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High throughput molecular and functional profiling of patients is a key driver of precision medicine. DNA and RNA characterization has been enabled at unprecedented cost and scale through rapid, disruptive progress in sequencing technology, but challenges persist in data management and interpretation. We analyze the state-of-the-art of large-scale unbiased sequencing in drug discovery and development, including technology, application, ethical, regulatory, policy and commercial considerations, and discuss issues of LUS implementation in clinical and regulatory practice. [ABSTRACT FROM AUTHOR]

Our theory of positively reinforced free-operant behavior (Perez & Dickinson, 2020) assumes that responding is controlled by two systems. One system is sensitive to the correlation between response and reinforcement rates and controls goal-directed behavior, whereas a habitual system learns by reward prediction error. We present an extension of this theory to the aversive domain that explains why free-operant avoidance responding increases with both the experienced rate of negative reinforcement and the difference between this rate and that programmed by the avoidance schedule. The theory also assumes that the habitual component is reinforced by the acquisition of aversive inhibitory properties by the feedback stimuli generated by responding, which then act as safety signals that reinforce habit performance. Our analysis suggests that the distinction between habitual and goal-directed control of rewarded behavior can also be applied to the aversive domain. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

The influence of the Rescorla-Wagner model cannot be overestimated, despite that (1) the model does not differ much computationally from its predecessors and competitors, and (2) its shortcomings are well-known in the learning community. Here we discuss the reasons behind its widespread influence in the cognitive and neural sciences, and argue that it is the constant search for general-process theories by learning scholars which eventually produced a model whose application spans many different areas of research to this day. We focus on the theoretical and empirical background of the model, the theoretical connections that it has with later developments across Marr's levels of analysis, as well as the broad variety of research that it has guided and inspired., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Understanding how culture evolves in society is an extremely difficult task. The bifocal stance theory (BST) deploys two copying strategies which can be linked to dual-system theories of behavior. BST would benefit from incorporating results from these theories, such as the evolution of attention to goals or steps of a behavioral sequence, and the role of the environment in prompting different copying strategies.

In the natural world, stimulus-outcome associations are often ambiguous, and most associations are highly complex and situation-dependent. Learning to disambiguate these complex associations to identify which specific outcomes will occur in which situations is critical for survival. Pavlovian occasion setters are stimuli that determine whether other stimuli will result in a specific outcome. Occasion setting is a well-established phenomenon, but very little investigation has been conducted on how occasion setters are disambiguated when they themselves are ambiguous (i.e., when they do not consistently signal whether another stimulus will be reinforced). In two preregistered studies, we investigated the role of higher-order Pavlovian occasion setting in humans. We developed and tested the first computational model predicting direct associative learning, traditional occasion setting (i.e., 1st-order occasion setting), and 2nd-order occasion setting. This model operationalizes stimulus ambiguity as a mechanism to engage in higher-order Pavlovian learning. Both behavioral and computational modeling results suggest that 2nd-order occasion setting was learned, as evidenced by lack and presence of transfer of occasion setting properties when expected and the superior fit of our 2nd-order occasion setting model compared to the 1st-order occasion setting or direct associations models. These results provide a controlled investigation into highly complex associative learning and may ultimately lead to improvements in the treatment of Pavlovian-based mental health disorders (e.g., anxiety disorders, substance use)., Competing Interests: The authors have declared that no competing interests exist.

Competing Interests: None disclosed.

Theories of learning distinguish between elemental and configural stimulus processing depending on whether stimuli are processed independently or as whole configurations. Evidence for elemental processing comes from findings of summation in animals where a compound of two dissimilar stimuli is deemed to be more predictive than each stimulus alone, whereas configural processing is supported by experiments using similar stimuli in which summation is not found. However, in humans the summation effect is robust and impervious to similarity manipulations. In three experiments in human predictive learning, we show that summation can be obliterated when partially reinforced cues are added to the summands in training and tests. This lack of summation only holds when the partially reinforced cues are similar to the reinforced cues (experiment 1) and seems to depend on participants sampling only the most salient cue in each trial (experiments 2a and 2b) in a sequential visual search process. Instead of attributing our and others' instances of lack of summation to the customary idea of configural processing, we offer a formal subsampling rule that might be applied to situations in which the stimuli are hard to parse from each other., (© 2022 Perez et al.; Published by Cold Spring Harbor Laboratory Press.)

Background: Individuals living with chronic spinal cord injury or disease (SCI/D) are at an increased risk of falling. However, little is known about the impact of falls and fall risk in the subacute phase of SCI/D, despite this being a time when fall prevention initiatives are delivered. Hence, we explored the impact of falls and fall risk in individuals with subacute SCI/D as they transitioned from inpatient rehabilitation to community living., Methods: This qualitative photo-elicitation study used an inductive thematic analysis. Eight individuals (7 male) undergoing inpatient rehabilitation at a Canadian tertiary rehabilitation hospital due to a new SCI/D participated. Six months following discharge, photo-elicitation interviewing was used to understand the impact of falls and fall risk. Over 7-14 days, participants completed a photo-assignment that involved taking photographs in response to questions, such as what increases/decreases your likelihood of falling? A semi-structured interview followed, in which participants described their photographs and discussed their experiences with falls, fall risk and fall prevention training., Results: Four themes were identified. 1) Risk factors and strategies identified through lived experience. Participants discovered their fall risk factors and fall prevention strategies through "trial and error". 2) Influences on the individual's perception of their fall risk. Prior experience with falls, including falls experienced by themselves as well as friends and family, influenced their perception of fall risk. 3) Experiencing life differently due to increased fall risk. A high fall risk reduced participation, increased negative emotions and decreased independence and quality of life. 4) Falls training in rehabilitation can be improved. Prior experiences with falls training varied; however, participants expressed a desire for comprehensive and individualized training., Conclusion: Although participants' experiences with falls and fall prevention varied, falls and the risk of falling can have a significant impact on the first year of living with a SCI/D., Competing Interests: The authors have declared that no competing interests exist.

It has been suggested that there are two distinct and parallel mechanisms for controlling instrumental behavior in mammals: goal-directed actions and habits. To gain an understanding of how these two systems interact to control behavior, it is essential to characterize the mechanisms by which the balance between these systems is influenced by experience. Studies in rodents have shown that the amount of training governs the relative expression of these two systems: Behavior is goal-directed following moderate training, but the more extensively an instrumental action is trained, the more it becomes habitual. It is less clear whether humans exhibit similar training effects on the expression of goal-directed and habitual behavior, as human studies have reported contradictory findings. To tackle these contradictory findings, we formed a consortium, where four laboratories undertook a preregistered experimental induction of habits by manipulating the amount of training. There was no statistical evidence for a main effect of the amount of training on the formation and expression of habits. However, exploratory analyses suggest a moderating effect of the affective component of stress on the impact of training over habit expression. Participants who were lower in affective stress appeared to be initially goal-directed, but became habitual with increased training, whereas participants who were high in affective stress were already habitual even after moderate training, thereby manifesting insensitivity to overtraining effects. Our findings highlight the importance of the role of moderating variables such as individual differences in stress and anxiety when studying the experimental induction of habits in humans., (© 2022 Pool et al.; Published by Cold Spring Harbor Laboratory Press.)

Contexts and discrete stimuli often hierarchically influence the association between a stimulus and outcome. This phenomenon, called occasion setting, is central to modulation-based Pavlovian learning. We conducted two experiments with humans in fear and appetitive conditioning paradigms, training stimuli in differential conditioning, feature-positive discriminations, and feature-negative discriminations. We also investigated the effects of trait anxiety and trait depression on these forms of learning. Results from both experiments showed that participants were able to successfully learn which stimuli predicted the electric shock and monetary reward outcomes. Additionally, as hypothesized, the stimuli trained as occasion setters had little-to-no effect on simple reinforced or non-reinforced stimuli, suggesting the former were indeed occasion setters. Lastly, in fear conditioning, trait anxiety was associated with increases in fear of occasion setter/conditional stimulus compounds; in appetitive conditioning, trait depression was associated with lower expectations of monetary reward for the trained negative occasion setting compound and transfer of the negative occasion setter to the simple reinforced stimulus. These results suggest that clinically anxious individuals may have enhanced fear of occasion setting compounds, and clinically depressed individuals may expect less reward with compounds involving the negative occasion setter., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Recent theories of instrumental behavior postulate that the correlation between response and reward rate is a critical factor in instrumental goal-directed performance. However, it is still not clear whether human actions can be sensitive to rate correlation. Using a novel within-subject design, participants were trained under ratio and interval contingencies of reinforcement matching both reward probabilities and reward rates between conditions. The impact of rate correlation on performance was evident in the higher performance observed under ratio contingencies for both types of matching. Moreover, there was no difference in performance between two classes of interval schedules with equivalent correlational properties but different reward probabilities. These results are discussed in terms of a recent dual-system model of instrumental behavior.

The American Medical Association (AMA) and National Institutes of Health (NIH) currently suggest that health care materials be written at a sixth-grade reading level. Our study investigates the readability of online information on Achilles rupture and reconstruction. Achilles tendon rupture, Achilles tendon repair , and Achilles tendon reconstruction were queried using advanced search functions of Google, Bing, and Yahoo!. Individual websites and text from the first 3 pages of results for each search engine were recorded and categorized as physician based, academic, commercial, government and nongovernmental organization, or unspecified. Individual readability scores were calculated via 6 different indices: Flesch-Kincaid grade level, Flesch Reading Ease, Gunning Fog, SMOG, Coleman-Liau index, and Automated Readability Index along with a readability classification score and average grade level. A total of 56 websites were assessed. Academic webpages composed the majority (51.8%), followed by physician-based sources (32.1%). The average overall grade level was 10.7 ± 2.54. Academic websites were written at the highest-grade level (11.5 ± 2.77), significantly higher than physician-based websites ( P = .040), and only 2 were written at, or below, a sixth-grade reading level. Currently, online information on Achilles tendon rupture and reconstruction is written at an inappropriately high reading level compared with recommendations from the AMA and NIH. Level of Evidence : Level IV .

Contemporary theories of instrumental performance assume that responding can be controlled by 2 behavioral systems, 1 goal-directed that encodes the outcome of an action, and 1 habitual that reinforces the response strength of the same action. Here we present a model of free-operant behavior in which goal-directed control is determined by the correlation between the rates of the action and the outcome whereas the total prediction error generated by contiguous reinforcement by the outcome controls habitual response strength. The outputs of these two systems summate to generate a total response strength. This cooperative model addresses the difference in the behavioral impact of ratio and interval schedules, the transition from goal-directed to habitual control with extended training, the persistence of goal-directed control under choice procedures and following extinction, among other phenomena. In these respects, this dual-system model is unique in its account of free-operant behavior. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

ALK, ROS1 and RET gene fusions are important predictive biomarkers for tyrosine kinase inhibitors in lung cancer. Currently, the gold standard method for gene fusion detection is Fluorescence In Situ Hybridization (FISH) and while highly sensitive and specific, it is also labour intensive, subjective in analysis, and unable to screen a large numbers of gene fusions. Recent developments in high-throughput transcriptome-based methods may provide a suitable alternative to FISH as they are compatible with multiplexing and diagnostic workflows. However, the concordance between these different methods compared with FISH has not been evaluated. In this study we compared the results from three transcriptome-based platforms (Nanostring Elements, Agena LungFusion panel and ThermoFisher NGS fusion panel) to those obtained from ALK, ROS1 and RET FISH on 51 clinical specimens. Overall agreement of results ranged from 86-96% depending on the platform used. While all platforms were highly sensitive, both the Agena panel and Thermo Fisher NGS fusion panel reported minor fusions that were not detectable by FISH. Our proof-of-principle study illustrates that transcriptome-based analyses are sensitive and robust methods for detecting actionable gene fusions in lung cancer and could provide a robust alternative to FISH testing in the diagnostic setting., Competing Interests: S.H., M.L. and O.P. are employees of Pfizer. Y.E. and D.I. are employees of Agena Bioscience.

Retroviral replicating vectors (RRVs) are a nonlytic alternative to oncolytic replicating viruses as anticancer agents, being selective both for dividing cells and for cells that have defects in innate immunity and interferon responsiveness. Tumor cells fit both these descriptions. Previous publications have described a prototype based on an amphotropic murine leukemia virus (MLV), encoding yeast cytosine deaminase (CD) that converts the prodrug 5-fluorocytosine (5-FC) to the potent anticancer drug, 5-fluorouracil (5-FU) in an infected tumor. We report here the selection of one lead clinical candidate based on a general design goal to optimize the genetic stability of the virus and the CD activity produced by the delivered transgene. Vectors were tested for titer, genetic stability, CD protein and enzyme activity, ability to confer susceptibility to 5-FC, and preliminary in vivo antitumor activity and stability. One vector, Toca 511, (aka T5.0002) encoding an optimized CD, shows a threefold increased specific activity in infected cells over infection with the prototype RRV and shows markedly higher genetic stability. Animal testing demonstrated that Toca 511 replicates stably in human tumor xenografts and, after 5-FC administration, causes complete regression of such xenografts. Toca 511 (vocimagene amiretrorepvec) has been taken forward to preclinical and clinical trials.

Patients with the most common and aggressive form of high-grade glioma, glioblastoma multiforme, have poor prognosis and few treatment options. In 2 immunocompetent mouse brain tumor models (CT26-BALB/c and Tu-2449-B6C3F1), we showed that a nonlytic retroviral replicating vector (Toca 511) stably delivers an optimized cytosine deaminase prodrug activating gene to the tumor lesion and leads to long-term survival after treatment with 5-fluorocytosine (5-FC). Survival benefit is dose dependent for both vector and 5-FC, and as few as 4 cycles of 5-FC dosing after Toca 511 therapy provides significant survival advantage. In the virally permissive CT26-BALB/c model, spread of Toca 511 to other tissues, particularly lymphoid tissues, is detectable by polymerase chain reaction (PCR) over a wide range of levels. In the Tu-2449-B6C3F1 model, Toca 511 PCR signal in nontumor tissues is much lower, spread is not always observed, and when observed, is mainly detected in lymphoid tissues at low levels. The difference in vector genome spread correlates with a more effective antiviral restriction element, APOBEC3, present in the B6C3F1 mice. Despite these differences, neither strain showed signs of treatment-related toxicity. These data support the concept that, in immunocompetent animals, a replicating retroviral vector carrying a prodrug activating gene (Toca 511) can spread through a tumor mass, leading to selective elimination of the tumor after prodrug administration, without local or systemic pathology. This concept is under investigation in an ongoing phase I/II clinical trial of Toca 511 in combination with 5-FC in patients with recurrent high-grade glioma (www.clinicaltrials.gov NCT01156584).

Objectives: RA is a common, relapsing autoimmune disease primarily affecting the joints. Fibroblast-like synovial (FLS) cells are thought to be responsible for pannus formation and secretion of factors that recruit leucocytes to affected joints, thereby promoting bone and cartilage destruction. Fibrocytes are multipotent circulating stem cells that may have a role in RA pathogenesis, perhaps as the precursors of the FLS cells, or by regulating FLS cell function., Methods: We utilized multidimensional phospho-specific flow cytometry to characterize the activation status of peripheral blood (PB) fibrocytes derived from human RA patients at different stages of disease and from mice with CIA., Results: Human PB fibrocytes from RA patients exhibited phosporylation activation of the p44/42 and p38 MAP kinases (MAPKs), and STAT3 (signal transducer and activator of transcription) and STAT-5 early in disease, within the first year of diagnosis. Similarly, in murine CIA, an increase in the total number of PB phosphoSTAT5-positive fibrocytes was observed at early time points in disease. Notably, in the affected paws of mice with CIA, we identified an increased number of fibrocytes, in contrast to the paws of control mice., Conclusions: These data suggest that activated fibrocytes may influence the disease process in RA and may serve as surrogate markers for disease in the PB of affected patients.

Background: The precise mechanisms involved in the initiation and progression of rheumatoid arthritis (RA) are not known. Early stages of RA often have non-specific symptoms, delaying diagnosis and therapy. Additionally, there are currently no established means to predict clinical responsiveness to therapy. Immune cell activation is a critical component therefore we examined the cellular activation of peripheral blood mononuclear cells (PBMCs) in the early stages of RA, in order to develop a novel diagnostic modality., Methods and Findings: PBMCs were isolated from individuals diagnosed with early RA (ERA) (n = 38), longstanding RA (n = 10), osteoarthritis (OA) (n = 19) and from healthy individuals (n = 10). PBMCs were examined for activation of 15 signaling effectors, using phosphorylation status as a measure of activation in immunophenotyped cells, by flow cytometry (phospho-flow). CD3+CD4+, CD3+CD8+ and CD20+ cells isolated from patients with ERA, RA and OA exhibited activation of multiple phospho-epitopes. ERA patient PBMCs showed a bias towards phosphorylation-activation in the CD4+ and CD20+ compartments compared to OA PBMCs, where phospho-activation was primarily observed in CD8+ cells. The ratio of phospho (p)-AKT/p-p38 was significantly elevated in patients with ERA and may have diagnostic potential. The mean fluorescent intensity (MFI) levels for p-AKT and p-H3 in CD4+, CD8+ and CD20+ T cells correlated directly with physician global assessment scores (MDGA) and DAS (disease activity score). Stratification by medications revealed that patients receiving leflunomide, systemic steroids or anti-TNF therapy had significant reductions in phospho-specific activation compared with patients not receiving these therapies. Correlative trends between medication-associated reductions in the levels of phosphorylation of specific signaling effectors and lower disease activity were observed., Conclusions: Phospho-flow analysis identified phosphorylation-activation of specific signaling effectors in the PB from patients with ERA. Notably, phosphorylation of these signaling effectors did not distinguish ERA from late RA, suggesting that the activation status of discrete cell populations is already established early in disease. However, when the ratio of MFI values for p-AKT and p-p38 is >1.5, there is a high likelihood of having a diagnosis of RA. Our results suggest that longitudinal sampling of patients undergoing therapy may result in phospho-signatures that are predictive of drug responsiveness.

MYC overexpression has been implicated in the pathogenesis of most types of human cancers. MYC is likely to contribute to tumorigenesis by its effects on global gene expression. Previously, we have shown that the loss of MYC overexpression is sufficient to reverse tumorigenesis. Here, we show that there is a precise threshold level of MYC expression required for maintaining the tumor phenotype, whereupon there is a switch from a gene expression program of proliferation to a state of proliferative arrest and apoptosis. Oligonucleotide microarray analysis and quantitative PCR were used to identify changes in expression in 3,921 genes, of which 2,348 were down-regulated and 1,573 were up-regulated. Critical changes in gene expression occurred at or near the MYC threshold, including genes implicated in the regulation of the G(1)-S and G(2)-M cell cycle checkpoints and death receptor/apoptosis signaling. Using two-dimensional protein analysis followed by mass spectrometry, phospho-flow fluorescence-activated cell sorting, and antibody arrays, we also identified changes at the protein level that contributed to MYC-dependent tumor regression. Proteins involved in mRNA translation decreased below threshold levels of MYC. Thus, at the MYC threshold, there is a loss of its ability to maintain tumorigenesis, with associated shifts in gene and protein expression that reestablish cell cycle checkpoints, halt protein translation, and promote apoptosis.

Statins are a class of drugs that inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMGcoA) reductase, a critical enzyme in the mevalonate pathway. Several reports document that statins may prevent different human cancers. However, whether or not statins can prevent cancer is controversial due to discordant results. One possible explanation for these conflicting conclusions is that only some tumors or specific statins may be effective. Here, we demonstrate in an in vivo transgenic model in which atorvastatin reverses and prevents the onset of MYC-induced lymphomagenesis, but fails to reverse or prevent tumorigenesis in the presence of constitutively activated K-Ras (G12D). Using phosphoprotein fluorescence-activated cell sorter (FACS) analysis, atorvastatin treatment was found to result in the inactivation of the Ras and ERK1/2 signaling pathways associated with the dephosphorylation and inactivation of MYC. Correspondingly, tumors with a constitutively activated K-Ras (G12D) did not exhibit dephosphorylation of ERK1/2 and MYC. Atorvastatin's effects on MYC were specific to the inhibition of HMGcoA reductase, as treatment with mevalonate, the product of HMG-CoA reductase activity, abrogated these effects and inhibited the ability of atorvastatin to reverse or suppress tumorigenesis. Also, RNAi directed at HMGcoA reductase was sufficient to abrogate the neoplastic properties of MYC-induced tumors. Thus, atorvastatin, by inhibiting HMGcoA reductase, induces changes in phosphoprotein signaling that in turn prevent MYC-induced lymphomagenesis.

Background: Leukocyte Function Antigen-1 (LFA-1) is a primary adhesion molecule that plays important roles in T cell activation, leukocyte recirculation, and trans-endothelial migration. By applying a multivariate intracellular phospho-proteomic analysis, we demonstrate that LFA-1 differentially activates signaling molecules., Results: Signal intensity was dependent on both ICAM ligand and LFA-1 concentration. In the presence of CD3 and CD28 stimulation, ICAM-2 and ICAM-3 decreased TGFbeta1 production more than ICAM-1. In long-term differentiation experiments, stimulation with ICAM-3, CD3, and CD28 generated IFNgamma producing CD4+CD45RO+CD62L-CD11aBrightCD27- cells that had increased expression of intracellular BCL2, displayed distinct chemokine receptor profiles, and exhibited distinct migratory characteristics. Only CD3/CD28 with ICAM-3 generated CD4+CD45RO+CD62L-CD11aBrightCD27- cells that were functionally responsive to chemotaxis and exhibited higher frequencies of cells that signaled to JNK and ERK1/2 upon stimulation with MIP3alpha. Furthermore, these reports identify that the LFA-1 receptor, when presented with multiple ligands, can result in distinct T cell differentiation states and suggest that the combinatorial integration of ICAM ligand interactions with LFA-1 have functional consequences for T cell biology., Conclusion: Thus, the ICAM ligands, differentially modulate LFA-1 signaling in T cells and potentiate the development of memory human T cells in vitro. These findings are of importance in a mechanistic understanding of memory cell differentiation and ex vivo generation of memory cell subsets for therapeutic applications.

Understanding a molecular basis for cellular function is a common goal of biomedicine. The complex and dynamic cellular processes underlying physiological processes become subtly or grossly perturbed in human disease. A primary objective is to demystify this complexity by creating and establishing relevant model systems to study important aspects of human disease. Although significant technological advancements over the last decade in both genomic and proteomic arenas have enabled progress, accessing the complexity of cellular interactions that occur in vivo has been a difficult arena in which to make progress. Moreover, there are extensive challenges in translating research tools to clinical applications. Flow cytometry, over the course of the last 40 years, has revolutionized the field of immunology, in both the basic science and clinical settings, as well as having been instrumental to new and exciting areas of discovery such as stem cell biology. Multiparameter machinery and systems exist now to access the heterogeneity of cellular subsets and enable phenotypic characterization and functional assays to be performed on material from both animal models and humans. This review focuses primarily on the development and application of using activation-state readouts of intracellular activity for phospho-epitopes. We present recent work on how a flow cytometric platform is used to obtain mechanistic insight into cellular processes as well as highlight the clinical applications that our laboratory has explored. Furthermore, this review discusses the challenges faced with processing high-content multidimensional and multivariate data sets. Flow cytometry, as a platform that is well situated in both the research and clinical settings, can contribute to drug discovery as well as having utility for both biomarker and patient-stratification.

Autoimmune disease pathologies are multifactorial with complex interactive networks of cells and chemical messengers that initiate cascades of aberrant cellular activity. Rheumatoid arthritis (RA) is a chronic inflammatory disease that is characterized by systemic inflammation, destruction of the joints, and production of autoantibodies recognizing dozens of putative autoantigens. The presence of autoreactive T cells in individuals leads to pathological autoimmunity by activating additional cellular constituents to mediate inflammation and joint destruction. The etiology of RA is unknown, and knowledge is lacking of the molecular mechanisms underlying the production and subsequent regulation of autoreactive T cells and predicting patient responses to treatments. Biochemical investigations into mechanisms of the disease have relied on animal models that are helpful in dissecting elements of the disease but that are not necessarily reflective of human RA development. The study of multiple activated signaling pathways in complex populations of cells, such as peripheral blood, at the single-cell level has not previously been possible. This article describes how intracellular phosphoepitope staining methodology in conjunction with surface-cell immunophenotyping can be used to deconvolute cellular subsets and allow functional characterization of patient-derived material. Multiparameter flow cytometric analysis allows for small subpopulations-representing different cellular subsets and differentiation or activation states-to be discerned and simultaneously assessed for intracellular biochemical activities. This article also describes how single-cell signal network analysis can be used to stratify patients and may be useful for understanding mechanisms of disease progression, treatment resistance, and development of diagnostic indicators.

This unit presents protocols for measuring intracellular phosphoepitopes by flow cytometry for biochemical investigations in both human and murine primary cells as well as in cell lines. Conventional methods that require cellular lysis cannot discriminate between proteins from different cellular subsets. Intracellular detection of phosphorylated and nonphosphorylated signaling molecules, on the other hand, has recently exposed the heterogeneity that can be observed upon signal transduction. Although staining methodologies for cytokines and cell cycle antigens have been appreciated for years, detection of phosphorylated molecules presents several new challenges, including generation of reagents and details of the staining technique. As these techniques are adapted to new applications, the protocols continue to be refined. This unit describes signal amplification of intracellular signals upon detergent-based permeabilizations, staining protocol for adherent cells, methodology for whole-blood staining, and multiparameter staining procedures for surface and intracellular antigens.

Leukocyte function antigen 1 (LFA-1) is essential for the formation of immune cell synapses and plays a role in the pathophysiology of various autoimmune diseases. We investigated the molecular details of LFA-1 activation during adhesion between cytotoxic cells and a target model leukemia cell. The cytolytic activity of a CD3-CD8+CD56+ natural killer (NK) subset was enhanced when LFA-1 was activated. In a comparison of LFA-1 ligands, intercellular adhesion molecule 2 (ICAM-2) and ICAM-3 promoted LFA-1-directed perforin release, whereas ICAM-1 had little effect. Ligand-induced LFA-1 clustering facilitated perforin release, demonstrating LFA-1 could regulate degranulation mechanisms. LFA-1 induced the activation of src family kinases, Vav1 and p44/42 mitogen-activated protein kinase (MAPK), in human CD56+ NK cells as evidenced by intracellular phospho-epitope measurements that correlated with effector-target cell binding and perforin-granzyme A-mediated cytolytic activity. These results identify novel, specific functional consequence of LFA-1-mediated cytolytic activity in perforin-containing human NK subsets.

Altered growth factor responses in phospho-protein-driven signaling networks are crucial to cancer cell survival and pathology. Profiles of cancer cell signaling networks might therefore identify mechanisms by which such cells interpret environmental cues for continued growth. Using multiparameter flow cytometry, we monitored phospho-protein responses to environmental cues in acute myeloid leukemia at the single cell level. By exposing cancer cell signaling networks to potentiating inputs, rather than relying upon the basal levels of protein phosphorylation alone, we could discern unique cancer network profiles that correlated with genetics and disease outcome. Strikingly, individual cancers manifested multiple cell subsets with unique network profiles, reflecting cancer heterogeneity at the level of signaling response. The results revealed a dramatic remodeling of signaling networks in cancer cells. Thus, single cell measurements of phospho-protein responses reveal shifts in signaling potential of a phospho-protein network, allowing for categorizing of cell network phenotypes by multidimensional molecular profiles of signaling.

Analysis of protein phosphorylation with flow cytometric techniques has emerged as a powerful tool in the field of immunological signaling, allowing cellular subsets in complex populations to be analyzed accurately and rapidly. In this review, we examine the development of phospho-epitope, or phospho-specific, flow cytometry and the premises upon which the technique is based. Phospho-specific flow cytometry is compared to traditional biochemical methods, and its advantages, such as single cell analysis, multiparameter data acquisition, rapid protocols, and the ability to analyze rare cell subsets, are detailed. We also discuss the many technical considerations that must be addressed when developing new antibodies or analyzing new epitopes including antigen accessibility, stability of the phospho-epitope, fluorophore selection, surface phenotype integrity, and antibody suitability for staining epitopes inside fixed and permeabilized cells. The methods that have been used to date are described in light of these technical considerations. The importance of developing bioinformatic platforms in parallel with these techniques is emphasized due to the large, multiparameter datasets that are rapidly accumulated and which require more efficient data viewing and complex clustering methods than currently available for flow cytometric data. Finally, we discuss the potential clinical applications of phospho-specific flow cytometry in analyzing immune cell development and antigen-specific immune responses, as well as pharmacodynamic profiling of disease states or drug efficacy and specificity against particular signaling proteins.

Flow cytometry offers the capability to assess the heterogeneity of cellular subsets that exist in complex populations, such as peripheral blood, based on immunophenotypes. We describe methodologies to measure phospho-epitopes in single cells as determinants of intracellular kinase activity. Multiparametric staining, using both surface and intracellular stains, allows for the study of discrete biochemical events in readily discernible lymphocyte subsets. As such, the usage of multiparameter flow cytometry to obtain proteomic information provides several major advantages: (1) the ability to perform multiparametric experiments to identify distinct signaling profiles in defined lymphocyte populations, (2) simultaneous correlation of multiple active kinases involved in signaling cascades, (3) profiling of active kinase states to identify signaling signatures of interest rapidly, and (4) biochemical access to rare cell subsets such as those from clinically derived samples or populations that comprise too few in numbers for conventional biochemical analysis.

Leukocyte functional antigen 1 (LFA-1), with intercellular adhesion molecule ligands, mediates T cell adhesion, but the signaling pathways and functional effects imparted by LFA-1 are unclear. Here, intracellular phosphoprotein staining with 13-dimensional flow cytometry showed that LFA-1 activation induced phosphorylation of the beta(2) integrin chain and release of Jun-activating binding protein 1 (JAB-1), and mediated signaling of kinase Erk1/2 through cytohesin-1. Dominant negatives of both JAB-1 and cytohesin-1 inhibited interleukin 2 production and impaired T helper type 1 differentiation. LFA-1 stimulation lowered the threshold of T cell activation. Thus, LFA-1 signaling contributes to T cell activation and effects T cell differentiation.

Using a muscle cell differentiation screen, we have identified myoseverin from a 2,6,9-trisubsituted purine library as a purine-based microtubule binding molecule [1]. Structure-activity relation studies of myoseverin identify positions N2 and N6 to be critical for inhibiting muscle differentiation. Inhibition of microtubule polymerization induced the reversion of terminally differentiated myotubes to mononucleated cells that were responsive to both growth and differentiation conditions, without any observable cytotoxicity. Comparison of myoseverin derivatives to taxol, vinblastine, nocodazole, and colchicine identify myoseverin's effect as being selectively reversible in addition to lacking the cytotoxic effects of these non-purine-based microtubule-disrupting molecules. Myoseverin, as a purine-based microtubule inhibitor, reverted terminal muscle-differentiated cells to a state that was responsive to environmental cues. These results suggest that myoseverin may have applications in muscle regeneration and stem cell differentiation.

Here, we show that motexafin gadolinium (Gd-Tex), a compound that promotes intracellular oxidative stress, selectively induces apoptosis in HIV-1-infected CD4(+) T cells in IL-2-stimulated cultures of peripheral blood mononuclear cells infected in vitro with HIV-1. This selective induction of apoptosis, which we detect by FACS analysis of intracellular HIV/p24 and concomitant surface and apoptosis marker expression, is abrogated by the glutathione precursor, N-acetyl-l-cysteine. Importantly, it occurs at Gd-Tex concentrations that are not cytotoxic to uninfected cells in the culture. These findings suggest that Gd-Tex may have therapeutic utility as an anti-HIV agent capable of selectively targeting and removing HIV-infected cells in an infected host.

Intracellular assays of signaling systems have been limited by an inability to correlate functional subsets of cells in complex populations on the basis of active kinase states. Such correlations could be important in distinguishing changes in signaling status that arise in rare cell subsets during functional activation or in disease manifestation. Here we demonstrate the ability to simultaneously detect activated kinase members of the mitogen-activated protein kinases family (p38 MAPK, p44/42 MAPK, JNK/SAPK), members of cell survival pathways (AKT/PKB), and members of T-cell activation pathways (TYK2), among others, in subpopulations of complex cell populations by multiparameter flow-cytometric analysis. We demonstrate the utility of these probes in identifying distinct signaling cascades for (1) both artificial and physiological stimulatory conditions of peripheral blood mononuclear cells (PBMCs), (2) cytokine stimulation in human memory and naïve lymphocyte subsets as identified by five differentiation markers, and (3) ordering of kinase activation in potential signaling hierarchies. Polychromatic flow-cytometric active kinase measurements demonstrate that multidimensional analysis of signaling pathways can provide functional signaling pathway assessment on a single-cell level and allow for potential correlation with biological and clinical parameters.

We identified intracellular adhesion molecule-2 (ICAM-2) in a genetic screen as an activator of the PI3K/AKT pathway leading to inhibition of apoptosis. ICAM-2 induced tyrosine phosphorylation of ezrin and PI3K kinase membrane translocation, resulting in phosphatidylinositol 3,4,5 production, PDK-1 and AKT activation, and subsequent phosphorylation of AKT targets BAD, GSK3, and FKHR. ICAM-2 clustering protected primary human CD19+ cells from TNFalpha- and Fas-mediated apoptosis as determined by single-cell analysis. ICAM-2 engagement by CD19+ cells of its natural receptor, LFA-1, on CD4+ naive cells specifically induced AKT activity in the absence of an MHC-peptide interaction. These results attribute a novel signaling function to ICAM-2 that might suggest mechanisms by which ICAM-2 signals intracellular communication at various immunological synapses.

HIV-1 budding appears to require Vps4 and Tsg101-two proteins that have links to endosomal sorting machinery. A picture emerges wherein divergent viruses recruit endosomal proteins like Tsg101 to gain access to ubiquitin processes that play a crucial role during viral budding.