Signaling via a CD27-TRAF2-SHP-1 axis during naive T cell activation promotes memory-associated gene regulatory networks.

The interaction of the tumor necrosis factor receptor (TNFR) family member CD27 on naive CD8+ T (Tn) cells with homotrimeric CD70 on antigen-presenting cells (APCs) is necessary for T cell memory fate determination. Here, we examined CD27 signaling during Tn cell activation and differentiation. In conjunction with T cell receptor (TCR) stimulation, ligation of CD27 by a synthetic trimeric CD70 ligand triggered CD27 internalization and degradation, suggesting active regulation of this signaling axis. Internalized CD27 recruited the signaling adaptor TRAF2 and the phosphatase SHP-1, thereby modulating TCR and CD28 signals. CD27-mediated modulation of TCR signals promoted transcription factor circuits that induced memory rather than effector associated gene programs, which are induced by CD28 costimulation. CD27-costimulated chimeric antigen receptor (CAR)-engineered T cells exhibited improved tumor control compared with CD28-costimulated CAR-T cells. Thus, CD27 signaling during Tn cell activation promotes memory properties with relevance to T cell immunotherapy.

Systemically administered low-affinity HER2 CAR T cells mediate antitumor efficacy without toxicity.

BACKGROUND: The paucity of tumor-specific targets for chimeric antigen receptor (CAR) T-cell therapy of solid tumors necessitates careful preclinical evaluation of the therapeutic window for candidate antigens. Human epidermal growth factor receptor 2 (HER2) is an attractive candidate for CAR T-cell therapy in humans but has the potential for eliciting on-target off-tumor toxicity. We developed an immunocompetent tumor model of CAR T-cell therapy targeting murine HER2 (mHER2) and examined the effect of CAR affinity, T-cell dose, and lymphodepletion on safety and efficacy. METHODS: Antibodies specific for mHER2 were generated, screened for affinity and specificity, tested for immunohistochemical staining of HER2 on normal tissues, and used for HER2-targeted CAR design. CAR candidates were evaluated for T-cell surface expression and the ability to induce T-cell proliferation, cytokine production, and cytotoxicity when transduced T cells were co-cultured with mHER2+ tumor cells in vitro. Safety and efficacy of various HER2 CARs was evaluated in two tumor models and normal non-tumor-bearing mice. RESULTS: Mice express HER2 in the same epithelial tissues as humans, rendering these tissues vulnerable to recognition by systemically administered HER2 CAR T cells. CAR T cells designed with single-chain variable fragment (scFvs) that have high-affinity for HER2 infiltrated and caused toxicity to normal HER2-positive tissues but exhibited poor infiltration into tumors and antitumor activity. In contrast, CAR T cells designed with an scFv with low-affinity for HER2 infiltrated HER2-positive tumors and controlled tumor growth without toxicity. Toxicity mediated by high-affinity CAR T cells was independent of tumor burden and correlated with proliferation of CAR T cells post infusion. CONCLUSIONS: Our findings illustrate the disadvantage of high-affinity CARs for targets such as HER2 that are expressed on normal tissues. The use of low-affinity HER2 CARs can safely regress tumors identifying a potential path for therapy of solid tumors that exhibit high levels of HER2.

Versatile Tissue-Injectable Hydrogels with Extended Hydrolytic Release of Bioactive Protein Therapeutics.

Hydrogels generally have broad utilization in healthcare due to their tunable structures, high water content, and inherent biocompatibility. FDA-approved applications of hydrogels include spinal cord regeneration, skin fillers, and local therapeutic delivery. Drawbacks exist in the clinical hydrogel space, largely pertaining to inconsistent therapeutic exposure, short-lived release windows, and difficulties inserting the polymer into tissue. In this study, we engineered injectable, biocompatible hydrogels that function as a local protein therapeutic depot with a high degree of user-customizability. We showcase a PEG-based hydrogel functionalized with bioorthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) handles for its polymerization and functionalization with a variety of payloads. Small-molecule and protein cargos, including chemokines and antibodies, were site-specifically modified with hydrolysable "azidoesters" of varying hydrophobicity via direct chemical conjugation or sortase-mediated transpeptidation. These hydrolysable esters afforded extended release of payloads linked to our hydrogels beyond diffusion; with timescales spanning days to months dependent on ester hydrophobicity. Injected hydrogels polymerize in situ and remain in tissue over extended periods of time. Hydrogel-delivered protein payloads elicit biological activity after being modified with SPAAC-compatible linkers, as demonstrated by the successful recruitment of murine T-cells to a mouse melanoma model by hydrolytically released murine CXCL10. These results highlight a highly versatile, customizable hydrogel-based delivery system for local delivery of protein therapeutics with payload release profiles appropriate for a variety of clinical needs.

The Sinorhizobium meliloti Nitrogen Stress Response Changes Radically in the Face of Concurrent Phosphate Stress.

Expression of hundreds of S. meliloti genes changed more than two-fold in response to either nitrogen or phosphate limitation. When these two stresses were applied together, stress responsive gene expression shifted dramatically. In particular, the nitrogen stress response in the presence of phosphate stress had only 30 of about 350 genes in common with the 280 genes that responded to nitrogen stress with adequate phosphate. Expression of sRNAs was also altered in response to these stresses. 82% of genes that responded to nitrogen stress also responded to phosphate stress, including 20 sRNAs. A subset of these sRNAs is known to be chaperoned by the RNA binding protein, Hfq. Hfq had previously been shown to influence about a third of the genes that responded to both nitrogen and phosphate stresses. Phosphate limitation influenced changes in gene expression more than nitrogen limitation and, when both stresses were present, phosphate stress sometimes reversed the direction of some of the changes induced by nitrogen stress. These nutrient stress responses are therefore context dependent.

Functional cognitive disorder: dementia's blind spot.

An increasing proportion of cognitive difficulties are recognized to have a functional cause, the chief clinical indicator of which is internal inconsistency. When these symptoms are impairing or distressing, and not better explained by other disorders, this can be conceptualized as a cognitive variant of functional neurological disorder, termed functional cognitive disorder (FCD). FCD is likely very common in clinical practice but may be under-diagnosed. Clinicians in many settings make liberal use of the descriptive term mild cognitive impairment (MCI) for those with cognitive difficulties not impairing enough to qualify as dementia. However, MCI is an aetiology-neutral description, which therefore includes patients with a wide range of underlying causes. Consequently, a proportion of MCI cases are due to non-neurodegenerative processes, including FCD. Indeed, significant numbers of patients diagnosed with MCI do not 'convert' to dementia. The lack of diagnostic specificity for MCI 'non-progressors' is a weakness inherent in framing MCI primarily within a deterministic neurodegenerative pathway. It is recognized that depression, anxiety and behavioural changes can represent a prodrome to neurodegeneration; empirical data are required to explore whether the same might hold for subsets of individuals with FCD. Clinicians and researchers can improve study efficacy and patient outcomes by viewing MCI as a descriptive term with a wide differential diagnosis, including potentially reversible components such as FCD. We present a preliminary definition of functional neurological disorder-cognitive subtype, explain its position in relation to other cognitive diagnoses and emerging biomarkers, highlight clinical features that can lead to positive diagnosis (as opposed to a diagnosis of exclusion), and red flags that should prompt consideration of alternative diagnoses. In the research setting, positive identifiers of FCD will enhance our recognition of individuals who are not in a neurodegenerative prodrome, while greater use of this diagnosis in clinical practice will facilitate personalized interventions.

Human heterochromatin protein 1α promotes nucleosome associations that drive chromatin condensation.

HP1(Hsα)-containing heterochromatin is located near centric regions of chromosomes and regulates DNA-mediated processes such as DNA repair and transcription. The higher-order structure of heterochromatin contributes to this regulation, yet the structure of heterochromatin is not well understood. We took a multidisciplinary approach to determine how HP1(Hsα)-nucleosome interactions contribute to the structure of heterochromatin. We show that HP1(Hsα) preferentially binds histone H3K9Me3-containing nucleosomal arrays in favor of non-methylated nucleosomal arrays and that nonspecific DNA interactions and pre-existing chromatin compaction promote binding. The chromo and chromo shadow domains of HP1(Hsα) play an essential role in HP1(Hsα)-nucleosome interactions, whereas the hinge region appears to have a less significant role. Electron microscopy of HP1(Hsα)-associated nucleosomal arrays showed that HP1(Hsα) caused nucleosome associations within an array, facilitating chromatin condensation. Differential sedimentation of HP1(Hsα)-associated nucleosomal arrays showed that HP1(Hsα) promotes interactions between arrays. These strand-to-strand interactions are supported by in vivo studies where tethering the Drosophila homologue HP1a to specific sites promotes interactions with distant chromosomal sites. Our findings demonstrate that HP1(Hsα)-nucleosome interactions cause chromatin condensation, a process that regulates many chromosome events.

Hypericum in infection: Identification of anti-viral and anti-inflammatory constituents.

The Iowa Center for Research on Botanical Dietary Supplements seeks to optimize Echinacea, Hypericum, and Prunella botanical supplements for human-health benefit, emphasizing antiviral, anti-inflammatory and anti-pain activities. This mini-review reports on ongoing studies on Hypericum. The Center uses the genetically diverse, well-documented Hypericum populations collected and maintained at the USDA-ARS North Central Regional Plant Introduction Station (NCRPIS), and the strength of research in synthetic chemistry at Iowa State University to tap natural diversity, to help discover key constituents and interactions among constituents that impact bioactivity and toxicity. The NCRPIS has acquired more than 180 distinct populations of Hypericum, with a focus on Hypericum perforatum L. (Hypericaceae), representing about 13% of currently recognized taxa. Center chemists have developed novel synthetic pathways for key flavones, acyl phloroglucinols, hyperolactones and a tetralin that have been found in Hypericum, and these compounds are used as standards and for bioactivity studies. Both light-dependent and light-independent anti-viral activities have been identified by using bioactivity-guided fractionation of H. perforatum and a HIV-1 infection test system. Our Center has focused on light-independent activity, potentially due to novel chemicals, and polar fractions are undergoing further fractionation. Anti-inflammatory activity has been found to be light-independent, and fractionation of a flavonoid-rich extract revealed four compounds (amentoflavone, chlorogenic acid, pseudohypericin and quercetin) that interacted in the light to inhibit lipopolysaccharide-induced prostaglandin E(2) activity. The Center continues to explore novel populations of H. perforatum and related species to identify constituents and interactions of constituents that contribute to potential health benefits related to infection.

Identification of light-independent inhibition of human immunodeficiency virus-1 infection through bioguided fractionation of Hypericum perforatum.

BACKGROUND: Light-dependent activities against enveloped viruses in St. John's Wort (Hypericum perforatum) extracts have been extensively studied. In contrast, light-independent antiviral activity from this species has not been investigated. RESULTS: Here, we identify the light-independent inhibition of human immunodeficiency virus-1 (HIV-1) by highly purified fractions of chloroform extracts of H. perforatum. Both cytotoxicity and antiviral activity were evident in initial chloroform extracts, but bioassay-guided fractionation produced fractions that inhibited HIV-1 with little to no cytotoxicity. Separation of these two biological activities has not been reported for constituents responsible for the light-dependent antiviral activities. Antiviral activity was associated with more polar subfractions. GC/MS analysis of the two most active subfractions identified 3-hydroxy lauric acid as predominant in one fraction and 3-hydroxy myristic acid as predominant in the other. Synthetic 3-hydroxy lauric acid inhibited HIV infectivity without cytotoxicity, suggesting that this modified fatty acid is likely responsible for observed antiviral activity present in that fraction. As production of 3-hydroxy fatty acids by plants remains controversial, H. perforatum seedlings were grown sterilely and evaluated for presence of 3-hydroxy fatty acids by GC/MS. Small quantities of some 3-hydroxy fatty acids were detected in sterile plants, whereas different 3-hydroxy fatty acids were detected in our chloroform extracts or field-grown material. CONCLUSION: Through bioguided fractionation, we have identified that 3-hydroxy lauric acid found in field grown Hypericum perforatum has anti-HIV activity. This novel anti-HIV activity can be potentially developed into inexpensive therapies, expanding the current arsenal of anti-retroviral agents.

The computerized object and abstract designs test (COAD): a pilot study of a new test of visual working memory.

OBJECTIVES: To investigate the clinical and theoretical validity of a new computerized test of visual working memory; the computerized object and abstract designs (COAD) test. The COAD test was designed to be consistent with Baddeley's inclusion of the 'episodic buffer' in the 'multicomponent model' of working memory. DESIGN AND METHOD: A cross-sectional design was used with two brain lesion groups (left N=9 and right hemisphere N=12) and a control group N=18. The participants had to complete the new test, along with standardized tests of visual working memory in current clinical use (visual patterns test and spatial span test). Differences between groups, as well as between tests were investigated. Correlations were performed across tests. Regression models were used to further evaluate the COAD clinical sensitivity in comparison with the other tests of visual working memory. RESULTS: Significant differences were observed, as predicted, between groups and between design types. The COAD test was significantly correlated with both the visual patterns Test and the spatial span test. The COAD test also proved to be more sensitive in detecting brain injury resulting in visual working memory deficits, than the standardized tests. DISCUSSION AND CONCLUSION: The results are discussed in relation to the COAD test's potential utility in the early detection of specific degenerative neurological disorders as well as the potential to be used in identifying deficits in visual working memory and in neurorehabilitation. It is concluded that the COAD test is a clinically valid psychometric test and is a more sensitive instrument than current standardized tests of visual working memory in clinical use.

Echinacea in infection.

Ongoing studies have developed strategies for identifying key bioactive compounds and chemical profiles in Echinacea with the goal of improving its human health benefits. Antiviral and antiinflammatory-antipain assays have targeted various classes of chemicals responsible for these activities. Analysis of polar fractions of E. purpurea extracts showed the presence of antiviral activity, with evidence suggesting that polyphenolic compounds other than the known HIV inhibitor, cichoric acid, may be involved. Antiinflammatory activity differed by species, with E. sanguinea having the greatest activity and E. angustifolia, E. pallida, and E. simulata having somewhat less. Fractionation and studies with pure compounds indicate that this activity is explained, at least in part, by the alkamide constituents. Ethanol extracts from Echinacea roots had potent activity as novel agonists of TRPV1, a mammalian pain receptor reported as an integrator of inflammatory pain and hyperalgesia and a prime therapeutic target for analgesic and antiinflammatory drugs. One fraction from E. purpurea ethanol extract was bioactive in this system. Interestingly, the antiinflammatory compounds identified to inhibit prostaglandin E(2) production differed from those involved in TRPV1 receptor activation.

Cognitive schemas, defence mechanisms and post-traumatic stress symptomatology.

OBJECTIVE: To identify specific ego defences and core schemas involved in post-traumatic stress disorder (PTSD) symptomatology. DESIGN: Stepwise regression with predictor models generated from ego defences and core schema variables. METHOD: Seventy-seven participants completed the Impact of Event Scale, the 40-item Defence Style Questionnaire and the Young Schema Questionnaire (short-form). RESULTS: Four core schemas (Defectiveness, Dependency, Enmeshment and Failure) and three defence mechanisms (Splitting, Rationalization and Projection) were found to be significant predictors in PTSD symptomatology. CONCLUSIONS: The identified core schema and ego defences are seen to be theoretically consistent with present cognitive and psychodynamic conceptualizations of PTSD. The implications for future research are discussed.

Inhibition of HIV-1 replication by P-TEFb inhibitors DRB, seliciclib and flavopiridol correlates with release of free P-TEFb from the large, inactive form of the complex.

BACKGROUND: The positive transcription elongation factor, P-TEFb, comprised of cyclin dependent kinase 9 (Cdk9) and cyclin T1, T2 or K regulates the productive elongation phase of RNA polymerase II (Pol II) dependent transcription of cellular and integrated viral genes. P-TEFb containing cyclin T1 is recruited to the HIV long terminal repeat (LTR) by binding to HIV Tat which in turn binds to the nascent HIV transcript. Within the cell, P-TEFb exists as a kinase-active, free form and a larger, kinase-inactive form that is believed to serve as a reservoir for the smaller form. RESULTS: We developed a method to rapidly quantitate the relative amounts of the two forms based on differential nuclear extraction. Using this technique, we found that titration of the P-TEFb inhibitors flavopiridol, DRB and seliciclib onto HeLa cells that support HIV replication led to a dose dependent loss of the large form of P-TEFb. Importantly, the reduction in the large form correlated with a reduction in HIV-1 replication such that when 50% of the large form was gone, HIV-1 replication was reduced by 50%. Some of the compounds were able to effectively block HIV replication without having a significant impact on cell viability. The most effective P-TEFb inhibitor flavopiridol was evaluated against HIV-1 in the physiologically relevant cell types, peripheral blood lymphocytes (PBLs) and monocyte derived macrophages (MDMs). Flavopiridol was found to have a smaller therapeutic index (LD50/IC50) in long term HIV-1 infectivity studies in primary cells due to greater cytotoxicity and reduced efficacy at blocking HIV-1 replication. CONCLUSION: Initial short term studies with P-TEFb inhibitors demonstrated a dose dependent loss of the large form of P-TEFb within the cell and a concomitant reduction in HIV-1 infectivity without significant cytotoxicity. These findings suggested that inhibitors of P-TEFb may serve as effective anti-HIV-1 therapies. However, longer term HIV-1 replication studies indicated that these inhibitors were more cytotoxic and less efficacious against HIV-1 in the primary cell cultures.

Manipulation of P-TEFb control machinery by HIV: recruitment of P-TEFb from the large form by Tat and binding of HEXIM1 to TAR.

Basal transcription of the HIV LTR is highly repressed and requires Tat to recruit the positive transcription elongation factor, P-TEFb, which functions to promote the transition of RNA polymerase II from abortive to productive elongation. P-TEFb is found in two forms in cells, a free, active form and a large, inactive complex that also contains 7SK RNA and HEXIM1 or HEXIM2. Here we show that HIV infection of cells led to the release of P-TEFb from the large form. Consistent with Tat being the cause of this effect, transfection of a FLAG-tagged Tat in 293T cells caused a dramatic shift of P-TEFb out of the large form to a smaller form containing Tat. In vitro, Tat competed with HEXIM1 for binding to 7SK, blocked the formation of the P-TEFb-HEXIM1-7SK complex, and caused the release P-TEFb from a pre-formed P-TEFb-HEXIM1-7SK complex. These findings indicate that Tat can acquire P-TEFb from the large form. In addition, we found that HEXIM1 binds tightly to the HIV 5' UTR containing TAR and recruits and inhibits P-TEFb activity. This suggests that in the absence of Tat, HEXIM1 may bind to TAR and repress transcription elongation of the HIV LTR.

Interplay between 7SK snRNA and oppositely charged regions in HEXIM1 direct the inhibition of P-TEFb.

Transcription elongation of eukaryotic genes by RNA polymerase II depends on the positive transcription elongation factor b (P-TEFb). When sequestered into the large complex, P-TEFb kinase activity is inhibited by the coordinate actions of 7SK small nuclear RNA (7SK snRNA) and hexamethylene bisacetamide (HMBA)-induced protein 1 (HEXIM1). We found that the basic region in HEXIM1 directs its nuclear import via two monopartite and two bipartite nuclear localization sequences. Moreover, the arginine-rich motif within it is essential for its binding to 7SK snRNA, P-TEFb, and inhibition of transcription. Notably, the basic region interacts with the adjacent acidic regions in the absence of RNA. The removal of the positive or negative charges from these regions in HEXIM1 leads to its sequestration into the large complex and inhibition of transcription independently of the arginine-rich motif. Finally, the removal of the negative charges from HEXIM1 results in its subnuclear localization into nuclear speckles. We propose a model where the interplay between 7SK snRNA and oppositely charged regions in HEXIM1 direct its binding to P-TEFb and subcellular localization that culminates in the inhibition of transcription.

Analysis of the large inactive P-TEFb complex indicates that it contains one 7SK molecule, a dimer of HEXIM1 or HEXIM2, and two P-TEFb molecules containing Cdk9 phosphorylated at threonine 186.

Positive transcription elongation factor b (P-TEFb) regulates eukaryotic gene expression at the level of elongation, and is itself controlled by the reversible association of 7SK RNA and an RNA-binding protein, HEXIM1 or HEXIM2. To further understand how P-TEFb is regulated, we analyzed the stoichiometry of all the known components of the large, inactive P-TEFb complex. Mutational analyses of a putative coiled coil region in the carboxyl-terminal portion of HEXIM1 revealed that the protein is a dimer in solution and remains a dimer after binding to 7SK. Although a HEXIM1 dimer contains two potential RNA binding motifs and ultimately recruits two P-TEFb molecules, it associates with only one molecule of RNA. The first 172 nucleotides of the 330-nucleotide 7SK are sufficient to bind HEXIM1 or HEXIM2, and then recruit and inhibit P-TEFb. Deletion of the first 121 amino acids of HEXIM1 allowed it to inhibit P-TEFb partially in the absence of 7SK RNA. Mutation of a conserved tyrosine (Tyr(271) in HEXIM1) to alanine or glutamate or mutation of a conserved phenylalanine (Phe(208)) to alanine, aspartate, or lysine, resulted in loss of inhibition of P-TEFb, but did not affect formation of the 7SK.HEXIM.P-TEFb complex. Analysis of T-loop phosphorylation in Cdk9 indicated that phosphorylation of Thr(186), but not Ser(175), was essential for kinase activity and for recruitment of P-TEFb to the 7SK.HEXIM complex. A model illustrates what is currently known about how HEXIM proteins, 7SK, and P-TEFb assemble to maintain an activated kinase in a readily available, but inactive form.

HEXIM2, a HEXIM1-related protein, regulates positive transcription elongation factor b through association with 7SK.

The kinase activity of positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 and cyclin T1 or T2, is required for the transition of RNA polymerase II into productive elongation. P-TEFb activity has been shown to be negatively regulated by association with the small nuclear RNA 7SK and the HEXIM1 protein. Here, we characterize HEXIM2, a previously predicted protein with sequence similarity to HEXIM1. HEXIM2 is expressed in HeLa and Jurkat cells, and glycerol gradient analysis and immunoprecipitations indicate that HEXIM2, like HEXIM1, has a regulated association with P-TEFb. As HEXIM1 is knocked down, HEXIM2 functionally compensates for its association with P-TEFb. Electrophoretic mobility shift assays and in vitro kinase assays demonstrate that HEXIM2 forms complexes containing 7SK and P-TEFb and, in conjunction with 7SK, inhibits P-TEFb kinase activity. Our results provide strong evidence that HEXIM2 is a regulator of P-TEFb function. Furthermore, our results support the idea that the utilization of HEXIM1 or HEXIM2 to bind and inhibit P-TEFb can be differentially regulated in vivo.

Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK9/cyclin T) inhibitor.

The positive transcription elongation factor b (P-TEFb) plays a pivotal role in productive elongation of nascent RNA molecules by RNA polymerase II. Core active P-TEFb is composed of CDK9 and cyclin T. In addition, mammalian cell extracts contain an inactive P-TEFb complex composed of four components, CDK9, cyclin T, the 7SK snRNA and the MAQ1/HEXIM1 protein. We now report an in vitro reconstitution of 7SK-dependent HEXIM1 association to purified P-TEFb and subsequent CDK9 inhibition. Yeast three-hybrid tests and gel-shift assays indicated that HEXIM1 binds 7SK snRNA directly and a 7SK snRNA-recognition motif was identified in the central part of HEXIM1 (amino acids (aa) 152-155). Data from yeast two-hybrid and pull-down assay on GST fusion proteins converge to a direct binding of P-TEFb to the HEXIM1 C-terminal domain (aa 181-359). Consistently, point mutations in an evolutionarily conserved motif (aa 202-205) were found to suppress P-TEFb binding and inhibition without affecting 7SK recognition. We propose that the RNA-binding domain of HEXIM1 mediates its association with 7SK and that P-TEFb then enters the complex through association with HEXIM1.