Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry.

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to assess calcium signaling by band-pass flow cytometry. This protocol is designed to measure calcium responses in primary murine T-cells using full spectrum flow cytometry. Total splenocytes are labeled with the ratiometric calcium indicator dye Indo-1, along with a panel of fluorochrome-conjugated antibodies to cell surface molecules. Leveraging the capabilities of full spectrum flow cytometry provides a platform for utilizing a wide array of cell surface stains in combination with Indo-1. Cells are then analyzed in real-time at 37 °C before and after the addition of an anti-CD3 antibody to stimulate the T-cell receptor. After unmixing the spectral signals, the ratio of calcium-bound to calcium-free Indo-1 is calculated and can be visualized over time for each gated population of splenocytes. This technique can allow for the simultaneous analysis of calcium responses in multiple cell populations.

Hierarchy of signaling thresholds downstream of the T cell receptor and the Tec kinase ITK.

The strength of peptide:MHC interactions with the T cell receptor (TCR) is correlated with the time to first cell division, the relative scale of the effector cell response, and the graded expression of activation-associated proteins like IRF4. To regulate T cell activation programming, the TCR and the TCR proximal interleukin-2-inducible T cell kinase (ITK) simultaneously trigger many biochemically separate signaling cascades. T cells lacking ITK exhibit selective impairments in effector T cell responses after activation, but under the strongest signaling conditions, ITK activity is dispensable. To gain insight into whether TCR signal strength and ITK activity tune observed graded gene expression through the unequal activation of distinct signaling pathways, we examined Erk1/2 phosphorylation or nuclear factor of activated T cells (NFAT) and nuclear factor (NF)-κB translocation in naïve OT-I CD8+ cell nuclei. We observed the consistent digital activation of NFAT1 and Erk1/2, but NF-κB displayed dynamic, graded activation in response to variation in TCR signal strength, tunable by treatment with an ITK inhibitor. Inhibitor-treated cells showed the dampened induction of AP-1 factors Fos and Fosb, NF-κB response gene transcripts, and survival factor Il2 transcripts. ATAC sequencing analysis also revealed that genomic regions most sensitive to ITK inhibition were enriched for NF-κB and AP-1 motifs. Specific inhibition of NF-κB during peptide stimulation tuned the expression of early gene products like c-Fos. Together, these data indicate a key role for ITK in orchestrating the optimal activation of separate TCR downstream pathways, specifically aiding NF-κB activation. More broadly, we revealed a mechanism by which variations in TCR signal strength can produce patterns of graded gene expression in activated T cells.

Activation of the Tec Kinase ITK Controls Graded IRF4 Expression in Response to Variations in TCR Signal Strength.

TCR signal strength is critical for CD8+ T cell clonal expansion after Ag stimulation. Levels of the transcription factor IRF4 control the magnitude of this process through the induction of genes involved in proliferation and glycolytic metabolism. The signaling mechanism connecting graded TCR signaling to the generation of varying amounts of IRF4 is not well understood. In this study, we show that Ag potency regulates the kinetics but not the magnitude of NFAT1 activation in single mouse CD8+ T cells. Consequently, T cells that transduce weaker TCR signals exhibit a marked delay in Irf4 mRNA induction, resulting in decreased overall IRF4 expression in individual cells and increased heterogeneity within the clonal population. We further show that the activity of the tyrosine kinase ITK acts as a signaling catalyst that accelerates the rate of the cellular response to TCR stimulation, controlling the time to onset of Irf4 gene transcription. These findings provide insight into the function of ITK in TCR signal transduction that ultimately regulates IRF4 expression levels in response to variations in TCR signal strength.

Peptide Antigen Concentration Modulates Digital NFAT1 Activation in Primary Mouse Naive CD8+ T Cells as Measured by Flow Cytometry of Isolated Cell Nuclei.

Circulating naive T cells exist in a quiescent state. After TCR contact with the cognate peptide presented by APCs in secondary lymphoid structures, T cells undergo a period of rapid transcriptional changes that set the stage for fate-determining effector or memory programming. We describe a novel method to analyze TCR signaling pathway activation in nuclei isolated from primary mouse naive T cells after stimulation with natural peptide Ags. We prelabeled cells with cell tracking dye to easily distinguish CD8+ T cell nuclei from APC nuclei by conventional flow cytometry. Using this approach, we observed clear digital activation of NFAT1 transcription factor in OT-I T cells stimulated with OVA peptide presented by bulk splenocytes. OVA concentration had discrete control over the fraction of the cells that translocated NFAT1, indicating that a distinct threshold amount of TCR signaling is required to switch on NFAT1 in naive T cells. This behavior was cell contact dependent and qualitatively more exact than the NFAT1 response in ionomycin-stimulated naive T cells. These data contribute to our understanding of the digital behavior of TCR signaling components documented in other studies and indicate how T cells might discriminate log-fold changes in Ag availability during an actual infection. Overall, these results highlight the potential of this coculture nuclei isolation protocol to address stimulation-dependent translocation of proteins in primary lymphocytes.

Multidomain Control Over TEC Kinase Activation State Tunes the T Cell Response.

Signaling through the T cell antigen receptor (TCR) activates a series of tyrosine kinases. Directly associated with the TCR, the SRC family kinase LCK and the SYK family kinase ZAP-70 are essential for all downstream responses to TCR stimulation. In contrast, the TEC family kinase ITK is not an obligate component of the TCR cascade. Instead, ITK functions as a tuning dial, to translate variations in TCR signal strength into differential programs of gene expression. Recent insights into TEC kinase structure have provided a view into the molecular mechanisms that generate different states of kinase activation. In resting lymphocytes, TEC kinases are autoinhibited, and multiple interactions between the regulatory and kinase domains maintain low activity. Following TCR stimulation, newly generated signaling modules compete with the autoinhibited core and shift the conformational ensemble to the fully active kinase. This multidomain control over kinase activation state provides a structural mechanism to account for ITK's ability to tune the TCR signal.

Patient predictors and utilization of health services within a medical home for homeless persons.

BACKGROUND: The Veterans Health Administration (VHA) established a patient-centered medical home model of care for veterans experiencing homelessness called a Homeless Patient Aligned Care Team (HPACT) to improve engagement with primary care and reduce utilization of hospital-based services. To evaluate the impact of the HPACT model, this study compares the number and type of health care visits in the 12 months before and after enrollment in HPACT at one VHA facility, and explores patient characteristics associated with increases and decreases in visits. METHODS: Chart reviews of VHA medical records were conducted for all patients enrolled in an HPACT in Pittsburgh, Pennsylvania, between May 2012 and December 2013 (N = 179). Multivariable mixed-effect logistic regressions estimated differences in having any visit in the 0-6 months and 7-12 months before and after HPACT enrollment, and multinomial logistic regressions predicted increases or decreases versus no change in number of visits over 12 months. RESULTS: Compared with 0-6 months prior to HPACT, patients were more likely to visit primary care in the 0-6 months (adjusted odds ratio [aOR] = 4.91, 95% confidence interval [CI] = 2.94-8.20) and 7-12 months (aOR = 2.30, 95% CI = 1.42-3.72) following HPACT. Patients were less likely to visit the emergency department (ED) or to be hospitalized in the 0-6 months (aOR = 0.57, 95% CI = 0.34-0.94; and aOR = 0.55, 95% CI = 0.25-0.76) and 7-12 months (aOR = 0.43, 95% CI = 0.33-0.91; and aOR = 0.45, 95% CI = 0.26-0.80) following HPACT. Patients were less likely to visit mental health (aOR = 0.35, 95% CI = 0.20-0.60) and addiction specialists (aOR = 0.39, 95% CI = 0.18-0.84) in the 7-12 months following HPACT. Overall, 59% of patients had increases in primary care visits following HPACT. Female patients and those with self-housing were less likely to have increases versus no change in primary care visits (adjusted relative risk ratio [aRRR] = 0.15, 95% CI = 0.03-0.74; and aRRR = 0.35, 95% CI = 0.14-0.90). CONCLUSIONS: An integrated HPACT model was successful in engaging homeless veterans in primary care for 1 year, potentially contributing to reductions in ED use. More tailored approaches may be needed for vulnerable populations experiencing homelessness, including homeless women.

Homeless veterans' experiences with substance use, recovery, and treatment through photo elicitation.

BACKGROUND: Homeless veterans often have addictions and comorbidities that complicate utilization of longitudinal health care services, such as primary care. An understanding of experiences of veterans enrolled in a Homeless Patient Aligned Care Team (H-PACT) may improve addiction treatment engagement in these settings. The authors aimed to describe H-PACT veterans' experiences with substance use (SU), substance use recovery (SUR), and substance use treatment (SUT). METHODS: Homeless veterans were recruited from a veteran primary care medical home clinic between September 2014 and March 2015. Twenty veterans were given digital cameras and prompts for taking photographs about their health and health care and participated in 2 photo elicitation interviews. For this secondary analysis, transcripts from the audio-recorded interviews were analyzed by 2 coders using qualitative content analysis. RESULTS: The majority of participants (75%, n = 15) discussed SU, SUR, and/or SUT in regards to their health and health care utilization. SU themes centered on disclosure of addiction or dependency; substances used; repercussions of SU; SU as a coping mechanism; and association of SU with military service. SUR themes included disclosure of length of sobriety; perceived facilitators of SUR in health, beliefs, social, environmental, financial, and creative pursuit domains; and perceived barriers to SUR in beliefs, social, and environmental domains. SUT themes focused on perceived facilitators of SUT in access to Department of Veterans Affairs (VA) and non-VA services and social domains and perceived barriers to SUT in the social domain. CONCLUSIONS: Providers seeking to elicit addiction-related clinical history and facilitate SUR and SUT might look to the current findings for guidance. Provider training in motivational interviewing may be warranted, which allows for an exploration of health-related consequences of SU and supports patients' self-efficacy.

Left, right, and meeting in the middle: Addressing addiction is something we can agree about.

The United States faces an addiction health crisis. Presidential election cycles in the United States are cause for creation of political party platforms. These platforms provide general stances and specific policies on a variety of issues. We undertook a review of the addiction policies of the 2016 Republican and Democratic platforms. Despite differences in focus, we found more similarities than differences between the two. We call upon those in political power to use every evidence-based policy at their disposal to promote addiction treatment and prevention.

T Cells and Gene Regulation: The Switching On and Turning Up of Genes after T Cell Receptor Stimulation in CD8 T Cells.

Signaling downstream of the T cell receptor (TCR) is directly regulated by the dose and affinity of peptide antigen. The strength of TCR signaling drives a multitude of T cell functions from development to differentiation. CD8 T cells differentiate into a diverse pool of effector and memory cells after activation, a process that is critical for pathogen clearance and is highly regulated by TCR signal strength. T cells rapidly alter their gene expression upon activation. Multiple signaling pathways downstream of the TCR activate transcription factors, which are critical for this process. The dynamics between proximal TCR signaling, transcription factor activation and CD8 T cell function are discussed here. We propose that inducible T cell kinase (ITK) acts as a rheostat for gene expression. This unique regulation of TCR signaling by ITK provides a possible signaling mechanism for the promotion of a diverse T cell repertoire in response to pathogen.

IRF4 Regulates the Ratio of T-Bet to Eomesodermin in CD8+ T Cells Responding to Persistent LCMV Infection.

CD8+ T cell exhaustion commonly occurs in chronic infections and cancers. During T cell exhaustion there is a progressive and hierarchical loss of effector cytokine production, up-regulation of inhibitory co-stimulatory molecules, and eventual deletion of antigen specific cells by apoptosis. A key factor that regulates T cell exhaustion is persistent TCR stimulation. Loss of this interaction results in restoration of CD8+ T cell effector functions in previously exhausted CD8+ T cells. TCR stimulation is also important for the differentiation of Eomeshi anti-viral CD8+ effector T cells from T-bethi precursors, both of which are required for optimal viral control. However, the molecular mechanisms regulating the differentiation of these two cell subsets and the relative ratios required for viral clearance have not been described. We show that TCR signal strength regulates the relative expression of T-bet and Eomes in antigen-specific CD8+ T cells by modulating levels of IRF4. Reduced IRF4 expression results in skewing of this ratio in the favor of Eomes, leading to lower proportions and numbers of T-bet+ Eomes- precursors and poor control of LCMV-clone 13 infection. Manipulation of this ratio in the favor of T-bet restores the differentiation of T-bet+ Eomes- precursors and the protective balance of T-bet to Eomes required for efficient viral control. These data highlight a critical role for IRF4 in regulating protective anti-viral CD8+ T cell responses by ensuring a balanced ratio of T-bet to Eomes, leading to the ultimate control of this chronic viral infection.

Impact of the Smoothened inhibitor, IPI-926, on smoothened ciliary localization and Hedgehog pathway activity.

A requisite step for canonical Hedgehog (Hh) pathway activation by Sonic Hedgehog (Shh) ligand is accumulation of Smoothened (Smo) to the primary cilium (PC). Activation of the Hh pathway has been implicated in a broad range of cancers, and several Smo antagonists are being assessed clinically, one of which is approved for the treatment of advanced basal cell carcinoma. Recent reports demonstrate that various Smo antagonists differentially impact Smo localization to the PC while still exerting inhibitory activity. In contrast to other synthetic small molecule Smo antagonists, the natural product cyclopamine binds to and promotes ciliary accumulation of Smo and "primes" cells for Hh pathway hyper-responsiveness after compound withdrawal. We compared the properties of IPI-926, a semi-synthetic cyclopamine analog, to cyclopamine with regard to potency, ciliary Smo accumulation, and Hh pathway activity after compound withdrawal. Like cyclopamine, IPI-926 promoted accumulation of Smo to the PC. However, in contrast to cyclopamine, IPI-926 treatment did not prime cells for hyper-responsiveness to Shh stimulation after compound withdrawal, but instead demonstrated continuous inhibition of signaling. By comparing the levels of drug-induced ciliary Smo accumulation with the degree of Hh pathway activity after compound withdrawal, we propose that a critical threshold of ciliary Smo is necessary for "priming" activity to occur. This "priming" appears achievable with cyclopamine, but not IPI-926, and is cell-line dependent. Additionally, IPI-926 activity was evaluated in a murine tumor xenograft model and a pharmacokinetic/pharmacodynamic relationship was examined to assess for in vivo evidence of Hh pathway hyper-responsiveness. Plasma concentrations of IPI-926 correlated with the degree and duration of Hh pathway suppression, and pathway activity did not exceed baseline levels out to 96 hours post dose. The overall findings suggest that IPI-926 possesses unique biophysical and pharmacological properties that result in Hh pathway inhibition in a manner that differentiates it from cyclopamine.

Medical consequences of marijuana use: a review of current literature.

With the advent of legalization of marijuana for medicinal and recreational purposes, and the increase use of marijuana, healthcare providers will be increasingly confronted with marijuana users as patients in clinical environments. While there is vast literature regarding the societal and mental health harms associated with marijuana use, there is a paucity of reviews of the potential consequences of marijuana use on physical health or medical conditions. We examine the recent literature on the physical harms associated with illicit and legal marijuana administration. We surveyed the peer-reviewed medical literature from 1998 to 2013 of studies assessing the association of marijuana use and physical diseases. We conclude that healthcare providers should be cognizant that the existing literature suggests that marijuana use can cause physical harm. However, evidence is needed, and further research should be considered, to prove causal associations of marijuana with many physical health conditions.

A scoping review of interdisciplinary collaboration in addictions education and training.

Developing a workforce of multidisciplinary healthcare professionals equipped with the knowledge and skills to collaboratively address the public health crisis of alcohol and other drug (AOD) use is critical for effectively identifying, preventing, and managing AOD conditions and their sequelae. Despite general enthusiasm for interdisciplinary education and training, little is known overall about the nature and outcomes of interdisciplinary collaboration in addictions education and training. We conducted a five-stage scoping review of the literature to provide an eight domain overview of the state of interdisciplinary collaboration in addictions education (ICAE). In our final review of 30 articles, we identified a lack of conceptual and terminological clarity around ICAE but a wide range of learners and professional collaborators in ICAE initiatives, which focused on a variety of AOD topics and used a constellation of didactic, interactive, and service-learning teaching strategies and formats. Although we found limited substantive educational or practice-oriented outcomes available for ICAE initiatives, learner and faculty feedback reflected high enthusiasm for ICAE and widespread perceptions of benefit for improved clinical care. Facilitators and barriers to the implementation of ICAE initiatives occurred at the level of the individual and the institution and ranged from pragmatic to conceptual. Emerging trends in ICAE initiatives included increased application of learning and implementation theory and extension of ICAE into research training. We conclude with recommendations to support ICAE as a new paradigm for addictions education for all health professionals.

Synthetic silvestrol analogues as potent and selective protein synthesis inhibitors.

Misregulation of protein translation plays a critical role in human cancer pathogenesis at many levels. Silvestrol, a cyclopenta[b]benzofuran natural product, blocks translation at the initiation step by interfering with assembly of the eIF4F translation complex. Silvestrol has a complex chemical structure whose functional group requirements have not been systematically investigated. Moreover, silvestrol has limited development potential due to poor druglike properties. Herein, we sought to develop a practical synthesis of key intermediates of silvestrol and explore structure-activity relationships around the C6 position. The ability of silvestrol and analogues to selectively inhibit the translation of proteins with high requirement on the translation-initiation machinery (i.e., complex 5'-untranslated region UTR) relative to simple 5'UTR was determined by a cellular reporter assay. Simplified analogues of silvestrol such as compounds 74 and 76 were shown to have similar cytotoxic potency and better ADME characteristics relative to those of silvestrol.

The calcineurin inhibitor tacrolimus activates the renal sodium chloride cotransporter to cause hypertension.

Calcineurin inhibitors (CNIs) are immunosuppressive drugs that are used widely to prevent rejection of transplanted organs and to treat autoimmune disease. Hypertension and renal tubule dysfunction, including hyperkalemia, hypercalciuria and acidosis, often complicate their use. These side effects resemble familial hyperkalemic hypertension, a genetic disease characterized by overactivity of the renal sodium chloride cotransporter (NCC) and caused by mutations in genes encoding WNK kinases. We hypothesized that CNIs induce hypertension by stimulating NCC. In wild-type mice, the CNI tacrolimus caused salt-sensitive hypertension and increased the abundance of phosphorylated NCC and the NCC-regulatory kinases WNK3, WNK4 and SPAK. We demonstrated the functional importance of NCC in this response by showing that tacrolimus did not affect blood pressure in NCC-knockout mice, whereas the hypertensive response to tacrolimus was exaggerated in mice overexpressing NCC. Moreover, hydrochlorothiazide, an NCC-blocking drug, reversed tacrolimus-induced hypertension. These observations were extended to humans by showing that kidney transplant recipients treated with tacrolimus had a greater fractional chloride excretion in response to bendroflumethiazide, another NCC-blocking drug, than individuals not treated with tacrolimus; renal NCC abundance was also greater. Together, these findings indicate that tacrolimus-induced chronic hypertension is mediated largely by NCC activation, and suggest that inexpensive and well-tolerated thiazide diuretics may be especially effective in preventing the complications of CNI treatment.

Do statins delay the incidence of ESRD in diabetic patients with moderate CKD?

BACKGROUND: There is little information on whether statins and LDL cholesterol are associated with the onset of end-stage renal disease (ESRD) in at-risk populations. METHODS: In a retrospective, single-center study of 156 patients with diabetes and CKD, we evaluated disease progression and incidence of ESRD in patients treated or not treated with statins. RESULTS: There were 81 and 75 patients in statin and no-statin groups respectively. Participants were followed for a median of 28 months (95% CI 19 to 34). Total cholesterol and LDL levels decreased significantly in the statin group and were associated with a significant decrease in the rate of eGFR decline (-6.0 vs. -9.8 mL/min/1.73m(2)/year, p=0.01). However, this difference was not statistically significant after consideration of propensity scores (p=0.43). Kaplan-Meier survival analyses demonstrated that the incidence of ESRD was significantly greater in the no-treatment group (HR=2.21, 95% CI 1.45-3.81, p=0.0007). LDL cholesterol, statins, eGFR, mean arterial pressure and ACE-I/ARB were significant predictors of ESRD in univariate analyses. However, stepwise Cox regression analyses using propensity scores only retained LDL and baseline eGFR as independent predictors of ESRD. CONCLUSIONS: Only LDL cholesterol but not statins were associated with ESRD progression in diabetic patients with moderate CKD after data adjustment for propensity scores. Prospective studies are needed to determine if LDL cholesterol mediates the effect of statins on disease progression in these patients.

Discovery of a potent and orally active hedgehog pathway antagonist (IPI-926).

Recent evidence suggests that blocking aberrant hedgehog pathway signaling may be a promising therapeutic strategy for the treatment of several types of cancer. Cyclopamine, a plant Veratrum alkaloid, is a natural product antagonist of the hedgehog pathway. In a previous report, a seven-membered D-ring semisynthetic analogue of cyclopamine, IPI-269609 (2), was shown to have greater acid stability and better aqueous solubility compared to cyclopamine. Further modifications of the A-ring system generated three series of analogues with improved potency and/or solubility. Lead compounds from each series were characterized in vitro and evaluated in vivo for biological activity and pharmacokinetic properties. These studies led to the discovery of IPI-926 (compound 28), a novel semisynthetic cyclopamine analogue with substantially improved pharmaceutical properties and potency and a favorable pharmacokinetic profile relative to cyclopamine and compound 2. As a result, complete tumor regression was observed in a Hh-dependent medulloblastoma allograft model after daily oral administration of 40 mg/kg of compound 28.