Sec22b and Stx4 Depletion Has No Major Effect on Cross-Presentation of PLGA Microsphere-Encapsulated Antigen and a Synthetic Long Peptide In Vitro.

The induction of CTL responses by vaccines is important to combat infectious diseases and cancer. Biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres and synthetic long peptides are efficiently internalized by professional APCs and prime CTL responses after cross-presentation of Ags on MHC class I molecules. Specifically, they mainly use the cytosolic pathway of cross-presentation that requires endosomal escape, proteasomal processing, and subsequent MHC class I loading of Ags in the endoplasmic reticulum (ER) and/or the endosome. The vesicle SNARE protein Sec22b has been described as important for this pathway by mediating vesical trafficking for the delivery of ER-derived proteins to the endosome. As this function has also been challenged, we investigated the role of Sec22b in cross-presentation of the PLGA microsphere-encapsulated model Ag OVA and a related synthetic long peptide. Using CRISPR/Cas9-mediated genome editing, we generated Sec22b knockouts in two murine C57BL/6-derived APC lines and found no evidence for an essential role of Sec22b. Although pending experimental evidence, the target SNARE protein syntaxin 4 (Stx4) has been suggested to promote cross-presentation by interacting with Sec22b for the fusion of ER-derived vesicles with the endosome. In the current study, we show that, similar to Sec22b, Stx4 knockout in murine APCs had very limited effects on cross-presentation under the conditions tested. This study contributes to characterizing cross-presentation of two promising Ag delivery systems and adds to the discussion about the role of Sec22b/Stx4 in related pathways. Our data point toward SNARE protein redundancy in the cytosolic pathway of cross-presentation.

Self-regulation in preschool: Are executive function and effortful control overlapping constructs?

The concepts of executive function (EF) and effortful control (EC) are strikingly similar. EF originate from neurocognitive research and are described as an accumulation of cognitive processes that serve the goal-oriented self-regulation (SR) of an individual. EC originates from temperament research and is defined as the efficiency of executive attention, including the ability to inhibit a dominant response, to activate a subdominant response, to proceed in a planned manner and to recognize conflicts or errors. The aim of this article was to examine the association between the constructs of EF and EC at the preschool-age. Eighty-eight children (49 female; M-age = 3.93 years, SD = .78) were tested with a computerized battery designed to assess EF at 3-6 years of age (EF Touch). Children's parents completed questionnaires assessing EF impairments (BRIEF-P) and EC (CBQ). Associations between the constructs and their conceptual overlap were analyzed using correlations and confirmatory factor analyses. We found significant correlations between EF and EC measures. A one-factor confirmatory model fitted the data very well and indicated that EF and EC are indeed overlapping and highly similar constructs. Therefore, our results show that measures of EC and EF have substantial overlap in preschoolers and suggest an integrated model of self-regulation.

Predicting ADHD symptoms and diagnosis at age 14 from objective activity levels at age 7 in a large UK cohort.

Hyperactivity is one of the three core symptoms in children with attention deficit hyperactivity disorder (ADHD). Diagnosing ADHD typically involves self-report, third party report and observations. Objective activity data can make a valuable contribution to the diagnostic process. Small actigraphy studies in clinical samples have shown that children with ADHD move more than children without ADHD. However, differences in physical activity between children with and without ADHD have not been assessed in large community samples or longitudinally. This study used data from the Millennium Cohort Study to test whether symptoms of ADHD (parent-rating Strengths and Difficulties Questionnaire) and ADHD diagnosis at age 14 (reported by parents) could be predicted from objective activity data (measured with actigraphs) at age 7 in N = 6675 children (final N = 5251). Regressions showed that less sedentary behavior at age 7 predicted more ADHD symptoms at age 14 (ß = - 0.002, CI - 0.004 to - 0.001). The result remained significant when controlled for ADHD symptoms at age 7, sex, BMI, month of birth, SES and ethnicity (ß = - 0.001, CI - 0.003 to - 0.0003). ADHD diagnosis at age 14 was also significantly predicted by less sedentary behavior at age 7 (ß = - 0.008). Our findings show that symptoms of ADHD can be predicted by objective activity data 5 years in advance and suggest that actigraphy could be a useful instrument aiding an ADHD diagnosis. Interestingly, the results indicate that the key difference between children with and without ADHD lies in reduced sedentary activity, i.e., times of rest.

Immunoproteasome inhibition induces plasma cell apoptosis and preserves kidney allografts by activating the unfolded protein response and suppressing plasma cell survival factors.

Chronic antibody-mediated rejection is the leading cause of allograft dysfunction and loss after kidney transplantation, and current immunosuppressive regimens fail to target the plasma cells that produce alloantibodies. We previously showed that treatment with the immunoproteasome inhibitor ONX 0914 prevented the expansion of plasma cells and prevented chronic allograft nephropathy and organ failure after kidney transplantation in rats, but the mechanism has remained elusive. In the current study, we confirmed a long-term reduction in alloantibody production and improvements in allograft histology in rats treated with ONX 0914 or with the broad-spectrum proteasome inhibitor bortezomib. Plasma cells from allotransplanted rats expressed immunoproteasomes at high levels. Immunoproteasome inhibition with ONX 0914 led to ubiquitin-conjugate accumulation, activation of the unfolded protein response, and induction of apoptosis in plasma cells. In addition, ONX 0914 suppressed the expression of adhesion molecules (VLA-4 and LFA-1), plasma cell survival factors (APRIL and IL-6), and IFN-γ-inducible chemokines in bone marrow, while the APRIL receptor BCMA, the IL-6 receptor, and the chemokine receptors CXCR4 and CXCR3 were down-regulated on plasma cells. Taken together, immunoproteasome inhibition blocked alloantibody production by inducing apoptosis of plasma cells through activating the unfolded protein response and suppressing plasma cell survival factors in the bone marrow.

Selective impairment of attentional set shifting in adults with ADHD.

BACKGROUND: Task switch protocols are frequently used in the assessment of cognitive control, both in clinical and non-clinical populations. These protocols frequently confound task switch and attentional set shift. The current study investigated the ability of adult ADHD patients to shift attentional set in the context of switching tasks. METHOD: We tested 38 adults with ADHD and 39 control adults with an extensive diagnostic battery and a task switch protocol without proactive interference. The experiment combined orthogonally task-switch vs. repetition, and attentional set shift vs. no shift. Each experimental stimulus had global and local features (Hierarchical/"Navon" stimuli), associated with corresponding attentional sets. RESULTS: ADHD patients were slower than controls in task switch trials with a simultaneous shift of attention between global/local attentional sets. This also correlated significantly with diagnostic scales for ADHD symptoms. The patients had more variable reaction times, but when the attentional set was kept constant neither were they significantly slower nor showed higher task switch costs. CONCLUSION: ADHD is associated with a deficit in flexible deployment of attention to varying sources of stimulus information.

Chronic stress suppresses anti-tumor TCD8+ responses and tumor regression following cancer immunotherapy in a mouse model of melanoma.

Animal tumor models and human cancer studies have provided convergent evidence that chronic psychological stress plays a decisive role in modulating anti-tumor T cell immunity. However, whether chronic stress also affects anti-cancer vaccine strategies that rely on the induction of functional tumor-specific TCD8+ cells has not been investigated yet. In this study we provide direct evidence that chronic stress suppresses the therapeutic efficacy of a biodegradable poly(d,l-lactide-co-glycolide) microsphere (PLGA-MS) based cancer vaccine in a murine melanoma model. Exposure of mice to social disruption stress (SDR), a well-established model mimicking psychological chronic stress in humans, significantly impaired tumor protection in response to cancer vaccination under both prophylactic and therapeutic conditions. Vaccine failure in stressed mice correlated with significantly reduced generation of interferon-γ (IFN-γ)-producing TCD8+ effectors and CTL-mediated killing. Phenotypic analysis of dendritic cells (DCs) revealed that both migratory and lymphoid-resident DCs failed to undergo full maturation upon antigen uptake. Notably, decreased DC maturation was associated with a significant impairment of peripheral DCs to migrate to draining LNs and to prime subsequent TCD8+ responses in vivo. In conclusion, chronic stress represents an important factor mediating immunosuppression in cancer-vaccinated hosts by impairing DC functions and subsequent TCD8+ priming. Potentially, the mechanistic insights gained in this study open new avenues in utilizing the full potential of anti-cancer vaccination strategies.

Suppression of prostate cancer and amelioration of the immunosuppressive tumor microenvironment through selective immunoproteasome inhibition.

New treatment options to battle hormone-refractory prostate carcinoma (PC) are a pressing medical need. Chronic inflammation has been implicated in PC etiology. The pro-inflammatory cytokines IL-6, IL-23 and IL-17 are key mediators to promote growth of PC. Here, we evaluate the potential of immunoproteasome inhibition for anti-inflammatory and direct anti-tumorigenic therapy of PC. The anti-tumor effect of immunoproteasome inhibitor ONX 0914 was tested in mouse and human PC cells and the in vivo therapeutic efficacy of immunoproteasome inhibition was analyzed in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice in preventive and therapeutic settings and in castration-resistant (CR)PC after castration. Inhibition of the immunoproteasome subunit LMP7 induced apoptotic cell death in PC cell lines. In TRAMP mice, ONX 0914-treatment resulted in significant inhibition of PC growth with a decreased frequency of malignant prostatic lesions and inhibition of metastasis formation. The number of immunosuppressive myeloid cells in PC was greatly reduced in response to ONX 0914. Thus, immunoproteasome inhibition shows remarkable efficacy against PC progression in vivo and impedes tumor recurrence in CRPC-TRAMP mice by blocking the immunosuppressive inflammatory response in the tumor microenvironment. In conclusion, we show that the immunoproteasome is a promising drug target for the treatment of PC.

Immune escape of colorectal tumours via local LRH-1/Cyp11b1-mediated synthesis of immunosuppressive glucocorticoids.

Control of tumour development and growth by the immune system critically defines patient fate and survival. What regulates the escape of colorectal tumours from destruction by the immune system remains currently unclear. Here, we investigated the role of intestinal synthesis of glucocorticoids in the tumour development during an inflammation-induced mouse model of colorectal cancer. We demonstrate that the local synthesis of immunoregulatory glucocorticoids has dual roles in the regulation of intestinal inflammation and tumour development. In the inflammation phase, LRH-1/Nr5A2-regulated and Cyp11b1-mediated intestinal glucocorticoid synthesis prevents tumour development and growth. In established tumours, however, tumour-autonomous Cyp11b1-mediated glucocorticoid synthesis suppresses anti-tumour immune responses and promotes immune escape. Transplantation of glucocorticoid synthesis-proficient colorectal tumour organoids into immunocompetent recipient mice resulted in rapid tumour growth, whereas transplantation of Cyp11b1-deleted and glucocorticoid synthesis-deficient tumour organoids was characterized by reduced tumour growth and increased immune cell infiltration. In human colorectal tumours, high expression of steroidogenic enzymes correlated with the expression of other immune checkpoints and suppressive cytokines, and negatively correlated with overall patients' survival. Thus, LRH-1-regulated tumour-specific glucocorticoid synthesis contributes to tumour immune escape and represents a novel potential therapeutic target.

PLGA-particle vaccine carrying TLR3/RIG-I ligand Riboxxim synergizes with immune checkpoint blockade for effective anti-cancer immunotherapy.

With emerging supremacy, cancer immunotherapy has evolved as a promising therapeutic modality compared to conventional antitumor therapies. Cancer immunotherapy composed of biodegradable poly(lactic-co-glycolic acid) (PLGA) particles containing antigens and toll-like receptor ligands induces vigorous antitumor immune responses in vivo. Here, we demonstrate the supreme adjuvant effect of the recently developed and pharmaceutically defined double-stranded (ds)RNA adjuvant Riboxxim especially when incorporated into PLGA particles. Encapsulation of Riboxxim together with antigens potently activates murine and human dendritic cells, and elevated tumor-specific CD8+ T cell responses are superior to those obtained using classical dsRNA analogues. This PLGA particle vaccine affords primary tumor growth retardation, prevention of metastases, and prolonged survival in preclinical tumor models. Its advantageous therapeutic potency was further enhanced by immune checkpoint blockade that resulted in reinvigoration of cytotoxic T lymphocyte responses and tumor ablation. Thus, combining immune checkpoint blockade with immunotherapy based on Riboxxim-bearing PLGA particles strongly increases its efficacy.

Temporal discounting in adolescents and adults with Tourette syndrome.

Tourette syndrome is a neurodevelopmental disorder associated with hyperactivity in dopaminergic networks. Dopaminergic hyperactivity in the basal ganglia has previously been linked to increased sensitivity to positive reinforcement and increases in choice impulsivity. In this study, we examine whether this extends to changes in temporal discounting, where impulsivity is operationalized as an increased preference for smaller-but-sooner over larger-but-later rewards. We assessed intertemporal choice in two studies including nineteen adolescents (age: mean[sd] = 14.21[±2.37], 13 male subjects) and twenty-five adult patients (age: mean[sd] = 29.88 [±9.03]; 19 male subjects) with Tourette syndrome and healthy age- and education matched controls. Computational modeling using exponential and hyperbolic discounting models via hierarchical Bayesian parameter estimation revealed reduced temporal discounting in adolescent patients, and no evidence for differences in adult patients. Results are discussed with respect to neural models of temporal discounting, dopaminergic alterations in Tourette syndrome and the developmental trajectory of temporal discounting. Specifically, adolescents might show attenuated discounting due to improved inhibitory functions that also affect choice impulsivity and/or the developmental trajectory of executive control functions. Future studies would benefit from a longitudinal approach to further elucidate the developmental trajectory of these effects.

Harnessing Dendritic Cells for Poly (D,L-lactide-co-glycolide) Microspheres (PLGA MS)-Mediated Anti-tumor Therapy.

With emerging success in fighting off cancer, chronic infections, and autoimmune diseases, immunotherapy has become a promising therapeutic approach compared to conventional therapies such as surgery, chemotherapy, radiation therapy, or immunosuppressive medication. Despite the advancement of monoclonal antibody therapy against immune checkpoints, the development of safe and efficient cancer vaccine formulations still remains a pressing medical need. Anti-tumor immunotherapy requires the induction of antigen-specific CD8+ cytotoxic T lymphocyte (CTL) responses which recognize and specifically destroy tumor cells. Due to the crucial role of dendritic cells (DCs) in initiating anti-tumor immunity, targeting tumor antigens to DCs has become auspicious in modern vaccine research. Over the last two decades, micron- or nanometer-sized particulate delivery systems encapsulating tumor antigens and immunostimulatory molecules into biodegradable polymers have shown great promise for the induction of potent, specific and long-lasting anti-tumor responses in vivo. Enhanced vaccine efficiency of the polymeric micro/nanoparticles has been attributed to controlled and continuous release of encapsulated antigens, efficient targeting of antigen presenting cells (APCs) such as DCs and subsequent induction of CTL immunity. Poly (D, L-lactide-co-glycolide) (PLGA), as one of these polymers, has been extensively studied for the design and development of particulate antigen delivery systems in cancer therapy. This review provides an overview of the current state of research on the application of PLGA microspheres (PLGA MS) as anti-tumor cancer vaccines in activating and potentiating immune responses attempting to highlight their potential in the development of cancer therapeutics.

The Association of Non-obscene Socially Inappropriate Behavior With Attention-Deficit/Hyperactivity Disorder Symptoms, Conduct Problems, and Risky Decision Making in a Large Sample of Adolescents.

Non-obscene socially inappropriate behavior (NOSI) is recognized as part of the tic disorder spectrum but has received little attention from researchers to date. A study in 87 patients with Tourette syndrome showed that comorbid attention-deficit/hyperactivity disorder (ADHD) and conduct disorder were also associated with an increase in socially inappropriate behavior. This study used data from the Millennium Cohort Study to investigate the relationship between NOSI and emotional symptoms, conduct problems, and hyperactivity/inattention as assessed by the Strengths and Difficulties Questionnaire (SDQ) in 1,280 youths, aged 14 years. Furthermore, the relationship between NOSI and decision-making processes as assessed by the Cambridge Gambling Task (CGT) was investigated. Hyperactivity/inattention and conduct problems were significantly associated with NOSI; emotional problems were not. Risk taking was significantly associated with misbehaving in lessons but not with being rude or noisy in public. The results replicate and confirm the association of NOSI with ADHD and conduct problems in a large sample, although it should be stressed that the size of the association was small. The results also suggest that some inappropriate behaviors are related to risk-taking behavior, while others are not.

Developing ADHD in preschool: Testing the dual pathway model of temperament.

The dual pathway model of Attention Deficit Hyperactivity Disorder (ADHD) suggests that effortful control and positive approach, or surgency, are independent pathways leading to ADHD. This model has been proven on the basis of temperament in school children, however not in preschool children. In this study we tested whether the dual pathway model of ADHD can be replicated in preschool children using temperamental measures. One hundred and nineteen children (59 girls, M-age = 4.97 years, SD = 0.96) participated in a study. Parents rated temperament on the Child Behavior Questionnaire (CBQ) and parents and teachers rated ADHD symptoms (SDQ). We found that effortful control and surgency independently predicted preschool ADHD symptoms but there were no moderations or mediations. Our findings support the dual pathway model of temperament but not compensatory models of ADHD. Ratings of temperament might be an important predictor of which child is at risk to develop clinical ADHD later on and therefore an important part of prevention.

Amelioration of autoimmunity with an inhibitor selectively targeting all active centres of the immunoproteasome.

BACKGROUND AND PURPOSE: Multicatalytic endopeptidase complex-like-1 (ß2i), low molecular mass polypeptide (LMP) 2 (ß1i) and LMP7 (ß5i) are the proteolytically active subunits of the immunoproteasome, a special type of proteasome mainly expressed in haematopoietic cells. Targeting LMP7 has been shown to be therapeutically effective in preclinical models of autoimmune diseases. In this study, we investigated the selectivity and biological activity of LU-005i, a recently described inhibitor of the immunoproteasome. EXPERIMENTAL APPROACH: The specificity of LU-005i and other immunoproteasome-selective inhibitors was characterized using fluorogenic peptide substrates. The effect of proteasome inhibition on cytokine release was investigated in endotoxin-stimulated mouse splenocytes or human peripheral blood mononuclear cells (PBMCs). The effect of proteasome inhibition on inflammatory bowel disease in the dextran sulfate sodium (DSS)-induced colitis model was assessed by measuring weight loss and colon length. KEY RESULTS: LU-005i is the first human and mouse immunoproteasome-selective inhibitor that targets all three proteolytically active immunoproteasome subunits. LU-005i inhibited cytokine secretion from endotoxin-stimulated mouse splenocytes or human PBMCs. Furthermore, differentiation of naïve T helper cells to T helper 17 cells was impaired in the presence of LU-005i. Additionally, LU-005i ameliorated DSS-induced colitis. CONCLUSION AND IMPLICATIONS: This study with a novel pan-immunoproteasome inhibitor substantiates that the immunoproteasome is a promising drug target for the treatment of inflammatory diseases and that exclusive inhibition of LMP7 is not necessary for therapeutic effectiveness. Our results will promote the design of new generations of immunoproteasome inhibitors with optimal therapeutic efficacy for clinical use in the treatment of autoimmunity and cancer.

Inhibition and deficiency of the immunoproteasome subunit LMP7 suppress the development and progression of colorectal carcinoma in mice.

New treatment options and drug targets for colorectal carcinoma are a pressing medical need. Inflammation and pro-inflammatory cytokines produced by Th1 and Th17 cells like IL-6, TNF, IL-17 and IL-23 promote the development and growth of colorectal cancer (CRC). The immunoproteasome is a proteasome subtype highly expressed in immune cells but also in the intestine. Since the immunoproteasome promotes Th1 and Th17 differentiation and pro-inflammatory cytokine production, we investigated here whether deficiency or inhibition of the immunoproteasome subunit LMP7 would interfere with CRC development and exacerbation in preventive and therapeutic mouse models. Treatment with the LMP7 inhibitor ONX 0914 blocked tumor initiation and progression in either chemically-induced (AOM/DSS) or transgenic mouse models (ApcMin/+) of colon carcinogenesis. ONX 0914 treatment strongly reduced tumor numbers and CRC-associated loss of body weight while the survival rates were significantly enhanced. Moreover, genetic LMP7 deficiency markedly reduced the tumor burden in AOM/DSS induced wild type and ApcMin/+ mice. In conclusion, we show that the immunoproteasome is involved in CRC development and progression and we identify LMP7 as a new potential drug target for the treatment of CRC.

The association of ADHD symptoms and reading acquisition during elementary school years.

The present longitudinal study aimed to investigate the influence of ADHD symptoms on reading development in elementary schoolchildren. To this end, repeated assessments of ADHD symptoms (teacher ratings of inattention, hyperactivity, and impulsivity) and reading achievement (standardized tests of decoding speed and text comprehension) were examined in a sample comprising 2,014 elementary schoolchildren at the end of Grades 1, 2, 3, respectively, and in the middle of Grade 4. Latent change score models revealed that the level of ADHD symptoms was associated with lower levels and less growth in decoding speed and text comprehension. Furthermore, individual differences in changes in ADHD symptoms and reading performance were negatively associated. Together, these results indicate commonalities in the development of ADHD symptomatology and reading achievement throughout elementary school. (PsycINFO Database Record

Coencapsulation of tumor lysate and CpG-ODN in PLGA-microspheres enables successful immunotherapy of prostate carcinoma in TRAMP mice.

Biodegradable poly(lactide-co-glycolide) (PLGA) microspheres (MS) deliver antigens and toll like receptor (TLR) ligands to antigen presenting cells (APC) in vitro and in vivo. PLGA-MS-microencapsulated model antigens are efficiently presented on MHC class I and II molecules of dendritic cells and stimulate strong cytotoxic and T helper cell responses enabling the eradication of pre-existing model tumors. The application of tumor lysates as a source of antigen for immunotherapy has so far not been very successful also due to a lack of suitable delivery systems. In this study we used PLGA-MS with co-encapsulated tumor lysates and CpG oligodeoxynucleotides (CpG-ODN) as well as microencapsulated polyI:C in order to elicit anti-tumor responses. Immunization of mice with such mixtures of MS yielded substantial cytotoxic T cell (CTL) responses and interfered with tumor growth in TRAMP mice, a pre-clinical transgenic mouse model of prostate carcinoma, which has previously resisted dendritic cell-based therapy. As an important step towards clinical application of PLGA-MS, we could show that γ-irradiation of PLGA-MS sterilized the MS, without reducing their efficacy in eliciting CTL and anti-tumor responses in subcutaneous tumor grafts. Since PLGA is approved for clinical application, sterilized PLGA-MS containing tumor lysates and TLR ligands hold promise as anti-tumor vaccines against prostate carcinoma in humans.