Programmed Cell Death-1 (PD-1) anchoring to the GPI-linked co-receptor CD48 reveals a novel mechanism to modulate PD-1-dependent inhibition of human T cells.

Activation of PD-1 by anchoring it to Antigen Receptor (AR) components or associated co-receptors represents an attractive approach to treat autoimmune conditions. In this study, we provide evidence that CD48, a common lipid raft and Src kinase-associated coreceptor, induces significant Src kinase-dependent activation of PD-1 upon crosslinking, while CD71, a receptor excluded from these compartments, does not. Functionally, using bead-conjugated antibodies we demonstrate that CD48-dependent activation of PD-1 inhibits proliferation of AR-induced primary human T cells, and similarly, PD-1 activation using PD-1/CD48 bispecific antibodies inhibits IL-2, enhances IL-10 secretion, and reduces NFAT activation in primary human and Jurkat T cells, respectively. As a whole, CD48-dependent activation of PD-1 represents a novel mechanism to fine tune T cell activation, and by functionally anchoring PD-1 with receptors other than AR, this study provides a conceptual framework for rational development of novel therapies that activate inhibitory checkpoint receptors for treatment of immune-mediated diseases.

Perinatal Depression: Challenges and Opportunities.

Perinatal depression (PND) is a major depressive episode during pregnancy or within 4 weeks after childbirth up to a year. Risk factors for PND include stressful life events, history of depression, poor social support, unplanned and unwanted pregnancies, poor relationship quality, current or previous abuse, and low socioeconomic status. This mental disorder has been shown to have negative effects on mothers' quality of life and their intimate relationships, birth outcomes, and breastfeeding likelihood, as well as long-term effects on children's cognitive and emotional development. To date, no nationally representative study has examined whether there are socioeconomic and/or racial/ethnic differences in PND. This study discusses the prevalence and risk factors for PND, as well as its health consequences for mothers and children, the reasons for its underreporting and undertreatment, the evidence for different screening instruments and different treatment options, and the existing supportive policies to address this disorder in the United States. We conclude with outlining next steps in addressing the gaps in the literature on PND.

Discovery and optimization of pyrazole amides as antagonists of CCR1.

A HTS screen for CCR1 antagonists afforded a novel sub-micromolar hit 5 containing a pyrazole core. In this report the design, optimization, and SAR of novel CCR1 antagonists based on a pyrazole core motif is presented. Optimization led to the advanced candidate compounds (S)-16q and (S)-16r with 250-fold improved CCR1 potency, excellent off-target selectivity and attractive drug-like properties.

BI 1002494, a Novel Potent and Selective Oral Spleen Tyrosine Kinase Inhibitor, Displays Differential Potency in Human Basophils and B Cells.

BI 1002494 [(R)-4-{(R)-1-[7-(3,4,5-trimethoxy-phenyl)-[1,6]napthyridin-5-yloxy]-ethyl}pyrrolidin-2-one] is a novel, potent, and selective spleen tyrosine kinase (SYK) inhibitor with sustained plasma exposure after oral administration in rats, which qualifies this molecule as a good in vitro and in vivo tool compound. BI 1002494 exhibits higher potency in inhibiting high-affinity IgE receptor-mediated mast cell and basophil degranulation (IC50 = 115 nM) compared with B-cell receptor-mediated activation of B cells (IC50 = 810 nM). This may be explained by lower kinase potency when the physiologic ligand B-cell linker was used, suggesting that SYK inhibitors may exhibit differential potency depending on the cell type and the respective signal transduction ligand. A 3-fold decrease in potency was observed in rat basophils (IC50 = 323 nM) compared with human basophils, but a similar species potency shift was not observed in B cells. The lower potency in rat basophils was confirmed in both ex vivo inhibition of bronchoconstriction in precision-cut rat lung slices and in reversal of anaphylaxis-driven airway resistance in rats. The different cellular potencies translated into different in vivo efficacy; full efficacy in a rat ovalbumin model (that contains an element of mast cell dependence) was achieved with a trough plasma concentration of 340 nM, whereas full efficacy in a rat collagen-induced arthritis model (that contains an element of B-cell dependence) was achieved with a trough plasma concentration of 1400 nM. Taken together, these data provide a platform from which different estimates of human efficacious exposures can be made according to the relevant cell type for the indication intended to be treated.

Weight loss goals among African-American women with type 2 diabetes in a behavioral weight control program.

African-American women with type 2 diabetes experience limited weight loss in behavioral weight control programs. Some research suggests that overly ambitious weight loss expectations may negatively affect weight losses achieved but it is unknown whether they affect weight loss among African-American women. The current study examined personal weight loss goals and expected satisfaction with a reasonable weight loss among African-American women with type 2 diabetes starting a behavioral obesity treatment. We also explored associations among these factors and weight loss treatment outcomes. Self-identified African-American women (N = 84) in a 24-session group program were assessed at baseline and 6-month follow-up. At baseline, women indicated weight loss goals of 14.1 ± 6.6 kg (14% of initial weight). They also reported relatively high expected satisfaction with a reasonable weight loss (7-10%). On average, participants lost 3.0 ± 3.9 kg (3% of initial weight) and attended 73 ± 21% of group sessions. Neither weight loss goals nor expected satisfaction with a reasonable weight loss was correlated with either actual weight loss outcome or attendance. Having higher personal weight loss goals was associated with lower expectations of satisfaction with a reasonable weight loss. This suggests that African-American women with type 2 diabetes enter treatment hoping to lose far more weight than they are likely to achieve. It is important to understand the psychosocial sequelae of failing to reach these goals on subsequent weight maintenance and future weight loss attempts within this population.

African Americans' responses to genetic explanations of lung cancer disparities and their willingness to participate in clinical genetics research.

PURPOSE: To assess whether reactions to genetic explanations for disparities in lung cancer incidence among family members of African American patients with lung cancer are associated with willingness to participate in clinical genetics research. METHODS: Data are reported for 67 self-identified African Americans aged 18 to 55 years who completed a telephone survey assessing reactions to explanations (i.e., genetics, toxin exposure, menthol cigarettes, and race-related stress) for lung cancer disparities. Majority were female (70%), current smokers (57%), and patients' biological relatives (70%). RESULTS: Family members rated the four explanations similarly, each as believable, fair, and not too worrisome. Participants also indicated a high level of willingness to participate in genetics research (M = 4.1 +/- 1.0; scale: 1-5). Endorsements of genetics explanations for disparities as believable and fair, and toxin exposure as believable were associated significantly with willingness to participate in genetics research. CONCLUSION: These results suggest that strategies to encourage African Americans' participation in genetics research would do well to inform potential participants of how their involvement might be used to better understand important environmental factors that affect health disparities.

Responses to online GSTM1 genetic test results among smokers related to patients with lung cancer: a pilot study.

Providing smokers with personal genetic test results indicating increased lung cancer risk may increase uptake of effective smoking cessation services. Using the internet may increase reach and enable real-time assessment of how people process genetic risk information away from the clinic setting. We therefore explored smokers' responses to Web-delivered GSTM1 genetic test results indicating higher or lower lung cancer risk. Participants were smokers (n = 44) biologically related to patients with newly diagnosed lung cancer. Measures were assessed at baseline, before and immediately after receipt of online genetic test results, and at 6-month follow-up. Outcomes included accurate comprehension of results, regret about being tested, cessation-related cognitions (e.g., perceived response efficacy), and uptake of free smoking cessation services (nicotine replacement therapy, printed self-help materials, telephone counseling sessions). Twenty-two "relative smokers" received a GSTM1-missing (higher risk) and 22 a GSTM1-present (lower risk) result. All relative smokers with GSTM1-missing results and 55% of those with GSTM1-present results accurately interpreted their results. No relative smokers regretted having taken the test. Relative smokers receiving GSTM1-missing results reported lower confidence that quitting could reduce lung cancer risk (perceived response efficacy) than those receiving GSTM1-present results. There were no other significant between-group differences. Uptake of smoking cessation services was high (e.g., 91% nicotine replacement therapy uptake). Genetic test results may not influence uptake of free smoking cessation services because of ceiling effects. Further research is needed to determine the risks and benefits of Web-based disclosure of genetic test results.

Stress, race, and body weight.

OBJECTIVE: Stress has been identified as a significant factor in health and in racial/ethnic health disparities. A potential mediator in these relationships is body weight. DESIGN: Cross-sectional and longitudinal relationships between stress, race, and body weight were examined in an ethnically diverse sample of overweight and obese women with Type 2 diabetes (n = 217) enrolled in a behavioral weight loss program. MAIN OUTCOME MEASURES: Stress (Perceived Stress Scale) was assessed at baseline only and body weight (body mass index) was assessed at baseline and 6 months. RESULTS: Stress was not related to baseline body weight. With every 1 unit lower scored on the baseline stress measure, women lost 0.10 kg +/- .04 more at 6 months (p < .05). When women were divided into tertiles based on baseline stress scores, those in the lowest stress group had significantly greater weight loss (5.2 kg +/- 4.9) compared with those in the highest stress group (3.0 kg +/- 4.0) (p < .05). There was a trend for African Americans to report higher levels of stress (20.7 +/- 8.8) than Whites (18.3 +/- 8.3) (p = .08). CONCLUSION: The association between higher stress and diminished weight loss has implications for enhancing weight loss programs for women with Type 2 diabetes.

Too many referrals of low-risk women for BRCA1/2 genetic services by family physicians.

The increasing availability and public awareness of BRCA1/2 genetic testing will increase women's self-referrals to genetic services. The objective of this study was to examine whether patient characteristics influence the referral decisions of family physicians when a patient requests BRCA1/2 genetic testing. Family physicians (n = 284) completed a Web-based survey in 2006 to assess their attitudes and practices related to the use of genetics in their clinical practice. Using a 2 x 2 x 2 factorial design, we tested the effects of a hypothetical patient's race, level of worry, and insurance status on the decisions of family physicians to refer her for BRCA1/2 testing. The patient was not appropriate for referral based on U.S. Preventive Services Task Force guidelines. No patient characteristics were associated with the family physicians' referral decisions. Although referral was not indicated, only 8% did not refer to genetic services; 92% referred for genetic services, and 50% referred to genetic counseling. Family physicians regarded it unlikely that the patient carried a mutation, but 65% of family physicians believed that if they refused to refer for genetic services it would harm their relationship with the patient. Despite scarce and costly genetic services, family physicians were likely to inappropriately refer a low-risk patient who requested BRCA1/2 testing. The implications of this inappropriate referral on women's screening behavior, genetic services, and health care costs are unknown. Clinicians and patients could benefit from education about the appropriate use of genetic services so that both are more comfortable with a decision against referral.

The feasibility of online genetic testing for lung cancer susceptibility: uptake of a web-based protocol and decision outcomes.

PURPOSE: To examine the feasibility of offering genetic susceptibility testing for lung cancer (GSTM1) via the Internet to smokers who were blood relatives of patients with lung cancer. Outcomes include proportion who logged on to the study website to consider testing, made informed decisions to log on and to be tested. METHODS: Baseline measures were assessed via telephone survey. Participants could choose to log on to the study website; those who did were offered testing. Informed decisions to log on and to be tested were indicated by concordance between the decision outcome and test-related attitudes and knowledge. RESULTS: Three hundred four relatives completed baseline interviews. One hundred sixteen eligible relatives expressed further interest in receiving information via the web. Fifty-eight logged on and 44 tested. Those logging on expressed greater quit motivation, awareness of cancer genetic testing, and were more likely to be daily Internet users than those who did not log on. Approximately half of the sample made informed decisions to log on and to be tested. CONCLUSION: Interest in a web-based protocol for genetic susceptibility testing was high. Internet-delivered decision support was as likely as other modalities to yield informed decisions. Some subgroups may need additional support to improve their decision outcomes.

Identification of a novel class of succinyl-nitrile-based Cathepsin S inhibitors.

The synthesis and in vitro activities of a series of succinyl-nitrile-based inhibitors of Cathepsin S are described. Several members of this class show nanomolar inhibition of the target enzyme as well as cellular potency. The inhibitors displaying the greatest potency contain N-alkyl substituted piperidine and pyrrolidine rings spiro-fused to the alpha-carbon of the P1 residue.

Discovery of potent and selective PKC-theta inhibitors.

An uHTS campaign was performed to identify selective inhibitors of PKC-theta. Initial triaging of the hit set based on selectivity and historical analysis led to the identification of 2,4-diamino-5-nitropyrimidines as potent and selective PKC-theta inhibitors. A homology model and initial SAR is presented demonstrating that a 2-arylalkylamino substituent in conjunction with suitable 4-diamino substituent are essential for achieving selectivity over many kinases. Additional hit to lead profiling is presented on selected compounds.

Involvement of GATA3 in protein kinase C theta-induced Th2 cytokine expression.

Protein kinase C theta (PKCtheta) is essential for T cell activation, as it is required for the activation of NF-kappaB and expression of IL-2. PKCtheta has also been shown to affect NFAT activation and Th2 differentiation. To better understand the role of PKCtheta in the regulation of T helper cells, we used PKCtheta-deficient DO11.10 transgenic T cells to study its role in vitro. DO11.10 Th1 cells deficient in PKCtheta produced significantly less TNF-alpha and IL-2. The expression of Th2 cytokines, including IL-4, IL-5, IL-10, IL-13 and IL-24 was significantly reduced in PKCtheta-deficient T cells. Moreover, the expression of the Th2 transcription factor, GATA3, was significantly reduced in PKCtheta-deficient T cells. Overexpression of GATA3 by retroviral infection in PKCtheta-deficient T cells resulted in increased expansion of IL-4-producing T cells and higher IL-4 production than that of wild type Th2 cells. IL-5, IL-10, IL-13 and IL-24 expressions were also rescued by GATA3 overexpression. Our observations suggest that PKCtheta regulates Th2 cytokine expression via GATA3.

An orally active reversible inhibitor of cathepsin S inhibits human trans vivo delayed-type hypersensitivity.

Cathepsin S is a major histocompatibility complex (MHC) class II associated invariant chain (Ii) degrading enzyme expressed in antigen presenting cells such as B cells and dendritic cells. This enzyme is essential for MHC class II associated antigen processing and presentation to CD4(+) T cells. Compound I, a selective, reversible and orally bioavailable, inhibitor of cathepsin S, with molecular IC(50)=9 nM, has been recently described. We have tested the effects of compound I in a trans vivo model of delayed-type hypersensitivity. Human peripheral blood mononuclear cells (7-10 x 10(6)) from tetanus-sensitized donors were co-injected with tetanus toxoid (0.25 Lf) into C57Bl/6 mouse footpads. At 24 h, significant footpad swelling (+0.024+/-0.001 cm) characterized by an influx of mouse neutrophils and monocytes was observed. Injection of peripheral blood mononuclear cells alone caused negligible swelling (0.002+/-0.0002 cm). Anti-human MHC class II (HLA-DR, DP, DQ) antibody (5 mg/kg, i.p.) inhibited the swelling 91+/-7%, thus demonstrating a role of human antigen presenting cells in this model. Compound I (10, 30, and 100 mg/kg, p.o.) inhibited the response with an ED50 of approximately 18 mg/kg. Compound III, a less active analogue (molecular IC50>20 microM) had no effect. Furthermore, pretreatment of peripheral blood mononuclear cells with 10 nM compound II, an irreversible inhibitor (molecular IC50=11 nM) inhibited swelling 87+/-4%. These findings support the role of cathepsin S in human delayed-type hypersensitivity. Inhibition of cathepsin S with compound I may be useful in the treatment of human autoimmune diseases like rheumatoid arthritis and multiple sclerosis.

The design of potent hydrazones and disulfides as cathepsin S inhibitors.

The design and synthesis of dipeptidyl disulfides and dipeptidyl benzoylhydrazones as selective inhibitors of the cysteine protease Cathepsin S are described. These inhibitors were expected to form a slowly reversible covalent adduct of the active site cysteine of Cathepsin S. Formation of the initial adduct was confirmed by mass spectral analysis. The nature and mechanism of these adducts was explored. Kinetic analysis of the benzoyl hydrazones indicate that these inhibitors are acting as irreversible inhibitors of Cathepsin S. Additionally, the benzoylhydrazones were shown to be potent inhibitors of Cathepsin S processing of Class II associated invariant peptide both in vitro and in vivo.

Design and synthesis of dipeptide nitriles as reversible and potent Cathepsin S inhibitors.

The specificity of the immune response relies on processing of foreign proteins and presentation of antigenic peptides at the cell surface. Inhibition of antigen presentation, and the subsequent activation of T-cells, should, in theory, modulate the immune response. The cysteine protease Cathepsin S performs a fundamental step in antigen presentation and therefore represents an attractive target for inhibition. Herein, we report a series of potent and reversible Cathepsin S inhibitors based on dipeptide nitriles. These inhibitors show nanomolar inhibition of the target enzyme as well as cellular potency in a human B cell line. The first X-ray crystal structure of a reversible inhibitor cocrystallized with Cathepsin S is also reported.