Immobilisation of enzymes to Perloza cellulose resin : this thesis was presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University /

The studies reported in this thesis describe the use of Perloza™ beaded cellulose resin as a solid support for enzyme immobilisation via covalent binding. The aim of the project was to extend the uses for Perloza™ and to compare the use of well known solid support activation chemistries with a recently developed one for Perloza™. Preparations such as these have potential industrial uses. Three attachment chemistries were studied. The first activation employed 1,1-carbodiimidazole (CDI) then direct attachment of enzyme. The second again used CDI activation followed by attachment of a 6-aminocaproic acid spacer arm and then the enzyme. The final method used was attachment of a diol and subsequent oxidation to an aldehyde. The diol/aldehyde method had the advantage over the CDI methods of being based on aqueous chemistries. The two CDI based methods require extensive use of dry organic solvents. The enzymes investigated in this study were trypsin, chymotrypsin. α-amylase, horseradish peroxidase (HRPO) and alcohol dehydrogenase (ADH). Trypsin was immobilised successtully by all three chemistries. All preparations retained significant activity after immobilisation at room temperature as judged by the chromogenic substrate specific for trypsin N-α-benzoyl-DL-arginine-p-nitroanilide.HC1 (BAPNA). Measurable activity was retained in different studies from between 2 to 7 days at 60°C. The activity of immobilised trypsin with a synthetic peptide substrate was comparable to the activity of free trypsin with the same substrate. Chymotrypsin was also successfully immobilised using all three chemistries. Each preparation showed significant retention of activity after immobilisation as judged by the chromogentic substrate N-glutaryl-L.-phenylalanine-p-nitroanilide (GAPNA). Stabilisation to heating at 60°C was less successful than with trypsin but significant activity was still retained for between 3 and 6 hours. The activity of immobilised preparations with a peptide substrate was comparable to free chymotrypsin. α-Amylase, horseradish peroxidase and alcohol dehydrogenase were studied less extensively than trypsin and chymotrypsin. Nevertheless all three enzymes were successfully immobilised onto Perloza™-CDI-ACA and Perloza™-Diol/Aldehyde. Difficulty was encountered in achieving significant levels of any enzyme immobilisation to Perloza™-CDI for all three enzymes. Subsequent activity assays showed HRPO and α-amylase retained significant activity on all three resin preparations. ADH showed no measurable activity on Perloza™-CDI and very little activity on Perloza™- CDI-ACA and Perloza™-Diol/Aldehyde. Investigations have shown that enzymes can be immobilised on Perloza™ with retention of significant amounts of normal activity at room temperature and improved stability compared with free enzyme at high temperature. Comparisons of the CDI activations with the diol/aldeyde chemistry showed better performance by the latter in trypsin immobilisation and similar performance for chymotrypsin immobilisation. Horseradish peroxidase and ™-amylase were successfully immobilised using CDI/ACA and diol/aldehyde chemistries with the CDI/ACA giving higher initial specific activities than the diol/aldehyde preparation. Alcohol dehydrogenase was also successfully immobilised but gave no measurable activity

Integrating approaches requires more than a division of labour: commentary on Wӧlfer & Hewstone

Wӧlfer and Hewstone (2015; hereafter W&H) argue that evolutionary psychology (EP) is useful for understanding sex differences in same-sex aggression, while social role theory (SRT) is best applied to sex differences in opposite-sex aggression. W&H tested this proposal using a rich dataset on high school students’ peer-reported aggression. They regressed classroom-level sex differences in same- and opposite-sex aggression onto five variables drawn from the two theoretical positions. Three variables (gender and masculinity norms, derived from SRT and body dimorphism, derived from EP) did not differ in their association with the two forms of aggression. Another variable (sex ratio: EP) was not interpretable because it was confounded with number of available targets, leaving a fifth (male hierarchy: EP) predicting sex differences in same-sex but not opposite-sex aggression. Our focus is not on the study itself, but on their proposal that theoretical disputes between EP and SRT can be resolved by assigning one form of aggression to EP and another to SRT. We believe that this argument mischaracterises both theories, reinforces the ‘evolutionary vs social’ divide, and falls short of integration.PostprintPeer reviewe

COVID-19 outcomes and vaccination in people with relapsing multiple sclerosis treated with ofatumumab

INTRODUCTION: The SARS-CoV-2 pandemic necessitated better understanding of the impact of disease-modifying therapies on COVID-19 outcomes and vaccination. We report characteristics of COVID-19 cases and vaccination status in ofatumumab-treated relapsing multiple sclerosis (RMS) patients. METHODS: COVID-19 data analyzed were from the ongoing, open-label, long-term extension phase 3b ALITHIOS study from December 2019 (pandemic start) and post-marketing cases from August 2020 (ofatumumab first approval) up to 25 September 2021. COVID-19 cases, severity, seriousness, outcomes, vaccination status, and breakthrough infection were evaluated. RESULTS: As of 25 September 2021, 245 of 1703 patients (14.4%) enrolled in ALITHIOS receiving ofatumumab (median exposure: 2.45 years) reported COVID-19 (confirmed: 210; suspected: 35). Most COVID-19 was of mild (44.1%) or moderate (46.5%) severity, but 9% had severe/life-threatening COVID-19. There were 24 serious cases (9.8%) with 23 patients hospitalized; 22 recovered and 2 died. At study cut-off, 241 patients (98.4%) had recovered or were recovering or had recovered with sequelae and 2 (0.8%) had not recovered. Ofatumumab was temporarily interrupted in 39 (15.9%) patients. Before COVID-19 onset, IgG levels were within the normal range in all COVID-19-affected patients, while IgM was \u3c 0.4 g/l in 23 (9.4%) patients. No patient had a reinfection. Overall, 559 patients were vaccinated (full, 476; partial, 74; unspecified, 9). Breakthrough infection was reported in 1.5% (7/476) patients, and 11 reported COVID-19 after partial vaccination. As of 25 September 2021, the Novartis Safety Database (~ 4713 patient-treatment years) recorded 90 confirmed COVID-19 cases receiving ofatumumab. Most cases were non-serious (n = 80), and ten were serious (1 medically significant, 9 hospitalized, 0 deaths). Among 36 of 90 cases with outcomes reported, 30 recovered and 6 did not recover. CONCLUSION: COVID-19 in RMS patients on ofatumumab was primarily of mild/moderate severity and non-serious in these observational data. Most recovered from COVID-19 without treatment interruption. Two people died with COVID-19. Breakthrough COVID-19 despite being fully/partially vaccinated was uncommon

Prognostic value of serum neurofilament light chain for disease activity and worsening in patients with relapsing multiple sclerosis: Results from the phase 3 ASCLEPIOS I and II trials

OBJECTIVE: This study aims to confirm the prognostic value of baseline serum neurofilament light chain (sNfL) for on-study disease activity and worsening in patients with relapsing MS (RMS). BACKGROUND: Previous DESIGN/METHODS: In this study, we report protocol-planned analyses from the pooled ASCLEPIOS I/II trials (N=1882). Per protocol, patients were stratified by median baseline sNfL levels (9.3 pg/ml) into high (\u3emedian) and low (≤median) categories to prognosticate: annualized rate of new/enlarging T2 (neT2) lesions in year 1 and 2, annualized relapse rate, annual percentage change in whole brain (WB) and regional brain volume [thalamus, white matter (WM), cortical gray matter (cGM)], and disability outcomes. Similar analyses were performed for the recently diagnosed (within 3 years), treatment-naive patients (no prior disease-modifying therapy) subgroup. RESULTS: High versus low sNfL at baseline was prognostic of increased on-study T2 lesion formation at year 1 (relative increase: ofatumumab +158%; teriflunomide +69%, both p\u3c0.001), which persisted in year 2 (+65%, p=0.124; +46%, p=0.003); of higher annual percentage change of WB volume (ofatumumab, -0.32% vs. -0.24%, p=0.044, and teriflunomide, -0.43% vs. -0.29%, p=0.002), thalamic volume (-0.56% vs. -0.31%, p=0.047 and -0.94% vs. -0.49%, p\u3c0.001), and WM volume (-0.30% vs. -0.19%, p=0.083 and -0.38% vs. -0.18%, p=0.003) but not of cGM volume (-0.39% vs. -0.32%, p=0.337 and -0.49% vs. -0.46%, p=0.563). A single sNfL assessment at baseline was not prognostic for on-study relapses or disability worsening. Results were similar in the subgroup of recently diagnosed, treatment-naive patients. CONCLUSION: This study confirms that baseline sNfL levels are prognostic of future on-study lesion formation and whole brain and regional atrophy in all RMS patients, including recently diagnosed, treatment-naive patients

Differential effects of anti-CD20 therapy on CD4 and CD8 T cells and implication of CD20-expressing CD8 T cells in MS disease activity

A small proportion of multiple sclerosis (MS) patients develop new disease activity soon after starting anti-CD20 therapy. This activity does not recur with further dosing, possibly reflecting deeper depletion of CD20-expressing cells with repeat infusions. We assessed cellular immune profiles and their association with transient disease activity following anti-CD20 initiation as a window into relapsing disease biology. Peripheral blood mononuclear cells from independent discovery and validation cohorts of MS patients initiating ocrelizumab were assessed for phenotypic and functional profiles using multiparametric flow cytometry. Pretreatment CD20-expressing T cells, especially CD2

Exploring the association between weight loss-inducing medications and multiple sclerosis: Insights from the FDA adverse event reporting system database

BACKGROUND: Several studies have demonstrated that early childhood and adolescent obesity are risk factors for multiple sclerosis (MS) susceptibility. Obesity is thought to share inflammatory components with MS through overproduction of pro-inflammatory adipokines (e.g., leptin) and reduction of anti-inflammatory adipokines (e.g, adiponectin). Recently, drug repurposing (i.e. identifying new indications for existing drugs) has garnered significant attention. The US Food and Drug Administration Adverse Event Reporting System (FAERS) database serves not only as a resource for mining adverse drug reactions and safety signals but also for identifying inverse associations and potential medication repurposing opportunities. OBJECTIVE: We aimed to explore the association between weight-loss-inducing drugs and MS using real-world reports from the FAERS database. DESIGN: Secondary analysis of existing data from the FAERS database. METHODS: We conducted a disproportionality analysis using the FAERS database between the fourth quarter of 2003 and the second quarter of 2023 to explore associations between MS and weight loss-inducing drugs. Disproportionality was quantified using the reporting odds ratio (ROR). An inverse association was defined when the upper limit of the 95% confidence interval for ROR was \u3c1. RESULTS: We found an inverse association between MS and anti-diabetic weight loss-inducing drugs including semaglutide (ROR: 0.238; 95% CI: 0.132-0.429), dulaglutide (ROR: 0.165; 95% CI: 0.109-0.248), liraglutide (ROR: 0.161; 95% CI: 0.091-0.284), empagliflozin (ROR: 0.234; 95% CI: 0.146-0.377), and metformin (ROR: 0.387; 95% CI: 0.340-0.440). No inverse associations were found for other weight loss-inducing drugs such as phentermine, bupropion, topiramate, zonisamide, and amphetamine. An exception was naltrexone (ROR: 0.556; 95% CI: 0.384-0.806). CONCLUSION: Our findings suggest a potential consideration for repurposing anti-diabetic weight loss-inducing drugs including semaglutide, dulaglutide, and liraglutide (glucagon-like peptide-1 receptor agonists), empagliflozin (sodium-glucose cotransporter-2 inhibitor), and metformin (biguanide), for MS. This warrants validation through rigorous methodologies and prospective studies

Blood neurofilament light levels predict non-relapsing progression following anti-CD20 therapy in relapsing and primary progressive multiple sclerosis: Findings from the ocrelizumab randomised, double-blind phase 3 clinical trials

BACKGROUND: Neurofilament light chain (NfL), a neuronal cytoskeletal protein that is released upon neuroaxonal injury, is associated with multiple sclerosis (MS) relapsing activity and has demonstrated some prognostic ability for future relapse-related disease progression, yet its value in assessing non-relapsing disease progression remains unclear. METHODS: We examined baseline and longitudinal blood NfL levels in 1421 persons with relapsing MS (RMS) and 596 persons with primary progressive MS (PPMS) from the pivotal ocrelizumab MS trials. NfL treatment-response and risk for disease worsening (including disability progression into the open-label extension period and slowly expanding lesions [SELs] on brain MRI) at baseline and following treatment with ocrelizumab were evaluated using time-to-event analysis and linear regression models. FINDINGS: In persons from the RMS control arms without acute disease activity and in the entire PPMS control arm, higher baseline NfL was prognostic for greater whole brain and thalamic atrophy, greater volume expansion of SELs, and clinical progression. Ocrelizumab reduced NfL levels vs. controls in persons with RMS and those with PPMS, and abrogated the prognostic value of baseline NfL on disability progression. Following effective suppression of relapse activity by ocrelizumab, NfL levels at weeks 24 and 48 were significantly associated with long-term risk for disability progression, including up to 9 years of observation in RMS and PPMS. INTERPRETATION: Highly elevated NfL from acute MS disease activity may mask a more subtle NfL abnormality that reflects underlying non-relapsing progressive biology. Ocrelizumab significantly reduced NfL levels, consistent with its effects on acute disease activity and disability progression. Persistently elevated NfL levels, observed in a subgroup of persons under ocrelizumab treatment, demonstrate potential clinical utility as a predictive biomarker of increased risk for clinical progression. Suppression of relapsing biology with high-efficacy immunotherapy provides a window into the relationship between NfL levels and future non-relapsing progression. FUNDING: F. Hoffmann-La Roche Ltd