TE cartilage. Finally, we review the arguments for developing and validating non-destructive testing methods for assessing TE products.

Efficient Lentiviral Transduction of Human Mesenchymal Stem Cells That Preserves Proliferation and Differentiation Capabilities

Long-term lentiviral transduction of human mesenchymal stem cells (hMSCs) greatly enhances the usefulness of these cells. However, such transduction currently requires the use of polybrene, which severely inhibits hMSC proliferation. In contrast, protamine sulfate at 100 μg/ml doubled transduction efficiencies without affecting proliferation or differentiation potential. Expression levels improved 2.2-fold with the addition of a woodchuck hepatitis post-transcriptional regulatory element. Further improvements in transduction efficiencies could be obtained by a modest increase in viral concentrations through increased viral titers or decreased transduction volumes without changing multiplicity of infection, by transducing over multiple days, or by culturing the cells in fibroblast growth factor-2. Centrifugation improved expression but had no effect on efficiency. Transgene expression was stable over 6 weeks in vitro and in vivo. Donor-to-donor and intradonor variability were observed in primary passage through passage 2 cultures, but not at passage 3. These results provide a better optimized approach for expanded use of hMSCs through genetic manipulation.

Chemical Characterization of Latent Fingerprints by Matrix-Assisted Laser Desorption Ionization, Time-of-Flight Secondary Ion Mass Spectrometry, Mega Electron Volt Secondary Mass Spectrometry, Gas Chromatography/Mass Spectrometry, X-ray Photoelectron Spectroscopy, and Attenuated Total Reflection Fourier Transform Infrared Spectroscopic Imaging: An Intercomparison

The first analytical intercomparison of fingerprint residue using equivalent samples of latent fingerprint residue and characterized by a suite of relevant techniques is presented. This work has never been undertaken, presumably due to the perishable nature of fingerprint residue, the lack of fingerprint standards, and the intradonor variability, which impacts sample reproducibility. For the first time, time-of-flight secondary ion mass spectrometry, high-energy secondary ion mass spectrometry, and X-ray photoelectron spectroscopy are used to target endogenous compounds in fingerprints and a method is presented for establishing their relative abundance in fingerprint residue. Comparison of the newer techniques with the more established gas chromatography/mass spectrometry and attenuated total reflection Fourier transform infrared spectroscopic imaging shows good agreement between the methods, with each method detecting repeatable differences between the donors, with the exception of matrix-assisted laser desorption ionization, for which quantitative analysis has not yet been established. We further comment on the sensitivity, selectivity, and practicability of each of the methods for use in future police casework or academic research.

Validation of a flow cytometry based G 2M delay cell cycle assay for use in evaluating the pharmacodynamic response to Aurora A inhibition

Pharmacodynamic assays are important aspects for understanding molecularly targeted anticancer agents to investigate the relationship between drug concentration (pharmacokinetics) and drug “effect” or biological activity. As new drug entities are developed that affect DNA cell cycle, a pharmacodynamic assay which measures cell cycle perturbation would be a valuable clinical trial tool. During recent years, flow cytometry has established itself as a useful method to determine the relative nuclear DNA content and percentage of cycling cells of biological specimens. However to date, the analytical validation of cytometry based assays is limited and there is no suitable guidance for method validation of flow cytometry based cell cycle assays. Here we report the validation of a flow cytometry based cell cycle G 2/M delay assay for use in evaluating the effect of investigational drug MLN8237, a small molecule inhibitor of a mitotic kinase Aurora A, for clinical trial use. The assay method was validated by examining assay robustness, repeatability, reproducibility, precision, and determining the cutoff for a true drug effect based on biostatistical analysis models. Experimental results show that the intra-assay repeatability was less than 20% with an intra-donor variability of less than 40%. The robustness of the assay was less than 30%. Since this is an ex-vivo stimulation assay, variability parameters were expected to be higher. Based on biostatistical modeling, an absolute change in %G 2M of 5.2% (95% CI) was needed in order to detect a true drug effect. Overall, the assay demonstrated acceptable variability to warrant further in vivo testing.

Expression of human FcγRIIIa as a GPI-linked molecule on CHO cells to enable measurement of human IgG binding

The efficacy of a therapeutic IgG molecule may be as dependent on the optimisation of the constant region to suit its intended indication as on the selection of its variable regions. A crucial effector function to be maximised or minimised is antibody-dependent cell-mediated cytotoxicity by natural killer cells. Traditional assays of ADCC activity suffer from considerable inter-donor and intra-donor variability, which makes the measurement of antibody binding to human FcγRIIIa, the key receptor for ADCC, an attractive alternative method of assessment. Here, we describe the development of cell lines and assays for this purpose. The transmembrane receptor, FcγRIIIa, requires co-expression with signal transducing subunits to prevent its degradation, unlike the homologous receptor FcγRIIIb that is expressed as a GPI-anchored molecule. Therefore, to simplify the production of cell lines as reliable assay components, we expressed FcγRIIIa as a GPI-anchored molecule. Separate, stable CHO cell lines that express either the 158F or the higher-affinity 158V allotype of FcγRIIIa were isolated using fluorescence-activated cell sorting. The identities of the expressed receptors were confirmed using a panel of monoclonal antibodies that distinguish between subclasses and allotypes of FcγRIII and the cell lines were shown to have slightly higher levels of receptor than FcγRIII-positive peripheral blood mononuclear cells. Because the affinity of FcγRIIIa for IgG is intermediate amongst the receptors that bind IgG, we were able to use these cell lines to develop flow cytometric assays to measure the binding of both complexed and monomeric immunoglobulin. Thus, by choosing the appropriate method, weakly- or strongly-binding IgG can be efficiently compared. We have quantified the difference in the binding of wildtype IgG1 and IgG3 molecules to the two functional allotypes of the receptor and report that the FcγRIIIa-158V–antibody interaction is 3- to 4-fold stronger that the interaction with FcγRIIIa-158F. Overall, these robust assays should be valuable for batch-testing clinical material as well as providing tools for improving the design of therapeutic IgG.

Human dendritic cell line models for DC differentiation and clinical DC vaccination studies

Dendritic cells (DC) are increasingly applied in the immunotherapy of cancer. As the development of a standardized DC vaccine product is often hampered by the limited availability of DC precursors and inter- and intra-donor variability, and the preparation of individual vaccines is labor-intensive, it would be preferable to use DC from a readily available and unlimited source, such as cell lines can provide. It has been described that leukemia-derived cell lines are able to differentiate into functional DC, creating possibilities for the development of highly reproducible DC vaccines and providing in vitro model systems for in-depth studies about DC physiology. This review discusses the different human DC cell line differentiation models described so far. Based on the available data, characteristics that determine the ability of leukemia cells to differentiate along the different precursor stages into functional DC will be formulated. In addition, evidence will be provided that the human CD34+ acute myeloid leukemia cell line MUTZ-3 provides DC that exhibit the functional properties that are crucial for the in vivo generation of CTL-mediated immunity and thus, currently, represents the most valuable, sustainable model system for myeloid DC differentiation and clinical DC vaccination studies.