PARP14 inhibition restores PD-1 immune checkpoint inhibitor response following IFNγ-driven acquired resistance in preclinical cancer models.

Resistance mechanisms to immune checkpoint blockade therapy (ICBT) limit its response duration and magnitude. Paradoxically, Interferon γ (IFNγ), a key cytokine for cellular immunity, can promote ICBT resistance. Using syngeneic mouse tumour models, we confirm that chronic IFNγ exposure confers resistance to immunotherapy targeting PD-1 (α-PD-1) in immunocompetent female mice. We observe upregulation of poly-ADP ribosyl polymerase 14 (PARP14) in chronic IFNγ-treated cancer cell models, in patient melanoma with elevated IFNG expression, and in melanoma cell cultures from ICBT-progressing lesions characterised by elevated IFNγ signalling. Effector T cell infiltration is enhanced in tumours derived from cells pre-treated with IFNγ in immunocompetent female mice when PARP14 is pharmacologically inhibited or knocked down, while the presence of regulatory T cells is decreased, leading to restoration of α-PD-1 sensitivity. Finally, we determine that tumours which spontaneously relapse in immunocompetent female mice following α-PD-1 therapy upregulate IFNγ signalling and can also be re-sensitised upon receiving PARP14 inhibitor treatment, establishing PARP14 as an actionable target to reverse IFNγ-driven ICBT resistance.

PARP7 negatively regulates the type I interferon response in cancer cells and its inhibition triggers antitumor immunity.

PARP7 is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates to change their function. Here, we identify PARP7 as a negative regulator of nucleic acid sensing in tumor cells. Inhibition of PARP7 restores type I interferon (IFN) signaling responses to nucleic acids in tumor models. Restored signaling can directly inhibit cell proliferation and activate the immune system, both of which contribute to tumor regression. Oral dosing of the PARP7 small-molecule inhibitor, RBN-2397, results in complete tumor regression in a lung cancer xenograft and induces tumor-specific adaptive immune memory in an immunocompetent mouse cancer model, dependent on inducing type I IFN signaling in tumor cells. PARP7 is a therapeutic target whose inhibition induces both cancer cell-autonomous and immune stimulatory effects via enhanced IFN signaling. These data support the targeting of a monoPARP in cancer and introduce a potent and selective PARP7 inhibitor to enter clinical development.

Targeting Calcium Release-activated Calcium Channel Is Not Sufficient to Prevent Rejection in Nonhuman Primate Kidney Transplantation.

BACKGROUND: Calcineurin inhibitors successfully control rejection of transplanted organs but also cause nephrotoxicity. This study, using a rhesus monkey renal transplantation model, sought to determine the applicability of a new immunomodulatory drug inhibiting the store-operated calcium release-activated calcium channel of lymphocytes to control transplant rejection without nephrotoxicity. METHODS: Animals underwent kidney transplantation and were treated with tacrolimus alone (n = 3), a CRACM1 inhibitor (PRCL-02) (n = 6) alone, or with initial tacrolimus monotherapy followed by gradual conversion at 3 weeks to PRCL-02 alone (n = 3). PRCL-02 was administered via a surgically inserted gastrostomy tube BID. RESULTS: Dose-related drug exposure in monkeys was established and renal transplants were then performed using PRCL-02 monotherapy. Oral dosing of PRCL-02 was well tolerated and resulted in suppressed T-cell proliferation in in vitro MLR comparable to animals in the tacrolimus control arm. Animals receiving tacrolimus monotherapy were e on day 100 without rejection. PRCL-02 monotherapy only marginally prolonged graft survival (MST = 13.16 d; group 2) compared with untreated controls. Animals treated initially with tacrolimus and converted to PRCL-02 monotherapy had a mean graft survival of 35.3 days which was prolonged compared with PRCL-02 monotherapy but not compared with the tacrolimus-treated group. Pharmacokinetic studies showed inconsistent drug exposures despite attempts to adjust dose and exposure which may have contributed to the rejections. CONCLUSIONS: We conclude that, in this nonhuman primate model of kidney transplantation, PRCL-02 demonstrated evidence of in vivo immunosuppressive activity but was inferior to tacrolimus treatment with respect to suppressing immune transplant rejection.

A Pilot Observational Study of an Acupressure/Acupuncture Intervention in Children with Autism Spectrum Disorder.

OBJECTIVES: To determine whether children with autism spectrum disorder (ASD) would tolerate an acupressure/acupuncture intervention and whether parents would adhere to a twice-weekly, 8-week intervention protocol. Second, to further understand best measures to use to capture impact of intervention on behavioral and regulatory functions. DESIGN: This is an observational pilot study with pre-, mid-, and postintervention measures. SETTINGS/LOCATION: The intervention was carried out in a private practice office in a large metropolitan area. SUBJECTS: A total of 10 children of ages 3-10 years with ASD and one of their parents participated. INTERVENTIONS: A total of 16 biweekly treatment sessions of acupressure and/or acupuncture were carried out by a licensed acupuncturist, and a daily home-based acupressure intervention was carried out by a parent. OUTCOME MEASURES: Attendance, tolerance of intervention, parent compliance with home program, and parent compliance in completing daily diary and five standardized measures of behavioral and regulatory functions pre-, mid-, and postintervention were recorded. RESULTS: The 10 children in this observational study, collectively, tolerated the intervention and parents adhered to the 16 sessions, biweekly protocol, and home protocol, as well as completing daily diary and five standardized measures at three different time intervals. The five measurements appeared to be sensitive to behavioral and regulatory functions that may improve with this type of intervention. CONCLUSIONS: The results of this observational pilot study suggest that acupressure/acupuncture is a feasible intervention for children with ASD that merits rigorous evaluation through a randomized controlled trial.

Autism Screening With Online Decision Support by Primary Care Pediatricians Aided by M-CHAT/F.

BACKGROUND AND OBJECTIVE: Autism spectrum disorders (ASDs) often go undetected in toddlers. The Modified Checklist for Autism in Toddlers (M-CHAT) With Follow-up Interview (M-CHAT/F) has been shown to improve detection and reduce over-referral. However, there is little evidence supporting the administration of the interview by a primary care pediatrician (PCP) during typical checkups. The goal of this study was to evaluate the feasibility, validity, and reliability of the M-CHAT/F by PCPs with online prompts at the time of a positive M-CHAT screen. DESIGN: Forty-seven PCPs from 22 clinics completed 197 M-CHAT/Fs triggered by positive M-CHAT screens via the same secure Web-based platform that parents used to complete M-CHATs before an 18- or 24-month well-child visit. A second M-CHAT/F was administered live or by telephone by trained research assistants (RAs) at the Kennedy Krieger Institute Center for Autism and Related Disorders. The Autism Diagnostic Observation Schedule, Second Edition, and the Mullen Scales of Early Learning were administered as criterion measures. Measures of agreement between PCPs and RAs were calculated, and measures of test performance compared. RESULTS: There was 86.6% agreement between PCPs and RAs, with a Cohen's κ of 0.72. Comparison of sensitivity, specificity, positive predictive value (PPV), and overall accuracy for M-CHAT/F between PCPs and RAs showed significant equivalence for all measures. Use of the M-CHAT/F by PCPs resulted in significant improvement in PPV compared with the M-CHAT alone. CONCLUSIONS: Minimally trained PCPs can administer the M-CHAT/F reliably and efficiently during regular well-child visits, increasing PPV without compromising detection.

The HSP90 Inhibitor Ganetespib Alleviates Disease Progression and Augments Intermittent Cyclophosphamide Therapy in the MRL/lpr Mouse Model of Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a complex, systemic autoimmune disease with a diverse range of immunological and clinical manifestations. The introduction of broad spectrum immunosuppressive therapies and better management of acute disease exacerbations have improved outcomes for lupus patients over recent years. However, these regimens are burdened by substantial toxicities and confer significantly higher risks of infection, thus there remains a significant and unmet medical need for alternative treatment options, particularly those with improved safety profiles. Heat shock protein 90 (HSP90) is a ubiquitously expressed molecular chaperone that acts as an important modulator of multiple innate and adaptive inflammatory processes. Of note, accumulating clinical and experimental evidence has implicated a role for HSP90 in the pathogenesis of SLE. Here we evaluated the potential of HSP90 as a therapeutic target for this disease using the selective small molecule inhibitor ganetespib in the well-characterized MRL/lpr autoimmune mouse model. In both the prophylactic and therapeutic dosing settings, ganetespib treatment promoted dramatic symptomatic improvements in multiple disease parameters, including suppression of autoantibody production and the preservation of renal tissue integrity and function. In addition, ganetespib exerted profound inhibitory effects on disease-related lymphadenopathy and splenomegaly, and reduced pathogenic T and B cell lineage populations in the spleen. Ganetespib monotherapy was found to be equally efficacious and tolerable when compared to an effective weekly dosing regimen of the standard-of-care immunosuppressive agent cyclophosphamide. Importantly, co-treatment of ganetespib with a sub-optimal, intermittent dosing schedule of cyclophosphamide resulted in superior therapeutic indices and maximal disease control. These findings highlight the potential of HSP90 inhibition as an alternative, and potentially complementary, strategy for therapeutic intervention in SLE. Such approaches may have important implications for disease management, particularly for limiting or preventing treatment-related toxicities, a major confounding factor in current SLE therapy.

The impact of social skills training for social anxiety disorder: a randomized controlled trial.

OBJECTIVE: Social anxiety disorder (SAD) impacts social, occupational and academic functioning. Although many interventions report change in social distress, improvement in social behavior remains under-addressed. This investigation examined the additive impact of social skills training (SST) for the treatment of SAD. METHOD: Using a sample of 106 adults who endorsed SAD across numerous social settings, participants were randomized to exposure therapy (imaginal and in vivo) alone, a combination of SST and exposure therapy known as Social Effectiveness Therapy (SET), or a wait list control. The assessment strategy included self-report measures, blinded clinical ratings and blinded assessment of social behavior. RESULTS: Both interventions significantly reduced distress in comparison to the wait list control and at post-treatment, 67% of patients treated with SET and 54% of patients treated with exposure therapy alone no longer met diagnostic criteria for SAD, a difference that was not statistically significant. When compared to exposure therapy alone, SET produced superior outcomes (p<.05) on measures of social skill and general clinical status. In addition to statistical significance, participants treated with SET or exposure reported clinically significant decreases on two measures of self-reported social anxiety and several measures of observed social behavior (all ps<.05). CONCLUSIONS: Both interventions produced efficacious treatment outcome, although SET may provide additional benefit on measures of social distress and social behavior.

Association between severity of behavioral phenotype and comorbid attention deficit hyperactivity disorder symptoms in children with autism spectrum disorders.

Autism spectrum disorder and attention deficit hyperactivity disorder are neurodevelopmental disorders that cannot be codiagnosed under existing diagnostic guidelines (Diagnostic and Statistical Manual of the American Psychiatric Association, 4th ed., text rev.). However, reports are emerging that attention deficit hyperactivity disorder is sometimes comorbid with autism spectrum disorder. In the current study, we examined rates of parent-reported clinically significant symptoms of attention deficit hyperactivity disorder in school-aged children (4-8 years) with autism spectrum disorder, most of whom were first enrolled in our research protocols as toddlers. Results revealed that children with autism spectrum disorder and attention deficit hyperactivity disorder had lower cognitive functioning, more severe social impairment, and greater delays in adaptive functioning than children with autism spectrum disorder only. Implications for clinical practice include the need to assess for attention deficit hyperactivity disorder symptoms at an early age in children diagnosed with autism spectrum disorder. Research is needed to determine efficacious interventions for young children with autism spectrum disorder with comorbid attention deficit hyperactivity disorder to optimize outcomes.

Characterization of a novel CRAC inhibitor that potently blocks human T cell activation and effector functions.

Store operated calcium entry (SOCE) downstream of T cell receptor (TCR) activation in T lymphocytes has been shown to be mediated mainly through the Calcium Release Activated Calcium (CRAC) channel. Here, we compared the effects of a novel, potent and selective CRAC current inhibitor, 2,6-Difluoro-N-{5-[4-methyl-1-(5-methyl-thiazol-2-yl)-1,2,5,6-tetrahydro-pyridin-3-yl]-pyrazin-2-yl}-benzamide (RO2959), on T cell effector functions with that of a previously reported CRAC channel inhibitor, YM-58483, and a calcineurin inhibitor Cyclosporin A (CsA). Using both electrophysiological and calcium-based fluorescence measurements, we showed that RO2959 is a potent SOCE inhibitor that blocked an IP3-dependent current in CRAC-expressing RBL-2H3 cells and CHO cells stably expressing human Orai1 and Stim1, as well as SOCE in human primary CD4(+) T cells triggered by either TCR stimulation or thapsigargin treatment. Furthermore, we demonstrated that RO2959 completely inhibited cytokine production as well as T cell proliferation mediated by TCR stimulation or MLR (mixed lymphocyte reaction). Lastly, we showed by gene expression array analysis that RO2959 potently blocked TCR triggered gene expression and T cell functional pathways similar to CsA and another calcineurin inhibitor FK506. Thus, both from a functional and transcriptional level, our data provide evidence that RO2959 is a novel and selective CRAC current inhibitor that potently inhibits human T cell functions.

Mitochondrial electron transport is the cellular target of the oncology drug elesclomol.

Elesclomol is a first-in-class investigational drug currently undergoing clinical evaluation as a novel cancer therapeutic. The potent antitumor activity of the compound results from the elevation of reactive oxygen species (ROS) and oxidative stress to levels incompatible with cellular survival. However, the molecular target(s) and mechanism by which elesclomol generates ROS and subsequent cell death were previously undefined. The cellular cytotoxicity of elesclomol in the yeast S. cerevisiae appears to occur by a mechanism similar, if not identical, to that in cancer cells. Accordingly, here we used a powerful and validated technology only available in yeast that provides critical insights into the mechanism of action, targets and processes that are disrupted by drug treatment. Using this approach we show that elesclomol does not work through a specific cellular protein target. Instead, it targets a biologically coherent set of processes occurring in the mitochondrion. Specifically, the results indicate that elesclomol, driven by its redox chemistry, interacts with the electron transport chain (ETC) to generate high levels of ROS within the organelle and consequently cell death. Additional experiments in melanoma cells involving drug treatments or cells lacking ETC function confirm that the drug works similarly in human cancer cells. This deeper understanding of elesclomol's mode of action has important implications for the therapeutic application of the drug, including providing a rationale for biomarker-based stratification of patients likely to respond in the clinical setting.

Social skills and social phobia: an investigation of DSM-IV subtypes.

Social phobia is characterized as pervasive social timidity in social settings. Although much is known about this disorder, aspects of its clinical presentation remain unexplored, in particular characteristics that distinguish the generalized and non-generalized subtypes. For example, it remains unclear whether patients with the non-generalized subtype display social skills deficits in social interactions, and if so, are these deficits clinically, as well as statistically, significant? In this study, adults with either the non-generalized (NGSP; n=60) or generalized (GSP; n=119) subtype of social phobia and adults with no psychological disorder (n=200) completed an extensive behavioral assessment of social skill and social anxiety. As expected, adults with NGSP and GSP reported equal distress and displayed similar rates of avoidance during an Impromptu Speech Task when compared to adults with no disorder. In contrast, the three groups were distinctly different when interacting with another person in various social situations. Adults with NGSP displayed social skill deficits when compared to individuals with no disorder, but they had fewer deficits than the GSP subtype. However, the identified skill deficits were clinically as well as statistically significant only for the GSP subtype. The results are discussed in terms of the contribution of skill deficits to the conceptualization and treatment of social phobia.

The impact of children with high-functioning autism on parental stress, sibling adjustment, and family functioning.

The article discusses a study conducted to investigate the impact of children with high-functioning autism (HFA) on parental stress, sibling adjustment, and family functioning; the study involves a sample of parents of 15 children with HFA and parents of 15 matched control children who completed questionnaires measuring the dependent variables. The results indicate parents of children with HFA experience significantly more parenting stress than parents of children with no psychological disorder, which was found to be directly related to characteristics of the children. The study further shows that the higher intellectual functioning in children with HFA does not compensate for the stress associated with parenting children with autism spectrum disorders. Because the intervention efforts directed at children with HFA will not eliminate the child's primary symptoms, treatment programs may need to address parental stress, which in turn will help optimize treatment outcome for the child and the family.

Social skills interventions for children with Asperger's syndrome or high-functioning autism: a review and recommendations.

This paper reviews the literature examining social skills training (SST) programs for youth with AS/HFA, with an emphasis on critically evaluating efficacy and highlighting areas of future research. The review highlights the disparity between SST programs described in the extant literature, including lack of a universal definition of social skills, various levels of intensity and duration of treatment, divergent theoretical backgrounds, and variety in services provided in clinic or classroom settings. Overall, it is clear that, despite their widespread clinical use, empirical support for SST programs for children with AS/HFA is minimal at this time. Based on this critical review, a "roadmap" for future research, consistent with recommendations put forth by a leading group of autism researchers, is presented.

Defective mast cell effector functions in mice lacking the CRACM1 pore subunit of store-operated calcium release-activated calcium channels.

CRACM1 (also called Orai1) constitutes the pore subunit of store-operated calcium release-activated calcium channels. A point mutation in the gene encoding CRACM1 is associated with severe combined immunodeficiency disease in humans. Here we generated CRACM1-deficient mice in which beta-galactosidase activity 'reported' CRACM1 expression. CRACM1-deficient mice were smaller in size. Mast cells derived from CRACM1-deficient mice showed grossly defective degranulation and cytokine secretion, and the allergic reactions elicited in vivo were inhibited in CRACM1-deficient mice. We detected robust CRACM1 expression in skeletal muscles and some regions of the brain, heart and kidney but not in the lymphoid regions of thymus and spleen. In contrast, we found CRACM2 expression to be much higher in mouse T cells. In agreement with those findings, the store-operated calcium influx and development and proliferation of CRACM1-deficient T cells was unaffected. Thus, CRACM1 is crucial in mouse mast cell effector function, but mouse T cell calcium release-activated calcium channels are functional in the absence of CRACM1.

Social anxiety disorder in childhood and adolescence: descriptive psychopathology.

Although the presentation of social anxiety disorder (SAD) in adults is well documented, less is known about its clinical manifestation in children and adolescents. To date, most studies have included combined samples of children and adolescents despite the fact that this age range represents an extensive period of growth and development. This study compares and contrasts the clinical presentation of SAD among children (ages 7-12) and adolescents (ages 13-17). One hundred and fifty children (n=74) and adolescents (n=76) with a primary diagnosis of SAD participated in the study. The assessment battery included clinical ratings and behavioral observation as well as parental and self-report. The results indicate that, although the symptom presentation of children and adolescents with primary SAD shares many features, children tend to present with a broader pattern of general psychopathology, while adolescents have a more pervasive pattern of social dysfunction and may be more functionally impaired as a result of their disorder. These findings suggest that interventions for SAD need to carefully consider clinical presentation of the disorder as it manifests in childhood and adolescence.

Differentiation and expansion of T cells with regulatory function from human peripheral lymphocytes by stimulation in the presence of TGF-{beta}.

T cells with immunoregulatory function have been described in human and mouse systems. In both systems these cells can be differentiated either in the thymus or from peripheral T cells. To date, more progress has been made in the study of murine regulatory T cells, because it has been very difficult to isolate human regulatory T cells of sufficient purity and in sufficient numbers to permit detailed examinations of their biochemistry. We report in this study that human T cells with regulatory function can be differentiated in vitro from naive (CD4(+)CD45RA(+)) cord blood or peripheral T cells by stimulation with anti-CD3 and anti-CD28 in the presence of TGF-beta. Cells derived in this manner express a surface phenotype (CD25(+), CD122(+), HLA-DR(+), glucocorticoid-induced TNF receptor-related gene(+), CD103(+), CTLA-4(+)) described for human and mouse regulatory T cells and express protein and message for the transcription factor forkhead/winged helix transcription factor (FOXP3). They produce primarily TGF-beta and IL-10, with lesser amounts of IFN-gamma and IL-13, when stimulated through their TCRs and are capable of inhibiting cytokine production and proliferation by stimulated naive T cells. Unlike Th1 and Th2 cells, these TGF-beta-derived regulatory T cells do not appear to be dependent on the protein kinase Ctheta; pathway of NF-kappaB activation for Ag-induced responses.

Computerized screening of substance abuse problems in a primary care setting: older vs. younger adults.

The purpose of the current study was to examine differences in responses of older adults (age 55 and above) and younger adults (ages 18 to 54) to the Drug Abuse Problem Assessment for Primary Care (DAPA-PC), a computerized drug and alcohol abuse screening instrument developed for primary care settings. Data were collected from a diverse population of 327 adults presenting for care at The George Washington University Medical Faculty Associates clinic in downtown Washington, DC. Results indicated that rates of drug and alcohol abuse were similar in both groups. However, older adults were less likely than younger adults to perceive their drug use as problematic. This finding has serious implications for older adults, who tend to be underrepresented in treatment programs. There is a need for screening seniors and identifying those who may be at risk for substance abuse problems. Differences in responses to alcohol and drug assessments by age should be taken into consideration when designing screening instruments. The results of this study suggest that the DAPA-PC would provide a useful instrument for screening older adults in a primary care setting.

Induction of immunological tolerance/hyporesponsiveness in baboons with a nondepleting CD4 antibody.

Tolerance induction with anti-CD4 Abs is well established in rodent transplant and autoimmune disease models, but has yet to be demonstrated in non-human primates or in clinical studies. In retrospect, failure of anti-CD4 Abs to induce tolerance in primates may be technical, a consequence of insufficient dosing and Ab properties influencing immunogenicity and cell depletion. To circumvent these possible limitations, we constructed a novel anti-CD4 mAb, TRX1, humanized to reduce immunogenicity and Fc-modified to prevent cell depletion. Using equine immune globulin (equine Ig) as a model Ag, we examined the tolerance-inducing capacity of TRX1 in baboons. During the induction phase, TRX1 inhibited the humoral response to equine Ig in a dose-dependent manner, with complete suppression of response at the highest dose tested (40 mg/kg). Upon challenge, anti-equine Ig responses were generated in baboons treated with 1 and 10 mg/kg doses of TRX1 and in control animals. In higher dosing cohorts (20 and 40 mg/kg), however, the immune response to equine Ig was modulated in seven of nine animals, including complete unresponsiveness to Ag challenges in two animals. Five of nine were hyporesponsive to equine Ig, generating titers 50- to 250-fold lower than control groups. Repeated challenge resulted in titers falling to baseline or near baseline, with two of five hyporesponsive animals becoming unresponsive to Ag. All animals responded to neoantigen immunization, indicating that the modified response to equine Ig was Ag specific. These studies demonstrate that anti-CD4 Ab-mediated, Ag-specific tolerance can be achieved in baboons without long term immune suppression.

Targeting CCR2 or CD18 inhibits experimental in-stent restenosis in primates: inhibitory potential depends on type of injury and leukocytes targeted.

A central role for leukocytes in neointimal hyperplasia after arterial injury is suspected. However, the relative importance of neutrophils and monocytes in balloon or stent-induced injury are not well understood, and mechanistic targeting of leukocyte recruitment or function is crude. We determined the temporal and spatial distribution of different leukocytes after balloon and stent-induced injury in primate iliac arteries. Based on these data, we targeted neutrophil and monocyte recruitment selectively after angioplasty or stent implantation and demonstrated that monocyte-specific blockade achieved via blockade of the MCP-1 receptor CCR2, was effective at reducing neointimal hyperplasia after stenting. In contrast, combined neutrophil and monocyte blockade achieved by targeting the leukocyte beta(2)-integrin beta-subunit CD18 was required to reduce neointimal hyperplasia after balloon injury. Distinct patterns of leukocyte infiltration in balloon versus stent-injured arteries predict distinct mechanisms for antiinflammatory strategies targeting neutrophils or monocytes in primates and may assist design of effective clinical strategies for optimizing vascular interventions.