Use of a Cyclic α-Alkylidene-ß-Diketone as a Cleavable Linker Strategy for Antibody-Drug Conjugates.

In the fast-evolving landscape of targeted cancer therapies, the revolutionary class of biotherapeutics known as antibody-drug conjugates (ADCs) are taking center stage. Most clinically approved ADCs utilize cleavable linkers to temporarily attach potent cytotoxic payloads to antibodies, allowing selective payload release under tumor-specific conditions. In this study, we explored the utilization of 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), a cyclic ß-diketone featuring an active alkylidene group, to develop a novel chemically labile linker. This linker was designed to exploit the difference in reduction potential between the intracellular compartment and plasma. Upon reduction of an azido trigger strategically installed neighboring the cyclic ß-diketone, the resulting nucleophilic primary amine reacts with the alkylidene group facilitated by a favorable ring closure reaction in accordance with Baldwin's rules. Consequently, this reaction enables the simultaneous release of the attached cytotoxic payload. The therapeutic utility of this novel linker strategy was demonstrated by separate conjugation of the linker to two epidermal growth factor receptor (EGFR)-targeting ligands to afford a peptide-drug conjugate and an ADC. This work comprises a significant contribution to the bioconjugation field by introducing the alkylidene cyclic ß-diketone as a tunable scaffold used for the temporary conjugation of therapeutic agents to peptides and proteins.

Self-determination theory and the smoking cessation process: Daily electronic self-reports can identify the initiation of quit attempts.

OBJECTIVES: To examine the motivational predictors of the smoking cessation process at the between-persons and within-persons levels. METHODS: Mediation analyses were conducted on self-report data (N = 236) that were collected using interval contingent sampling over a 39-day study period. RESULTS: There was a high rate of attrition, as nearly 50% of participants were lost to follow-up. There were credible indirect effects of autonomous self-regulation on smoking behavior on the next day and seven-day abstinence through perceived competence and medication use. At the between-persons level, these models explained 17% of the variance in smoking behavior on the next day and 31% of the variance in seven-day abstinence; at the within-persons level, these estimates were 39% and 57%, respectively. CONCLUSIONS: Day-to-day changes in autonomous self-regulation, perceived competence, and medication use are important initiators of the smoking cessation process. PRACTICE IMPLICATIONS: Smokers might be more likely to make a quit attempt if practitioners "tune into" the day-to-day fluctuations of their patients' motivation for stopping smoking, perhaps using an electronic platform to assess and compare smokers' current reports to their previous experiences. Such "motivational attunement" can afford practitioners an opportunity to provide need support when patients are willing and able to initiate a quit attempt.

A randomised controlled trial of long NY-ESO-1 peptide-pulsed autologous dendritic cells with or without alpha-galactosylceramide in high-risk melanoma.

AIM: We have previously reported that polyfunctional T cell responses can be induced to the cancer testis antigen NY-ESO-1 in melanoma patients injected with mature autologous monocyte-derived dendritic cells (DCs) loaded with long NY-ESO-1-derived peptides together with α-galactosylceramide (α-GalCer), an agonist for type 1 Natural Killer T (NKT) cells. OBJECTIVE: To assess whether inclusion of α-GalCer in autologous NY-ESO-1 long peptide-pulsed DC vaccines (DCV + α-GalCer) improves T cell responses when compared to peptide-pulsed DC vaccines without α-GalCer (DCV). DESIGN, SETTING AND PARTICIPANTS: Single-centre blinded randomised controlled trial in patients ≥ 18 years old with histologically confirmed, fully resected stage II-IV malignant cutaneous melanoma, conducted between July 2015 and June 2018 at the Wellington Blood and Cancer Centre of the Capital and Coast District Health Board. INTERVENTIONS: Stage I. Patients were randomised to two cycles of DCV or DCV + α-GalCer (intravenous dose of 10 × 106 cells, interval of 28 days). Stage II. Patients assigned to DCV + α-GalCer were randomised to two further cycles of DCV + α-GalCer or observation, while patients initially assigned to DCV crossed over to two cycles of DCV + α-GalCer. OUTCOME MEASURES: Primary: Area under the curve (AUC) of mean NY-ESO-1-specific T cell count detected by ex vivo IFN-γ ELISpot in pre- and post-treatment blood samples, compared between treatment arms at Stage I. Secondary: Proportion of responders in each arm at Stage I; NKT cell count in each arm at Stage I; serum cytokine levels at Stage I; adverse events Stage I; T cell count for DCV + α-GalCer versus observation at Stage II, T cell count before versus after cross-over. RESULTS: Thirty-eight patients gave written informed consent; 5 were excluded before randomisation due to progressive disease or incomplete leukapheresis, 17 were assigned to DCV, and 16 to DCV + α-GalCer. The vaccines were well tolerated and associated with increases in mean total T cell count, predominantly CD4+ T cells, but the difference between the treatment arms was not statistically significant (difference - 6.85, 95% confidence interval, - 21.65 to 7.92; P = 0.36). No significant improvements in T cell response were associated with DCV + α-GalCer with increased dosing, or in the cross-over. However, the NKT cell response to α-GalCer-loaded vaccines was limited compared to previous studies, with mean circulating NKT cell levels not significantly increased in the DCV + α-GalCer arm and no significant differences in cytokine response between the treatment arms. CONCLUSIONS: A high population coverage of NY-ESO-1-specific T cell responses was achieved with a good safety profile, but we failed to demonstrate that loading with α-GalCer provided an additional advantage to the T cell response with this cellular vaccine design. CLINICAL TRIAL REGISTRATION: ACTRN12612001101875. Funded by the Health Research Council of New Zealand.

Honey bee colony loss linked to parasites, pesticides and extreme weather across the United States.

Honey bee (Apis mellifera) colony loss is a widespread phenomenon with important economic and biological implications, whose drivers are still an open matter of investigation. We contribute to this line of research through a large-scale, multi-variable study combining multiple publicly accessible data sources. Specifically, we analyzed quarterly data covering the contiguous United States for the years 2015-2021, and combined open data on honey bee colony status and stressors, weather data, and land use. The different spatio-temporal resolutions of these data are addressed through an up-scaling approach that generates additional statistical features which capture more complex distributional characteristics and significantly improve modeling performance. Treating this expanded feature set with state-of-the-art feature selection methods, we obtained findings that, nation-wide, are in line with the current knowledge on the aggravating roles of Varroa destructor and pesticides in colony loss. Moreover, we found that extreme temperature and precipitation events, even when controlling for other factors, significantly impact colony loss. Overall, our results reveal the complexity of biotic and abiotic factors affecting managed honey bee colonies across the United States.

Glycolipid-peptide conjugate vaccines elicit CD8+ T-cell responses and prevent breast cancer metastasis.

Objectives: Metastasis is the principal cause of breast cancer mortality. Vaccines targeting breast cancer antigens have yet to demonstrate clinical efficacy, and there remains an unmet need for safe and effective treatment to reduce the risk of metastasis, particularly for people with triple-negative breast cancer (TNBC). Certain glycolipids can act as vaccine adjuvants by specifically stimulating natural killer T (NKT) cells to provide a universal form of T-cell help. Methods: We designed and made a series of conjugate vaccines comprising a prodrug of the NKT cell-activating glycolipid α-galactosylceramide covalently linked to tumor-expressed peptides, and assessed these using E0771- and 4T1-based breast cancer models in vivo. We employed peptides from the model antigen ovalbumin and from clinically relevant breast cancer antigens HER2 and NY-ESO-1. Results: Glycolipid-peptide conjugate vaccines that activate NKT cells led to antigen-presenting cell activation, induced inflammatory cytokines, and, compared with peptide alone or admixed peptide and α-galactosylceramide, specifically enhanced CD8+ T-cell responses against tumor-associated peptides. Primary tumor growth was delayed by vaccination in all tumor models. Using 4T1-based cell lines expressing HER2 or NY-ESO-1, a single administration of the relevant conjugate vaccine prevented tumor colonisation of the lung following intravenous inoculation of tumor cells or spontaneous metastasis from breast, respectively. Conclusion: Glycolipid-peptide conjugate vaccines that activate NKT cells prevent lung metastasis in breast cancer models and warrant investigation as adjuvant therapies for high-risk breast cancer.

Quantifying Cell-Derived Changes in Collagen Synthesis, Alignment, and Mechanics in a 3D Connective Tissue Model.

Dysregulation of extracellular matrix (ECM) synthesis, organization, and mechanics are hallmark features of diseases like fibrosis and cancer. However, most in vitro models fail to recapitulate the three-dimensional (3D) multi-scale hierarchical architecture of collagen-rich tissues and as a result, are unable to mirror native or disease phenotypes. Herein, using primary human fibroblasts seeded into custom fabricated 3D non-adhesive agarose molds, a novel strategy is proposed to direct the morphogenesis of engineered 3D ring-shaped tissue constructs with tensile and histological properties that recapitulate key features of fibrous connective tissue. To characterize the shift from monodispersed cells to a highly-aligned, collagen-rich matrix, a multi-modal approach integrating histology, multiphoton second-harmonic generation, and electron microscopy is employed. Structural changes in collagen synthesis and alignment are then mapped to functional differences in tissue mechanics and total collagen content. Due to the absence of an exogenously added scaffolding material, this model enables the direct quantification of cell-derived changes in 3D matrix synthesis, alignment, and mechanics in response to the addition or removal of relevant biomolecular perturbations. To illustrate this, the effects of nutrient composition, fetal bovine serum, rho-kinase inhibitor, and pro- and anti-fibrotic compounds on ECM synthesis, 3D collagen architecture, and mechanophenotype are quantified.

Shared Decision Making in Health Care: Theoretical Perspectives for Why It Works and For Whom.

Applying both theoretical perspectives and empirical evidence, we address 2 key questions regarding shared decision making (SDM): 1) When should SDM be more patient driven, and when should it be more provider driven? and 2) Should health care providers match their SDM style/strategy to patient needs and preferences? Self-determination theory, for example, posits a distinction between autonomy and independence. A patient may autonomously seek their health care provider's input and guidance, perhaps due to low perceived competence, low coping resources, or high emotional arousal. Given their need state, they may autonomously require nonindependence. In this case, it may be more patient centered and need supportive to provide more provider-driven care. We discuss how other patient characteristics such as personality attributes, motivational state, and the course of illness and other parameters such as time available for an encounter may inform optimal provider decision-making style and strategy. We conclude that for some types of patients and clinical circumstances, a more provider-driven approach to decision making may be more practical, ethical, and efficacious. Thus, while all decision making should be patient centered (i.e., it should consider patient needs and preferences), it does not always have to be patient driven. We propose a flexible model of SDM whereby practitioners are encouraged to tailor their decision making behaviors to patient needs, preferences, and other attributes. Studies are needed to test whether matching decision-making behavior based on patient states and traits (i.e., achieving concordance) is more effective than simply providing all patients with the same type of decision making, which could be tested using matching/mismatching designs.

Tobacco Dependence Treatment: Examining Cessation Effectiveness in Oncology Settings.

The Wilmot Cancer Institute launched the Tobacco Dependence Treatment Program in 2015. Formal program evaluation consisted of 324 patients who presented for at least one visit to assess quit rates. The secondary aim was to ascertain the effectiveness of guideline recommendations that four or more visits would be beneficial in an outpatient oncology tobacco treatment program to promote success in smoking cessation. The first 32 months of program data revealed that there were significantly improved quit rates for those who were seen for four or more visits compared to those seen for three or fewer visits.

A case of metastatic malignant granular cell tumour of the scalp.

Granular cell tumours of the scalp are rare. Malignant transformation of these tumours is even more uncommon, making the diagnosis exceedingly difficult. The recommended treatment of surgical excision with negative margins is not easily achieved in this location, given the anatomy of the scalp.

A case of naevus sebaceus of Jadassohn of the scalp in Jamaica.

Naevus sebaceus (NS), also referred to as NS of Jadassohn, is a rare non-melanocytic congenital cutaneous hamartoma with mainly sebaceous differentiation. NS has pluripotent potential with the possible evolution of benign and/or malignant neoplastic transformation. Literature of clinical audit and retrospective analyses conclude that there is no need for prophylactic excision except for cases in which malignant transformation is suspected. Although malignant transformation is rare, there are psychosocial issues with which to contend. We present a case of a 5 year-old girl with a cerebriform mass to her right parietal scalp, which was present at birth.

TLR2 agonists and their structure-activity relationships.

Agonists of Toll-like Receptor 2 (TLR2) are attractive synthetic targets due to their use as adjuvants in immunotherapies to treat various diseases notably, cancer. An indepth understanding of TLR2 agonist structure-activity relationships is therefore advantageous for the methodical design of vaccines targetting the TLR2 machinery. This review aims to collate and discuss the literature regarding synthetic studies towards TLR2 agonists and the structure-activity relationships thereof. It is hoped that interested readers will gain a holistic understanding of this topic, and will prompt further efforts towards finding effective agonists of TLR2.

A randomized placebo-controlled trial of bupropion for Cancer-related fatigue: Study design and procedures.

BACKGROUND: Cancer-related fatigue is a significant problem and is associated with poor quality of life. Behavioral interventions include exercise and cognitive-behavioral therapy, which survivors may be unwilling or unable to adopt. Pharmacologic interventions (e.g., selective serotonin reuptake inhibitors) have been disappointing. One potential therapy is the antidepressant bupropion, a norepinephrine-dopamine reuptake inhibitor that targets both inflammation and the hypothalamic-pituitary-adrenal axis. The current study is intended to provide a rigorous test of the efficacy and tolerability of bupropion for cancer-related fatigue. METHODS: A randomized, double-blind, placebo-controlled trial will examine the effects of bupropion on cancer-related fatigue. The trial will be conducted nationwide through the University of Rochester Medical Center (URMC) National Cancer Institute Community Oncology Research Program (NCORP). Disease-free breast cancer survivors (n = 422) who completed chemotherapy and/or radiotherapy 12-60 months previously and report significant fatigue will be randomized 1:1 to receive bupropion (300 mg/day) or placebo. Outcomes will be assessed at baseline and the 12-week follow-up. The primary outcome, fatigue, will be measured with the Functional Assessment of Chronic Illness Therapy - Fatigue (FACIT-F). Secondary outcomes include quality of life, depression, and drug tolerability. Exploratory outcomes include cognition and symptomatology. Potential biological mechanisms and genetic moderators of cancer-related fatigue will also be explored. DISCUSSION: This study is the first placebo-controlled trial to our knowledge to evaluate bupropion for cancer-related fatigue. Positive results could revolutionize the treatment of cancer-related fatigue, as bupropion is safe, inexpensive, widely-available, and may be more tolerable and acceptable for many patients than current, limited treatment options.

Synthesis and Evaluation of Novel TLR2 Agonists as Potential Adjuvants for Cancer Vaccines.

Cancer immunotherapy has gained increasing attention due to its potential specificity and lack of adverse side effects when compared to more traditional modes of treatment. Toll-like receptor 2 (TLR2) agonists are lipopeptides possessing the S-[2,3-bis(palmitoyloxy)propyl]-l-cysteine (Pam2Cys) motif and exhibit potent immunostimulatory effects. These agonists offer a means of providing "danger signals" in order to activate the immune system toward tumor antigens. Thus, the development of TLR2 agonists is attractive in the search of potential immunostimulants for cancer. Existing SAR studies of Pam2Cys with TLR2 indicate that the structural requirements for activity are, for the most part, very intolerable. We have investigated the importance of stereochemistry, the effect of N-terminal acylation, and homologation between the two ester functionalities in Pam2Cys-conjugated lipopeptides on TLR2 activity. The R diastereomer is significantly more potent than the S diastereomer and N-terminal modification generally lowers TLR2 activity. Most notably, homologation gives rise to analogues which are comparatively active to the native Pam2Cys containing constructs.

Implications of insular cortex laterality for treatment of nicotine addiction.

BACKGROUND: Damage to the insula disrupts nicotine-induced cravings and is associated with greater odds of cessation. The role of laterality in regulating these changes is unclear. Neuroimaging studies in cigarette smokers show left hemispheric activation during a period of forced withdrawal and right hemispheric activation after having just smoked. Among current smokers hospitalized for stroke involving their insula, we compared left versus right insular damage and its effect on smoking outcomes. METHODS: A total of 37 smokers hospitalized with unilateral insular strokes (14 right, 23 left) were administered questionnaires to assess urge (Questionnaire on Smoking Urges) before (retrospectively) and during hospitalization and 3 months post-stroke, withdrawal during hospitalization (Wisconsin Smoking Withdrawal Scale), and prolonged abstinence at 3 months post-stroke. Crude and adjusted linear regression models were performed controlling for baseline covariates. RESULTS: Right and left insular-damaged smokers experienced a significant decrease in urge from baseline to hospitalization and three-month follow-up (p < 0.01). Smokers with left-sided insular infarcts relative to right-sided experienced a larger decrease in acute urge (adjusted ß=-1.16, 95% CI: -2.59, 0.27, p = 0.11) but not chronically (adjusted ß=-0.06, 95% CI: -1.53, 1.40, p = 0.93). Left-sided insular damage was also associated with significantly fewer and less severe withdrawal symptoms during hospitalization (adjusted ß=-3.52, 95% CI: -7.01, -0.04, p = 0.05). No differences were noted between groups for prolonged abstinence (p = 0.50). CONCLUSIONS: Left insular adaptations are suggestive to have an impact on acute changes in urge and withdrawal more so than the right insula, however lateral asymmetries did not exist for long-term changes.

Glucoregulatory activity of vesiculin in insulin sensitive and resistant mice.

Pancreatic islet-derived peptide hormones play key roles in the maintenance of systemic energy homeostasis and glucose balance and defects in their regulation are strongly implicated in the pathogenesis of obesity and diabetes. Peptides have also been used as lead compounds for therapeutics targeting metabolic disease. It is therefore important to understand the activity and function of islet hormones in both their target tissues and the whole organism. Insulin-like growth factor II (IGF-II) is an insulin homolog secreted by the islet ß-cells. Vesiculin is a newly discovered peptide hormone, processed from IGF-II and secreted from islet ß-cells in response to glucose. We postulated that vesiculin might act to regulate systemic glucose metabolism. Here we report our original investigations of vesiculin's activity in relation to glucoregulation. Vesiculin and IGF-II displayed similar dose-response relationships for lowering blood glucose in insulin-responsive FVB/n mice. By contrast, the ability of IGF-II to lower blood glucose was blunted in insulin-resistant triprolyl human-amylin transgenic mice, whereas vesiculin's ability to lower blood glucose remained unaffected. We also confirmed the ability of vesiculin to bypass insulin resistance in a second mouse model. In vitro analysis of signalling by vesiculin and IGF-II indicates that, like IGF-II, vesiculin signals through the IR/ IGF1R. Overall, we show that removal of only four amino acids from IGF-II has generated a peptide hormone with different bioactivity relevant to blood-glucose regulation. Investigating the differences among vesiculin, IGF-II and insulin signalling and activity may provide new insights into insulin resistance and potentially inform the design of novel therapeutics.

Enhancing T cell responses and tumour immunity by vaccination with peptides conjugated to a weak NKT cell agonist.

Activated NKT cells can stimulate antigen-presenting cells leading to enhanced peptide antigen-specific immunity. However, administration of potent NKT cell agonists like α-galactosylceramide (α-GalCer) can be associated with release of high levels of cytokines, and in some situations, hepatotoxicity. Here we show that it is possible to provoke sufficient NKT cell activity to stimulate strong antigen-specific T cell responses without these unwanted effects. This was achieved by chemically conjugating antigenic peptides to α-galactosylphytosphingosine (α-GalPhs), an NKT cell agonist with very weak activity based on structural characterisation and biological assays. Conjugation improved delivery to antigen-presenting cells in vivo, while use of a cathepsin-sensitive linker to release the α-GalPhs and peptide within the same cell promoted strong T cell activation and therapeutic anti-tumour responses in mice. The conjugates activated human NKT cells and enhanced human T cell responses to a viral peptide in vitro. Accordingly, we have demonstrated a means to safely exploit the immunostimulatory properties of NKT cells to enhance T cell activation for virus- and tumour-specific immunity.

Does E-cigarette Use at Baseline Influence Smoking Cessation Rates among 2-Year College Students?

INTRODUCTION: This study evaluates the impact of baseline e-cigarette use on smoking cessation rates in a national sample of two-year college student smokers. METHODS: Participants were 1400 students from over 60 two-year colleges across 25 states who were current smokers enrolled in a web-assisted tobacco intervention (WATI) trial. Survey data were collected at baseline, 1-, 6-, and 12-months, with primary outcomes evaluated at 6-months. RESULTS: At 6-months, baseline e-cigarette users were more likely to report cessation of traditional cigarettes compared to non-users (OR 1.39, 95% CI 1.002-1.92). Cessation was also associated with higher baseline confidence in quitting and greater time to first cigarette in the morning. Baseline e-cigarette use was not found to be associated with self-reported cessation of all nicotine/tobacco products (OR 1.09, 95% CI 0.75-1.58) nor biochemically verified cessation of all nicotine/tobacco products (OR 0.83, 95% CI 0.47-1.47). Higher confidence was again associated with both self-reported and biochemically verified cessation of all nicotine/tobacco products. Female gender was associated only with biochemically verified cessation of all nicotine/tobacco products at 6-months. CONCLUSIONS: Two-year college students represent a priority population for cessation interventions. The findings from this study highlight the complexities of evaluating the impact of e-cigarette use on cessation.

Genetic testing for a patient with suspected familial hypercholesterolaemia.

Familial hypercholesterolaemia (FH) is a genetic condition that results in elevated low-density lipoprotein (LDL) cholesterol (LDL-C) levels with consequent increased risk for premature cardiovascular disease events. Although it is considered an autosomal-dominant genetic condition, the underlying genetic causes of FH can be complex. Currently most guidelines rely on clinical criteria to diagnose FH. But this approach has some pitfalls. We present a patient who was not formally diagnosed with FH using commonly used and well-accepted clinical criteria but via genetic testing was found to have a mutation for this disorder. This case brings to fore the challenges clinicians face in diagnosing and managing such unusual cases optimally. Through this case report, we hope to stimulate a debate among clinicians as well as other stakeholders regarding the need to develop more efficient ways of selecting patients for genetic testing in response to elevated LDL levels.

A phase I vaccination study with dendritic cells loaded with NY-ESO-1 and α-galactosylceramide: induction of polyfunctional T cells in high-risk melanoma patients.

Vaccines that elicit targeted tumor antigen-specific T-cell responses have the potential to be used as adjuvant therapy in patients with high risk of relapse. However, the responses induced by vaccines in cancer patients have generally been disappointing. To improve vaccine function, we investigated the possibility of exploiting the immunostimulatory capacity of type 1 Natural killer T (NKT) cells, a cell type enriched in lymphoid tissues that can trigger improved antigen-presenting function in dendritic cells (DCs). In this phase I dose escalation study, we treated eight patients with high-risk surgically resected stage II-IV melanoma with intravenous autologous monocyte-derived DCs loaded with the NKT cell agonist α-GalCer and peptides derived from the cancer testis antigen NY-ESO-1. Two synthetic long peptides spanning defined immunogenic regions of the NY-ESO-1 sequence were used. This therapy proved to be safe and immunologically effective, inducing increases in circulating NY-ESO-1-specific T cells that could be detected directly ex vivo in seven out of eight patients. These responses were achieved using as few as 5 × 105 peptide-loaded cells per dose. Analysis after in vitro restimulation showed increases in polyfunctional CD4+ and CD8+ T cells that were capable of manufacturing two or more cytokines simultaneously. Evidence of NKT cell proliferation and/or NKT cell-associated cytokine secretion was seen in most patients. In light of these strong responses, the concept of including NKT cell agonists in vaccine design requires further investigation.