Tyrosine Sulfation at Antibody Light Chain CDR-1 Increases Binding Affinity and Neutralization Potency to Interleukine-4.

Structure and function of therapeutic antibodies can be modulated by a variety of post-translational modifications (PTM). Tyrosine (Tyr) sulfation is a type of negatively charged PTM that occurs during protein trafficking through the Golgi. In this study, we discovered that an anti-interleukin (IL)-4 human IgG1, produced by transiently transfected HEK293 cells, contained a fraction of unusual negatively charged species. Interestingly, the isolated acidic species exhibited a two-fold higher affinity to IL-4 and a nearly four-fold higher potency compared to the main species. Mass spectrometry (MS) showed the isolated acidic species possessed an +80-Dalton from the expected mass, suggesting an occurrence of Tyr sulfation. Consistent with this hypothesis, we show the ability to control the acidic species during transient expression with the addition of Tyr sulfation inhibitor sodium chlorate or, conversely, enriched the acidic species from 30% to 92% of the total antibody protein when the IL-4 IgG was co-transfected with tyrosylprotein sulfotransferase genes. Further MS and mutagenesis analysis identified a Tyr residue at the light chain complementarity-determining region-1 (CDRL-1), which was sulfated specifically. These results together have demonstrated for the first time that Tyr sulfation at CDRL-1 could modulate antibody binding affinity and potency to a human immune cytokine.

Results of Leveraging Pharmaceutical Patient Assistance Programs to Expand Access to High Cost Medications in a Student-Run Free Clinic.

High costs make many medications inaccessible to patients in the United States. Uninsured and underinsured patients are disproportionately affected. Pharmaceutical companies offer patient assistance programs (PAPs) to lower the cost-sharing burden of expensive prescription medications for uninsured patients. PAPs are used by various clinics, particularly oncology clinics and those caring for underserved communities, to expand patients' access to medications. Prior studies describing the implementation of PAPs in student-run free clinics have demonstrated cost-savings during the first few years of using PAPs. However, there is a lack of data regarding the efficacy and cost savings of longitudinal use of PAPs across several years. This study describes the growth of PAP use at a student-run free clinic in Nashville, Tennessee over ten years, demonstrating that PAPs can be used reliably and sustainably to expand patients' access to expensive medications. From 2012 to 2021, we increased the number of medications available through PAPs from 8 to 59 and the number of patient enrollments from 20 to 232. In 2021, our PAP enrollments demonstrated potential cost savings of over $1.2 million. Strategies, limitations, and future directions of PAP use are also discussed, highlighting that PAPs can be a powerful tool for free clinics in serving underserved communities.

ERG promotes the maintenance of hematopoietic stem cells by restricting their differentiation.

The balance between self-renewal and differentiation is crucial for the maintenance of hematopoietic stem cells (HSCs). Whereas numerous gene regulatory factors have been shown to control HSC self-renewal or drive their differentiation, we have relatively few insights into transcription factors that serve to restrict HSC differentiation. In the present work, we identify ETS (E-twenty-six)-related gene (ERG) as a critical factor protecting HSCs from differentiation. Specifically, loss of Erg accelerates HSC differentiation by >20-fold, thus leading to rapid depletion of immunophenotypic and functional HSCs. Molecularly, we could demonstrate that ERG, in addition to promoting the expression of HSC self-renewal genes, also represses a group of MYC targets, thereby explaining why Erg loss closely mimics Myc overexpression. Consistently, the BET domain inhibitor CPI-203, known to repress Myc expression, confers a partial phenotypic rescue. In summary, ERG plays a critical role in coordinating the balance between self-renewal and differentiation of HSCs.

Rotational thromboelastometry (ROTEM® ) in gestational diabetes mellitus and coagulation in healthy term pregnancy: A prospective observational study in Australia.

BACKGROUND: Rotational thromboelastometry (ROTEM® ) is a point-of-care test of coagulation. ROTEM® -defined hypercoagulability has been identified in pregnant women and in non-pregnant patients with diabetes mellitus. Pregnancy is known to be a hypercoagulable state, but the influence of gestational diabetes mellitus (GDM) on coagulation is unknown. AIM: The aim of this study was to assess the combined effect of pregnancy and GDM on coagulation using ROTEM® and to compare this to healthy pregnant women presenting for elective caesarean delivery. MATERIALS AND METHODS: Ethics approval was granted for recruitment of women presenting for elective caesarean delivery. Women with pre-existing conditions affecting coagulation were excluded. Group N included health pregnant women at term and Group G included pregnant women at term with GDM. Data regarding GDM management and glycaemic control were collected. Poor glycaemic control was defined by markers of accelerated fetal growth and elevated fasting or postprandial blood glucose levels. The ROTEM® parameters (extrinsically activated thromboelastometric test (EXTEM) / fibrin polymerisation test (FIBTEM) amplitude at five minutes, coagulation time, maximum clot firmness and clot formation time) were compared between the two groups using Student's t-test. RESULTS: There were 75 women in Group N and 21 women in Group G. Mean age and median body mass index values were comparable for both groups. There were no statistical differences found between the EXTEM and FIBTEM parameters analysed for the two groups. CONCLUSIONS: There was no association between GDM and increased hypercoagulability as demonstrated by ROTEM® parameters in healthy pregnant women presenting for elective caesarean delivery at term.

Nasal interferon responses to community rhinovirus infections are similar in controls and children with asthma.

BACKGROUND: Rhinovirus (RV) is the main cause of asthma exacerbations in children. Some studies reported that persons with asthma have attenuated interferon (IFN) responses to experimental RV infection compared with healthy individuals. However, responses to community-acquired RV infections in controls and children with asthma have not been compared. OBJECTIVE: To evaluate nasal cytokine responses after natural RV infections in people with asthma and healthy children. METHODS: We compared nasal cytokine expression among controls and children with asthma during healthy, virus-negative surveillance weeks and self-reported RV-positive sick weeks. A total of 14 controls and 21 patients with asthma were studied. Asthma disease severity was based on symptoms and medication use. Viral genome was detected by multiplex polymerase chain reaction. Nasal cytokine protein levels were determined by multiplex assays. RESULTS: Two out of 47 surveillance weeks tested positive for RV, illustrating an asymptomatic infection rate of 5%. A total of 38 of 47 sick weeks (81%) tested positive for the respiratory virus. Of these, 33 (87%) were positive for RV. During well weeks, nasal interleukin 8 (IL-8), IL-12, and IL-1ß levels were higher in children with asthma than controls. Compared with healthy virus-negative surveillance weeks, IL-8, IL-13, and interferon beta increased during colds only in patients with asthma. In both controls and children with asthma, the nasal levels of interferon gamma, interferon lambda-1, IL-1ß, IL-8, and IL-10 increased during RV-positive sick weeks. During RV infection, IL-8, IL-1ß, and tumor necrosis factor-α levels were strongly correlated. CONCLUSION: In both controls and patients with asthma, natural RV infection results in robust type II and III IFN responses.

Pediatric liver transplantation in Australia and New Zealand: The case for a collaborative anesthetic database.

BACKGROUND: Liver transplantation is conducted with strict oversight of organizational structure and clinical practice. However, specific regulations pertaining to the delivery of anesthetic services are lacking and consideration of departmental structure and mechanisms for quality control must occur at a local level. Busy centers collect and process sufficient data to guide this process but those with low case loads may not generate enough data for useful analysis. In Australia and New Zealand, pediatric liver transplants are performed at only four locations. As these operations are not equally distributed geographically or temporally there are periods of low activity at some centers. As anesthesia affects patient outcome, quality assurance activities are important in this setting. AIMS: Provide a global overview of the structure and function of liver transplantation networks. Identify issues related to provision of pediatric anesthetic services with specific reference to Australasia. Examine anesthetic data from a single pediatric center to illustrate benefits and limitations of such activity. METHODS: Pediatric liver transplant centers from Australia and New Zealand were surveyed to determine the organizational and logistical issues related to a liver transplant service. An audit of 15 years of liver transplants from a single center was conducted for benchmarking purposes and to identify changes in anesthetic practice over time. RESULTS: Pediatric liver transplants performed in Queensland from January 2005 to December 2019 were reviewed. Changes in transfusion practice reflected international trends. Morbidity and mortality were comparable to international data. Important complications such as hepatic artery and portal vein thrombosis were uncommon and did not generate enough data for further analysis. CONCLUSIONS: Combining the anesthetic liver transplant data from all sites in a single registry would expand data collection and generate broadly applicable findings. We propose the establishment of an Australasian pediatric anesthetic liver transplant database.

We're all in this together, but for different reasons: Social values and social actions that affect COVID-19 preventative behaviors.

We examined how personal values, beliefs and concerns about COVID-19, and socio-demographics, relate to two important COVID-19 preventative behaviors: willingness to get vaccinated for COVID-19 and social distancing, in 1413 Australian adults. As expected, social focus values influenced the extent of compliance with these preventative behaviors, even when controlling for beliefs and concerns about COVID-19 and socio-demographics. We also examined the persuasiveness of four different value-expressive messages promoting social distancing, in a subsample of 737 Australian adults. We found that the message expressing self-transcendence values was ranked most persuasive by 77% of respondents. However, as hypothesized, personal values were related to message persuasiveness. As the importance ascribed to social focus values increased, the likelihood that the self-transcendence message was ranked as most persuasive increased. In contrast, the likelihood that the openness to change message was ranked as most persuasive increased for those who ascribed lesser importance to social focus values. Our findings can help the framing of government messaging around preventative behaviors, including maintaining social distancing in vaccinated populations who may still spread the disease, and preventing COVID-19 spread by or to vaccine refusers.

MK-8719, a Novel and Selective O-GlcNAcase Inhibitor That Reduces the Formation of Pathological Tau and Ameliorates Neurodegeneration in a Mouse Model of Tauopathy.

Deposition of hyperphosphorylated and aggregated tau protein in the central nervous system is characteristic of Alzheimer disease and other tauopathies. Tau is subject to O-linked N-acetylglucosamine (O-GlcNAc) modification, and O-GlcNAcylation of tau has been shown to influence tau phosphorylation and aggregation. Inhibition of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc moieties, is a novel strategy to attenuate the formation of pathologic tau. Here we described the in vitro and in vivo pharmacological properties of a novel and selective OGA inhibitor, MK-8719. In vitro, this compound is a potent inhibitor of the human OGA enzyme with comparable activity against the corresponding enzymes from mouse, rat, and dog. In vivo, oral administration of MK-8719 elevates brain and peripheral blood mononuclear cell O-GlcNAc levels in a dose-dependent manner. In addition, positron emission tomography imaging studies demonstrate robust target engagement of MK-8719 in the brains of rats and rTg4510 mice. In the rTg4510 mouse model of human tauopathy, MK-8719 significantly increases brain O-GlcNAc levels and reduces pathologic tau. The reduction in tau pathology in rTg4510 mice is accompanied by attenuation of brain atrophy, including reduction of forebrain volume loss as revealed by volumetric magnetic resonance imaging analysis. These findings suggest that OGA inhibition may reduce tau pathology in tauopathies. However, since hundreds of O-GlcNAcylated proteins may be influenced by OGA inhibition, it will be critical to understand the physiologic and toxicological consequences of chronic O-GlcNAc elevation in vivo. SIGNIFICANCE STATEMENT: MK-8719 is a novel, selective, and potent O-linked N-acetylglucosamine (O-GlcNAc)-ase (OGA) inhibitor that inhibits OGA enzyme activity across multiple species with comparable in vitro potency. In vivo, MK-8719 elevates brain O-GlcNAc levels, reduces pathological tau, and ameliorates brain atrophy in the rTg4510 mouse model of tauopathy. These findings indicate that OGA inhibition may be a promising therapeutic strategy for the treatment of Alzheimer disease and other tauopathies.

IL-1ß prevents ILC2 expansion, type 2 cytokine secretion, and mucus metaplasia in response to early-life rhinovirus infection in mice.

BACKGROUND: Early-life wheezing-associated respiratory infection with human rhinovirus (RV) is associated with asthma development. RV infection of 6-day-old immature mice causes mucous metaplasia and airway hyperresponsiveness which is associated with the expansion of IL-13-producing type 2 innate lymphoid cells (ILC2s) and dependent on IL-25 and IL-33. We examined regulation of this asthma-like phenotype by IL-1ß. METHODS: Six-day-old wild-type or NRLP3-/- mice were inoculated with sham or RV-A1B. Selected mice were treated with IL-1 receptor antagonist (IL-1RA), anti-IL-1ß, or recombinant IL-1ß. RESULTS: Rhinovirus infection induced Il25, Il33, Il4, Il5, Il13, muc5ac, and gob5 mRNA expression, ILC2 expansion, mucus metaplasia, and airway hyperresponsiveness. RV also induced lung mRNA and protein expression of pro-IL-1ß and NLRP3 as well as cleavage of caspase-1 and pro-IL-1ß, indicating inflammasome priming and activation. Lung macrophages were a major source of IL-1ß. Inhibition of IL-1ß signaling with IL-1RA, anti-IL-1ß, or NLRP3 KO increased RV-induced type 2 cytokine immune responses, ILC2 number, and mucus metaplasia, while decreasing IL-17 mRNA expression. Treatment with IL-1ß had the opposite effect, decreasing IL-25, IL-33, and mucous metaplasia while increasing IL-17 expression. IL-1ß and IL-17 each suppressed Il25, Il33, and muc5ac mRNA expression in cultured airway epithelial cells. Finally, RV-infected 6-day-old mice showed reduced IL-1ß mRNA and protein expression compared to mature mice. CONCLUSION: Macrophage IL-1ß limits type 2 inflammation and mucous metaplasia following RV infection by suppressing epithelial cell innate cytokine expression. Reduced IL-1ß production in immature animals provides a mechanism permitting asthma development after early-life viral infection.

Value Profiles During Middle Childhood: Developmental Processes and Social Behavior.

Little is known about how children's value priorities develop over time. This study identifies children's value priority profiles and follows their development during middle childhood. Australian children (N = 609; ages 5-12 at Time 1) reported their values over 2 years. Latent Transition Analysis indicated four profiles: Social-Focus, Self-Focus, Growth-Focus and Undifferentiated. Within person development was characterized by profile stability or transfer to the Social-Focus profile. Younger children were more likely to have an Undifferentiated profile (or Self-Focus among boys) than older ones. Girls were more likely to have a Social-Focus profile or transfer to it, and less likely to have a Self- or Growth-Focus profile than boys. Social-Focus profile membership over time predicted more prosocial and less aggressive behavior.

The influence of obesity on coagulation in healthy term pregnancy as assessed by rotational thromboelastometry.

BACKGROUND: Rotational thromboelastometry (ROTEM® ) is a point-of-care coagulation test which has been used to demonstrate hypercoagulability in pregnant populations and obese populations. AIM: The aim of this study was to assess the combined effect of pregnancy and obesity on coagulation using ROTEM® in healthy pregnant women of varying body mass indices (BMIs) presenting for elective caesarean delivery. MATERIALS AND METHODS: Ethics approval was granted for recruitment of women presenting for elective caesarean delivery. Women with any condition affecting coagulation were excluded. The ROTEM® parameters of extrinsically activated thromboelastometric test / fibrin polymerisation test (EXTEM/FIBTEM) amplitude at five minutes (A5), coagulation time (CT), maximum clot firmness (MCF) and clot formation time (CFT) were compared between three different groups: normal weight, overweight and obese women. RESULTS: One hundred and eighty-five women presenting for elective caesarean delivery met inclusion criteria and were divided into three groups; normal weight (BMI < 25 kg/m2 , n = 86), overweight (BMI 25-29.9 kg/m2 , n = 54) and obese (BMI ≥ 30 kg/m2 , n = 45). They had a mean (SD) age of 32.7 ± 5.0 years and the median (interquartile range) BMI of 21.9 kg/m2 (20.5-23.0), 27.0 kg/m2 (26.0-28.5), 36.0 kg/m2 (32.2-41.8) for the normal weight, overweight and obese groups respectively. Forty-one (22.2%) women were nulliparous. Across the three groups for FIBTEM A5 (P = 0.018), FIBTEM MCF (P = 0.032), FIBTEM CFT (P = 0.047) and EXTEM MCF (P = 0.015) there was evidence of increasing coagulability with increasing BMI. However, following Bonferroni correction, this was no longer significant. CONCLUSIONS: There is no association between BMI and ROTEM® parameters in pregnant women presenting for elective caesarean delivery at term.

Flexibility to contingency changes distinguishes habitual and goal-directed strategies in humans.

Decision-making in the real world presents the challenge of requiring flexible yet prompt behavior, a balance that has been characterized in terms of a trade-off between a slower, prospective goal-directed model-based (MB) strategy and a fast, retrospective habitual model-free (MF) strategy. Theory predicts that flexibility to changes in both reward values and transition contingencies can determine the relative influence of the two systems in reinforcement learning, but few studies have manipulated the latter. Therefore, we developed a novel two-level contingency change task in which transition contingencies between states change every few trials; MB and MF control predict different responses following these contingency changes, allowing their relative influence to be inferred. Additionally, we manipulated the rate of contingency changes in order to determine whether contingency change volatility would play a role in shifting subjects between a MB and MF strategy. We found that human subjects employed a hybrid MB/MF strategy on the task, corroborating the parallel contribution of MB and MF systems in reinforcement learning. Further, subjects did not remain at one level of MB/MF behaviour but rather displayed a shift towards more MB behavior over the first two blocks that was not attributable to the rate of contingency changes but rather to the extent of training. We demonstrate that flexibility to contingency changes can distinguish MB and MF strategies, with human subjects utilizing a hybrid strategy that shifts towards more MB behavior over blocks, consequently corresponding to a higher payoff.

Assessing pharmacy student experience with, knowledge of and attitudes towards harm reduction: illuminating barriers to pharmacist-led harm reduction.

BACKGROUND: As the burden from the opioid epidemic continues to increase in the state of Minnesota and across the nation, the University of Minnesota College of Pharmacy seeks to design an innovative, comprehensive harm reduction curriculum in order to better train student pharmacists to serve the varied needs of the greater community. This study examines incoming individuals' baseline knowledge of and attitudes toward harm reduction in order to better inform curriculum planning and to ultimately produce pharmacists capable of impacting the devastating effects of the opioid crisis. METHODS: Incoming first-year pharmacy students took a survey focused on their knowledge of opioid overdose and the drug naloxone and also provided written reflections on their perceptions of harm reduction. Data was coded using consensual qualitative research (CQR) into appropriate domains. RESULTS: Pharmacy students beginning their professional education revealed a lack of knowledge of proper response to an overdose situation, with 18.56% unfamiliar with the opioid antagonist drug naloxone. Close to 10% (9.58%) of students expressed unwillingness to do anything other than call an ambulance during an overdose event, while 8.98% were either unsure or felt that they would not feel compelled to do something to help. Qualitative coding revealed many barriers to students' becoming capable harm reductionists, including lack of knowledge of substance use, addiction, and harm reduction, in addition to the presence of bias and stigma. CONCLUSION: In order to interrupt the cycle of misinformation and stigma within the larger community and the subgroup of medical providers, gaps in student knowledge must be addressed in meaningful, specific ways over the course of their pharmacy education. Evaluating baseline knowledge and beliefs informs the design of a flexible, action-oriented curriculum to produce well-trained pharmacists ready to engage in finding solutions to the opioid crisis.

Design and synthesis of water soluble ß-aminosulfone analogues of SCH 900229 as γ-secretase inhibitors.

In this paper we describe our strategy to improve the aqueous solubility of SCH 900229, a potent PS1-selective γ-secretase inhibitor for the treatment of Alzheimer's disease. Incorporation of ionizable amino groups into the side chain terminal generates water soluble ß-aminosulfone analogues of SCH 900229 that maintain robust in vitro potency and in vivo efficacy.

Therapeutic activity of an interleukin-4/interleukin-13 dual antagonist on oxazolone-induced colitis in mice.

Interleukin-4 (IL-4) and IL-13 are critical drivers of immune activation and inflammation in ulcerative colitis, asthma and other diseases. Because these cytokines may have redundant function, dual targeting holds promise for achieving greater efficacy. We have recently described a bifunctional therapeutic targeting IL-4 and IL-13 developed on a novel protein scaffold, generated by combining specific binding domains in an optimal configuration using appropriate linker regions. In the current study, the bifunctional IL-4/IL-13 antagonist was evaluated in the murine oxazolone-induced colitis model, which produces disease with features of ulcerative colitis. The bifunctional IL-4/IL-13 antagonist reduced body weight loss throughout the 7-day course of the model, and ameliorated the increased colon weight and decreased colon length that accompany disease. Colon tissue gene expression was modulated in accordance with the treatment effect. Concentrations of serum amyloid P were elevated in proportion to disease severity, making it an effective biomarker. Serum concentrations of the bifunctional IL-4/IL-13 antagonist were inversely proportional to disease severity, colon tissue expression of pro-inflammatory genes, and serum amyloid P concentration. Taken together, these results define a panel of biomarkers signifying engagement of the IL-4/IL-13 pathway, confirm the T helper type 2 nature of disease in this model, and demonstrate the effectiveness of dual cytokine blockade.

Quantitative analysis of target coverage and germinal center response by a CXCL13 neutralizing antibody in a T-dependent mouse immunization model.

PURPOSE: Study the impact of CXCL13 neutralization on germinal center (GC) response in vivo, and build quantitative relationship between target coverage and pharmacological effects at the target tissue. METHODS: An anti-CXCL13 neutralizing monoclonal antibody was dosed in vivo in a T-dependent mouse immunization (TDI) model. A quantitative site-of-action (SoA) model was developed to integrate antibody PK and total CXCL13 levels in serum and spleen towards estimating target coverage as a function of dose. To aid in the SoA model development, a radio-labeled study using [I(125)] CXCL13 was conducted in mice. Model estimated target coverage was linked to germinal center response using a sigmoidal inhibitory effect model. RESULTS: In vivo studies demonstrated that CXCL13 inhibition led to an architectural change in B-cell follicles, dislocation of GCs and a significant reduction in the GC absolute numbers per square area (GC/mm(2)). The SoA modeling analysis indicated that ~79% coverage in spleen was required to achieve 50% suppression of GC/mm(2). The 3 mg/kg dose with 52% spleen coverage resulted in no PD suppression, whereas 30 mg/kg with 93% coverage achieved close to maximum PD suppression, highlighting the steepness of PD response. CONCLUSIONS: This study showcases an application of SoA modeling towards a quantitative understanding of CXCL13 pharmacology.

Discovery of potent iminoheterocycle BACE1 inhibitors.

The synthesis of a series of iminoheterocycles and their structure-activity relationships (SAR) as inhibitors of the aspartyl protease BACE1 will be detailed. An effort to access the S3 subsite directly from the S1 subsite initially yielded compounds with sub-micromolar potency. A subset of compounds from this effort unexpectedly occupied a different binding site and displayed excellent BACE1 affinities. Select compounds from this subset acutely lowered Aß40 levels upon subcutaneous and oral administration to rats.

Clinical characteristics associated with Spitz nevi and Spitzoid malignant melanomas: the Yale University Spitzoid Neoplasm Repository experience, 1991 to 2008.

BACKGROUND: Spitz nevi and Spitzoid malignant melanomas are uncommon and may be difficult to distinguish histopathologically. Identification of clinical features associated with these lesions may aid in diagnosis. OBJECTIVE: We sought to identify clinical characteristics associated with Spitz nevi and Spitzoid malignant melanomas. METHODS: We conducted a retrospective cohort study of Spitz nevi and Spitzoid malignant melanomas from the Yale University Spitzoid Neoplasm Repository diagnosed from years 1991 through 2008. Descriptive statistics and multivariate logistic regression were used to compare select patient- and tumor-level factors associated with each lesion. RESULTS: Our cohort included 484 Spitz nevi and 54 Spitzoid malignant melanomas. Spitz nevi were more common (P = .03) in females (65%; n = 316) compared with Spitzoid malignant melanomas (50%; n = 27), occurred more frequently in younger patients (mean age at diagnosis 22 vs 55 years; P < .001), and more likely presented as smaller lesions (diameter 7.6 vs 10.5 mm; P < .001). Increasing age (odds ratio 1.16, 95% CI [1.09, 1.14]; P< .001) and male gender (odds ratio 2.77, 95% CI [1.17, 6.55]; P< .02) predicted Spitzoid malignant melanoma diagnosis. LIMITATIONS: Small sample size, unmeasured confounding, and restriction to a single institution may limit the accuracy and generalizability of our findings. CONCLUSIONS: Age and gender help predict diagnosis of Spitz nevi and Spitzoid malignant melanomas.