Round-bottom 96-well Plate Research Articles

Abstract 4141: Quantitative measurement of 3D tumor spheroid invasion using live cell time-lapse imaging

Abstract 90% of the death of cancer individuals is related to cancer metastasis1. The invasion and migration of cancer cells into surrounding tissues and chambers is the first step of metastasis2. To have a better and more in-depth understanding of the pathophysiological activities involved in metastatic cancer, accurate and reliable methods for evaluating cell invasion are urgently needed. Compared to the 2D cell model, the 3D system mimics the natural physiological properties and conditions, such as structure, physiology, biological signals of living tissues, cell-matrix interactions4. In this study, we followed the commonly used protocol for 3D tumor invasion assay3 to generate spheroids in Ultra-Low-attachment (ULA) round bottom 96-well plate and subsequently embedded them in Matrigel® (5 mg/mL). After polymerization, the Matrigel that contains tumor spheroid was submerged in 100 μL cell culture media. The invasion of the 3D spheroid in the presence or absence of drug treatment was monitored and quantified using the brightfield and fluorescence live cell time-lapse image acquisition and image analysis functions of the Agilent xCELLigence RTCA eSight. The progress of spheroid invasion and the drug-inhibitory effects on tumor invasion were quantified by tracking and calculating the area of invasive protrusion from the spheroid. Our data show that 1) different types of cancer cells had different metastatic potentials demonstrated by the level of invadopodium extending into the Matrigel at desired time points; 2) the invasion of 3D HT1080 spheroid was inhibited by cytochalasin D (Cyto D), an inhibitor of actin polymerization, and GM6001, a potent broad-spectrum inhibitor of matrix metalloproteinases (MMP), in a dose-dependent manner; 3) Cyto D exhibited higher efficacy of inhibiting tumor invasion than GM6001. In summary, our approach by combining the 3D tumor spheroid invasion model with the RTCA eSight’s capability of live cell imaging and automatic quantification of the area of invasive protrusion can facilitate and enhance the development of new treatments at the preclinical stage in the future. Citation Format: Grace Yang, Tian Wang, Peifang Ye, Xiaoyu Zhang. Quantitative measurement of 3D tumor spheroid invasion using live cell time-lapse imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4141.

Influence of polyamines of bacterial origin on the production of key cytokines in the culture of human mononuclear leukocytes

To date, participation of biogenic polyamines has been studied in details, with respect to regulation of microbial gene expression, interrelations between bacteria, development of their persistence state. Opportunity of their use as markers of human pathological conditions is being actively evaluated. The aim of our study was to assess the effect of bacterial diamines, i.e., cadaverine and putrescine, upon production of key cytokines (IFNγ and IL-4) in the culture of human mononuclear leukocytes. We studied leukocytes of peripheral venous blood obtained from 18 healthy male volunteers (mean age 24.0±0.6 years). The leukocytes were isolated by means of gradient centrifugation using a Ficoll-Verografin mixture. For the cultivation of lymphocytes, a micro-method and plastic round-bottom 96-well plates were used. Concanavalin A at a concentration of 5 μg/ml was used as a T cell mitogen. Polyamines were used at final concentrations of 5, 25, 50, 75 and 100 μM/L. The cultivation was carried out in humidified atmosphere with 5% CO2, at 37 °C for 72 hours. At the end of incubation, the culture medium was collected and frozen for subsequent quantitative enzyme immunoassays of cytokine concentrations (IFNγ and IL-4) (Russia). Viable cells were counted using Goryaev chamber after staining with 0.1% trypan blue solution. Statistical analysis was performed using Student’s t test or Mann–Whitney test. Addition of cadaverine at all concentrations reduced IFNγ production in the culture of mitogen-activated cells. When culturing leukocytes supplied with putrescine (5 to 50 μM/L), a dose-dependent decrease of IFNγ was observed. Upon further increase of putrescine concentrations, the IFNγ production is restored to the values of the control samples. Direct toxic effect of polyamines upon the cells was revealed. Both the diamines, at the doses of 50, 75, and 100 μM/L, caused increase of IL-4 production by the mitogen-activated cells. Such changes can be associated both with direct cytotoxic effect of cadaverine and putrescine, being mediated by changes of some metabolic pathways. In addition, the effects of polyamines upon monocytes present in culture can include their anti-inflammatory state, e.c., an increased IL-4 production. In general, cadaverine and putrescine, produced by microorganisms of various taxonomic groups, regulate the effectiveness of compensatory-adaptive reactions that ensure adaptation of microbial populations to changing or unfavorable environmental conditions.

A Chondrosphere-Based Scaffold Free Approach to Manufacture an In Vitro Articular Cartilage Model.

In vitro engineering of human articular cartilage (AC) is a regenerative medicine challenge. The main objective of this study was the development of a repeatable scaffold-free in vitro model of chondrocyte spheroid-based treatments of cartilage defects, to allow for systematic study and further optimization of this type of treatment. Human articular chondrocytes (HC) and immortalized mesenchymal cells differentiated in chondrocytes (Y201-Cs) were cultured in round-bottom 96-well plates to produce multicellular spheroids and their growth kinetics, and viability was evaluated over 7 days of culture. Then, the spheroids were assembled and cultured for 21 days on a gelatin-coated poly(lactic-co-glycolic acid) electrospun membrane (10 spheroids/cm2), following a protocol in line with the clinically approved Chondrosphere® (CO.DON AG) technique. Both HC and Y201-C cells formed compact and viable spheroids after 7 days of culture with a reduction of diameter over the 7 days from 1300 ± 150 μm to 600 ± 90 μm and from 1250 ± 60 μm to 800 ± 20 μm for HC and Y201-C, respectively. When the spheroids were transferred onto the support membrane, these adhered on the membrane itself and fused themselves, producing collagen type II (COL2A1) and aggrecan (ACAN), according to gene expression and glycosaminoglycans quantification analyses. We detected higher expression of COL2A1 in HC cells, while the Y201-C constructs were characterized by an increased ACAN expression. The approach we presented allows a standardizable production of spheroids with predictable geometry and the creation of a reproducible scaffold-free in vitro AC-like construct showing high expression of chondrogenic markers, using both HC and Y201-C. In addition, the bankable Y201-C cells provide an effective base model for experimentation with the spheroid approach to further enhance the process. Impact statement This is first work on the development of a repeatable scaffold-free in vitro model based on an optimized protocol in line with a recent clinically approved Chondrosphere® (CO.DON AG) technique. In addition, we demonstrated that a bankable cell type (Y201-C) could produce an engineered cartilage-like construct, giving a repeatable model as a key tool for experimentation of therapeutic treatment ahead of studies with heterogeneous cell populations.

Effect of Anisotropic Aggregates Comprising Pegylated Block Copolymers on Cell Growth and Aggregation

In recent years, effects of shape of nanomaterials on biological response has attracted attention. For example, rod or fibrous polymeric micelles show a longer circulation time in blood and preferential accumulation to tumor cells than a spherical counterpart.1) On the other hand, fibrous cationic molecular assemblies exhibit higher or/and broader spectrum of antimicrobial activity than a spherical counterpart2). In addition, nano/micro-topology of substrates for tissue engineering is now recognized as a tool of regulating cellular responses such as adhesion, extension, aggregation, and differentiation. However, there have not been active systems to control such cellular responses by shape of molecular assemblies. Previously, we have developed amphiphilic pegylated diblock copolymers conjugating poly(L-lactide) (PLLA), poly(e-caprolactone) (PCL), or poly(trimethylene carbonate) (PTMC), forming anisotropic aggregates in water by introduction of a supramolecular assembling motif in the block junction. The size and length of the anisotropic aggregates are varied depending on the type of polymer used in hydrophobic segments. In this context, we explore cell culture of normal human dermal fibroblasts (NHDFs) in the presence of these anisotropic rod/fiber-like aggregates to evaluate the effect of shape of aggregates in culture media on cellular responses. The polymer aggregates were formulated by membrane dialysis. Typically, the block copolymer (40 mg) was dissolved in DMF (1 mL) and stirred overnight at room temperature. Nile red (NR) as a fluorescent probe was dissolved in DMF to have the concentration of 0.02 mg/mL. Then, the DMF solution of NR (1 mL) was added to the block copolymer solution and stirred for 2 hours. After that, the solution was transferred to a dialysis tube (Spectra/Por®7 Dialysis Membrane) with molecular cut off (MWCO) of 2,000 and dialyzed against ultrapure water for two days. Finally, the suspension was diluted to have the polymer concentration of 10 mg/mL (1.0 wt%) NHDFs were seeded on poly(ethylene terephthalate) (PET) plates embedded in a 24-well polystyrene plate at a cell density of 0.5 × 104 cells/cm2 in a cell culture medium (950 µL). The polymer suspension (50 µL) was then added to adjust the final polymer concentration to 0.05 wt%. The cells were incubated at 37 °C in 5% CO2 for 24 h and 48 h. At a certain time of incubation, cells were washed and stained with phalloidin and DAPI to visualize actin and nuclei in the observation by a confocal laser scanning microscope (CLSM). Furthermore, as three-dimension cell culture, NHDFs were first treated with CellTrackerTM to stain cytoplasm. Then, the cell suspension with a density of 1.6 × 104 cells/well including 0.05 wt% of the polymer aggregates was loaded in PrimeSurface\U0001f12c a round-bottom 96-well plates. The cells were incubated at 37 °C in 5% CO2 for 2, 6, and 12 h. Afterwards, cells were washed, stained with DAPI, and observed on a CLSM and a phase-contrast microscope. References 1)D. Li et al., Adv. Funct. Mater., 2016, 26, 66–79. 2)K.Fukushima et al., ACS Nano, 2012, 6, 9191-9199.

Allergen-specific immunotherapy modulates the balance of circulating Tfh and Tfr cells

Abstract Follicular helper T cells (Tfh cells) are a CD4 T cell subset essential for germinal center formation and B cell responses, although their role in allergy and allergen-specific immunotherapy (AIT) is unclear. Here, we show that AIT-treated patients have a higher ratio of regulatory Tfh cells (Tfr) to follicular T cells (Tfh) and hypothesize an IL-2 dependent mechanism.

High-Throughput Validation of Mutations Identified in Primary Leukemia Cells

Next generation sequencing has enabled the cataloguing of mutations found in many different cancers. The mutational burden for any given leukemia patient can range from 2 to hundreds of non-somatic mutations and it can therefore be challenging to separate drivers from the passengers. In performing deep sequencing on 139 primary leukemia samples using a custom capture library of ~1,800 genes including all kinases, phosphatases, and a variety of growth factors, cytokine receptors, and adapter proteins, we identified 3,390 non-somatic unique SNV and indel mutations in 1,286 genes. Many of the changes identified were non-recurrent or low frequency mutations found a single patient or a small numbers of patients.To determine the functional consequences, each mutation must be created and tested in vitro. We sought to develop a high throughput method to: 1) prioritize mutations for validation, 2) create mutations for testing, and 3) assess the transformative capacity of each mutation using a BaF3 growth factor independence assay. Using the following heuristic we ranked and prioritized mutations for further validation: i) include only high quality sequence variant cells; ii) exclude mutations found in >50% of samples iii) include mutations not found in dbSNP or 1000 genomes, or in 1000 genomes with a global mutant allele frequency less than or equal to 0.1; and iv) include mutations found dbSNP but not in 100 genomes with a cohort frequency equal to or less than <5%. Variants were ranked using HitWalker (Bottomly et al. 2013 PubMed ID: 23303510). HitWalker uses random walks with restarts algorithm to rank mutations based on proximity to known functionally important targets. These functionally important targets were identified using data from small molecule inhibitor screens performed on freshly isolated leukemia cells from each patient sample. We then prioritized 784 mutations for creation based on a HiWalker rank of 1-20.After mutations were ranked and prioritized, an R-based program, PrimeR, was used to automate mutagenesis primer design. Primers were batch ordered and arranged in 96-well plates based on primer Tm to enable the use of gradient PCR. Mutagenesis was performed on cDNA Entry clones purchased from commercial sources. Greater than 50% of the initial mutagenesis reactions were successful in producing mutagenized plasmids with no non-specific mutations. Confirmed mutations and their wild type counter parts were transferred into retroviral vector destination clones using Gateway LR Clonase II (ThermoFisher Scientific). Following LR Clonase reactions, we repeated sequencing and found no non-specific mutations and opted to identify successful constructs solely by restriction digestion/DNA gel analysis in future runs.IL-3 withdrawl assays were performed using round bottom 96-well plates, enabling removal of IL-3 from the cell media. Cells were split equally into 5 flat bottomed 96-well tissue culture plates, and covered with breathable seals and incubated at 37°C. Every 4 days MTS was added to a single plate according to the manufacturers specification and a cell viability assay was performed. Wells possessing an absorbance of 0.100 or higher compared to the blanks were considered to contain mutations of interest, and were subsequently repeated using traditional growth factor independence assays in 25mL flasks. Using this method we have screened 86 mutations along with their wildtype controls and identified 10 mutations with transformative capacity in 7 genes. All transforming mutations had a HitWalker rank of 1-3 supporting the use of HitWalker in prioritizing mutations for further validation. DisclosuresDruker:Dana-Farber Cancer Institute: Patents & Royalties: Millipore royalties via Dana-Farber Cancer Institute; Array: Patents & Royalties; Curis: Patents & Royalties; Pfizer: Patents & Royalties; CTI: Consultancy, Equity Ownership; Cylene: Consultancy, Equity Ownership; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Agios: Honoraria; Ambit BioSciences: Consultancy; AstraZeneca: Consultancy; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses; Roche: Consultancy; ARIAD: Patents & Royalties: inventor royalties paid by Oregon Health & Science University for licenses, Research Funding; BMS: Research Funding; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Dana-Farber Cancer Institute: Patents & Royalties: Millipore royalties via Dana-Farber Cancer Institute; Array: Patents & Royalties; Curis: Patents & Royalties; Pfizer: Patents & Royalties.

Conopeptide ρ-TIA Defines a New Allosteric Site on the Extracellular Surface of the α1B-Adrenoceptor

The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey β(1)-adrenergic receptor crystal structure, we modeled the α(1B)-adrenoceptor (α(1B)-AR) to help identify the allosteric site for ρ-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ρ-TIA, we identified 14 residues on the ECS of the α(1B)-AR that influenced ρ-TIA binding. Double mutant cycle analysis and docking confirmed that ρ-TIA binding was dominated by a salt bridge and cation-π between Arg-4-ρ-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-π interaction between Trp-3-ρ-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-ρ-TIA and Val-197, Trp-3-ρ-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for α(1)-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs.

Interleukin-15 enhances rituximab-dependent cytotoxicity against chronic lymphocytic leukemia cells and overcomes transforming growth factor beta-mediated immunosuppression

Chemoimmunotherapy with anti-CD20 monoclonal antibody rituximab is increasingly used for the treatment of patients with chronic lymphocytic leukemia (CLL). Antibody-dependent cytotoxicity (ADCC) is one of the most important mechanisms of action of rituximab against B-cell malignancies. We studied ways to increase the cytotoxic effect of rituximab on CLL cells by enhancing ADCC. Peripheral blood mononuclear cell (PBMC) or purified natural killer (NK) cells from healthy donors were activated with interleukin-15 (IL-15) and cultured with rituximab-coated CLL cells, and ADCC was evaluated using a (51)chromium release assay. The IL-15 significantly enhanced invitro ADCC against CLL cells, and this effect was mainly mediated by NK cells. The IL-15 treated effector cells with the low affinity FcγRIIIA receptor (158FF) had an ADCC comparable to those with the high affinity FcγRIIIA form (158VF). Inaddition, IL-15 enhanced rituximab-mediated ADCC of CLL cells in the presence of transforming growth factor-beta. The IL-15 increases rituximab-mediated ADCC against CLL, and supports the use of such agents with the goal of improving clinical response to chemoimmunotherapy in patients with CLL.

High Mobility Group Box 1 Release from Hepatocytes during Ischemia and Reperfusion Injury Is Mediated by Decreased Histone Deacetylase Activity

The mobilization and extracellular release of nuclear high mobility group box-1 (HMGB1) by ischemic cells activates inflammatory pathways following liver ischemia/reperfusion (I/R) injury. In immune cells such as macrophages, post-translational modification by acetylation appears to be critical for active HMGB1 release. Hyperacetylation shifts its equilibrium from a predominant nuclear location toward cytosolic accumulation and subsequent release. However, mechanisms governing its release by parenchymal cells such as hepatocytes are unknown. In this study, we found that serum HMGB1 released following liver I/R in vivo is acetylated, and that hepatocytes exposed to oxidative stress in vitro also released acetylated HMGB1. Histone deacetylases (HDACs) are a family of enzymes that remove acetyl groups and control the acetylation status of histones and various intracellular proteins. Levels of acetylated HMGB1 increased with a concomitant decrease in total nuclear HDAC activity, suggesting that suppression in HDAC activity contributes to the increase in acetylated HMGB1 release after oxidative stress in hepatocytes. We identified the isoforms HDAC1 and HDAC4 as critical in regulating acetylated HMGB1 release. Activation of HDAC1 was decreased in the nucleus of hepatocytes undergoing oxidative stress. In addition, HDAC1 knockdown with siRNA promoted HMGB1 translocation and release. Furthermore, we demonstrate that HDAC4 is shuttled from the nucleus to cytoplasm in response to oxidative stress, resulting in decreased HDAC activity in the nucleus. Together, these findings suggest that decreased nuclear HDAC1 and HDAC4 activities in hepatocytes following liver I/R is a mechanism that promotes the hyperacetylation and subsequent release of HMGB1.

Evolutionary and Functional Analysis of Celiac Risk Loci Reveals SH2B3 as a Protective Factor against Bacterial Infection

Celiac disease (CD) is an intolerance to dietary proteins of wheat, barley, and rye. CD may have substantial morbidity, yet it is quite common with a prevalence of 1%-2% in Western populations. It is not clear why the CD phenotype is so prevalent despite its negative effects on human health, especially because appropriate treatment in the form of a gluten-free diet has only been available since the 1950s, when dietary gluten was discovered to be the triggering factor. The high prevalence of CD might suggest that genes underlying this disease may have been favored by the process of natural selection. We assessed signatures of selection for ten confirmed CD-associated loci in several genome-wide data sets, comprising 8154 controls from four European populations and 195 individuals from a North African population, by studying haplotype lengths via the integrated haplotype score (iHS) method. Consistent signs of positive selection for CD-associated derived alleles were observed in three loci: IL12A, IL18RAP, and SH2B3. For the SH2B3 risk allele, we also show a difference in allele frequency distribution (Fst) between HapMap phase II populations. Functional investigation of the effect of the SH2B3 genotype in response to lipopolysaccharide and muramyl dipeptide revealed that carriers of the SH2B3 rs3184504*A risk allele showed stronger activation of the NOD2 recognition pathway. This suggests that SH2B3 plays a role in protection against bacteria infection, and it provides a possible explanation for the selective sweep on SH2B3, which occurred sometime between 1200 and 1700 years ago.

N- and O-Glycans Modulate Galectin-1 Binding, CD45 Signaling, and T Cell Death

Galectin-1, a beta-galactoside-binding protein highly expressed in the thymus, induces apoptosis of specific thymocyte subsets and activated T cells. Galectin-1 binds to N- and O-glycans on several glycoprotein receptors, including CD7, CD43, and CD45. Here we show that galectin-1 signaling through CD45, which carries both N- and O-glycans, is regulated by CD45 isoform expression, core 2 O-glycan formation and the balance of N-glycan sialylation. Regulation of galectin-1 T cell death by O-glycans is mediated through CD45 phosphatase activity. While galectin-1 signaling in cells expressing low molecular weight isoforms of CD45 requires expression of core 2 O-glycans (high affinity ligands for galectin-1), galectin-1 signaling in cells expressing a high molecular weight isoform of CD45 does not require core 2 O-glycans, suggesting that a larger amount of core 1 O-glycans (low affinity ligands for galectin-1) is sufficient to overcome lack of core 2 O-glycans. Furthermore, regulation of galectin-1 signaling by alpha2,6-sialylation of N-glycans is not solely dependent on CD45 phosphatase activity and can be modulated by the relative expression of enzymes that attach sialic acid in an alpha2,6- or alpha2,3-linkage. Thus, N- and O-glycans modulate galectin-1 T cell death by distinct mechanisms, and different glycosylation events can render thymocytes susceptible or resistant to galectin-1.

The Effect of Serum on the PEG-Based Crosslinker-Induced Spheroid Formation of Pancreatic β-Cell

A polymeric crosslinker poly(ethylene glycol) derivative with dioleyl groups as hydrophobic group at both ends was developed for promoting cellular spheroid formation. Our approach to bridge cells was based on the crosslinking of cell membrane using a crosslinker via the hydrophobic interaction. Using the crosslinker, spheroid formation of pancreatic β-cell line RIN in a round bottom 96-well plate was evaluated, especially in effect of serum on spheroid formation. In the presence or absence of serum, the size of prepared spheroid was found to decrease with increasing culture time and with increasing crosslinker concentration. However, the medium without serum proved to be a favorable condition for promoting cell aggregation because in this case spheroid with smaller size could be obtained. It was clarified that spheroid formation and insulin secretion of the spheroid prepared by the crosslinker were enhanced, especially in the medium without serum.

Xerogel Optical Sensor Films for Quantitative Detection of Nitroxyl

Xerogel sensing films were synthesized via sol-gel chemistry were used to fabricate optical nitroxyl (HNO) sensors [corrected] Selective detection of HNO in solution was achieved by monitoring the rates of manganese(III) meso-tetrakis(4-sulfonatophenyl) porphyrinate (MnIIITPPS) reductive nitrosylation in the anaerobic interior of aminoalkoxysilane-derived xerogel films. Nitroxyl permeability in sensor films deposited in round-bottom 96-well plates was enhanced via incorporation of trimethoxysilyl-terminated poly(amidoamine-organosilicon) dendrimers in the xerogel network. The selectivity of MnIIITPPS for HNO, the overall sensitivity, and the working dynamic range of the resulting sensors were characterized. The HNO-sensing microtiter plates were used to quantify pH-dependent HNO generation by the recently described HNO-donor sodium 1-(isopropylamino)diazene-1-ium-1,2-diolate (IPA/NO), and compare HNO production efficiency between IPA/NO and Angeli's salt, a traditional HNO-donor.

Residues Distant from the Active Site Influence Protein-tyrosine Phosphatase 1B Inhibitor Binding

Regions of protein-tyrosine phosphatase (PTP) 1B that are distant from the active site yet affect inhibitor binding were identified by a novel library screen. This screen was based on the observation that expression of v-Src in yeast leads to lethality, which can be rescued by the coexpression of PTP1B. However, this rescue is lost when yeast are grown in the presence of PTP1B inhibitors. To identify regions of PTP1B (amino acids 1-400, catalytic domain plus 80-amino acid C-terminal tail) that can affect the binding of the difluoromethyl phosphonate (DFMP) inhibitor 7-bromo-6-difluoromethylphosphonate 3-naphthalenenitrile, a library coexpressing PTP1B mutants and v-Src was generated, and the ability of yeast to grow in the presence of the inhibitor was evaluated. PTP1B inhibitor-resistant mutations were found to concentrate on helix alpha7 and its surrounding region, but not in the active site. No resistant amino acid substitutions were found to occur in the C-terminal tail, suggesting that this region has little effect on active-site inhibitor binding. An in-depth characterization of a resistant substitution localizing to region alpha7 (S295F) revealed that this change minimally affected enzyme catalytic activity, but significantly reduced the potency of a panel of structurally diverse DFMP PTP1B inhibitors. This loss of inhibitor potency was found to be due to the difluoro moiety of these inhibitors because only the difluoro inhibitors were shifted. For example, the inhibitor potency of a monofluorinated or non-fluorinated analog of one of these DFMP inhibitors was only minimally affected. Using this type of library screen, which can scan the nearly full-length PTP1B sequence (catalytic domain and C-terminal tail) for effects on inhibitor binding, we have been able to identify novel regions of PTP1B that specifically affect the binding of DFMP inhibitors.

Cytotoxic T lymphocyte (CTL) adherence assay (CAA): a non-radioactive assay for murine CTL recognition of peptide-MHC class I complexes

Cytotoxic T lymphocytes (CTL) form an important immune surveillance system against intracellular pathogens. Here we describe a simple, visual assay for identifying peptides specifically recognized by CTL, based on the discovery that CTL develop increased adhesive properties upon TCR triggering. Several CTL lines were shown to pellet to the bottom of a round bottom 96-well plate in the absence of peptide. In contrast, these same CTL lines incubated with their cognate peptide, allowing them to present peptide to each other, adhered to the sides of the well and were readily distinguished by macroscopic visual examination of the plate after 4–5 h or overnight incubation. This CTL adherence assay (CAA) demonstrated peptide specificity and MHC restriction, and was titratable with peptide concentration. With this technique, a minimal-sized, malaria CTL epitope was correctly identified from a panel of overlapping nonamers, although the adherence pattern of two mono-substituted, variant peptides was less predictive of lytic activity. Also, substitutions in an HIV-1 envelope CTL epitope that reduced lytic activity were correctly predicted. Inhibitors of RNA and protein synthesis, upon preincubation, abrogated the adherence, indicating, at minimum, a need for live cells. Wortmannin, a PI-3 kinase inhibitor, inhibited the peptide specific adherence, consistent with a role for TCR or integrin signal transduction in CAA. Other cytoskeletal and metabolic inhibitors had no effect. Adherence of the T cells may involve low affinity, nonspecific interactions since wells coated with FCS, BSA or milk powder all produced an effective CAA in the presence of peptide under serum free conditions. Consequently, CAA may represent a rapid, simple method for screening large numbers of peptides to find cytolytic epitopes for a given CTL line and may identify additional epitopes causing T cell activation and adherence but not cytolytic activity.

Clinical application of responder cell frequency estimates with four years of follow up

Limiting dilution cultures have been used to estimate the frequency of T cells which respond to antigen stimulation in vitro, but nothing is known of the reproducibility of this assay when applied to human blood. We developed a simplified form of limiting dilution culture in which blood mononuclear cells were diluted in round bottom 96-well plates and cultured for 10 days. The assay was used to estimate the frequency of blood mononuclear cells proliferating in response to varicella-zoster virus antigen. 250 subjects aged 25–87 years were studied: 95 of these subjects had annual measurements repeated over a 4 year period. The coefficients of variation for intra-assay replicates was 5%, and for inter-assay comparisons was 17.6% and 26% for tests at a 1 or 3 month interval respectively. For subjects studied at 1 year intervals the coefficient of variation was 42% with the total variability equally distributed between the variation between subjects and the variation within subjects. These data provide the first quantitative data to validate limiting dilution cultures for the long term in vitro measurement of human T cell responses.

Fast ELISA for measuring serum antibody responses

A method which speeds up the enzyme-linked immunosorbent assay (ELISA) is described. The procedure uses a modified Falcon fast assay screening system (Becton Dickinson Labware, Lincoln Park, NJ) and Falcon round-bottom 96-well plates. Antigen is adsorbed onto beads which extend from a lid and fit into 96-well plates. The beads are washed in a trough and reacted to antibody in the round-bottom plate. The labor required to wash the plates after coating with antigen, antibody or conjugate is thereby reduced. Greater flexibility and accuracy result, especially with the use of more than one 96-well plate. In this study, naturally occuring human IgG antibody responses to two isolated bacterial antigens were measured in over 200 subjects. It was found that numerical taxonomy could be used to split out the high IgG responders. The IgM response to one of the antigens was less variable and not significantly related to the IgG response. The fast ELISA is as useful to operate as the standard ELISA, but less stressful on the operator and more rapid.